Anti-cd79b antibodies and immunoconjugates and methods for using them

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. There are presented versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a specified amino acid sequence and at least one free cysteine amino acid residue specified in A118C (according to the European Numeration) in the heavy chain and V205C (according to the Kabat numeration) in the light chain. There are disclosed: versions of a conjugate compound of the antibody and a drug preparation, wherein the antibody is bond to the drug preparation through free cysteine; an antibody-based pharmaceutical compound for treating cancer; method for detecting CD79b or cancer cells, as well as a method for inhibiting cell proliferation using the conjugate compound. What is described is a method for producing the conjugate compound.

EFFECT: invention can find further application in the therapy of CD79b-associated cancer diseases, including treating haemopoietic tumours in mammals.

70 cl, 20 tbl, 9 ex, 51 dwg

 

Cross-reference to related applications

Under this non-provisional application filed in accordance with article 37, § 1.53(b) of the code of Federal Regulations (CFR), the priority of provisional patent application U.S. No. 61/025137, filed January 31, 2008, provisional patent application U.S. reg. No. 61/032790, filed February 29, 2008, and provisional patent application U.S. reg. No. 61/054709, filed may 20, 2008, in accordance with article 35, § 119(e) of the Code of laws of the United States, with a description of each of these applications is fully incorporated into the present application by reference.

The scope to which the invention relates

The present invention relates to compositions that can be used for the treatment of hematopoietic tumor in mammals and to methods of using such compositions.

Prior art

In the USA a malignant tumor (cancer), after heart disease, are the second leading disease, leading to death (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer is characterized by an increase in the number of abnormal, or neoplastic cells derived from normal tissue, which proliferum with the formation of the tumor mass and invasion of adjacent tissues these neoplastic tumor cells, with the formation of malignant cells which eventually spread�safety through the bloodstream or lymphatic system to regional lymph nodes and in the peripheral region on the mechanism, called metastasis. When cancer cells proliferate under conditions in which normal cells can not grow. Itself cancer the disease manifests in different forms for a wide range, characterized by different degrees of invasiveness and aggressiveness.

Cancers involving cells generated in the process of hematopoiesis, i.e. the process by which the formed cellular elements of blood, such as lymphocytes, leukocytes, platelets, erythrocytes and natural killer cells are called cancer of the haematopoietic system. Lymphocytes that can be detected in the blood and in the lymphatic tissues and play a crucial role in the immune response, are divided into two major classes: b-lymphocytes (b cells) and T lymphocytes (T cells), which mediate humoral and cell-mediated immune response, respectively.

B-cells Mature in the bone marrow and leave the marrow, by expressing on their surface the antigen-binding antibody. After the first contact "untrained" b-cells with the antigen, in which a membrane-bound antibody is specific, the cells start to divide rapidly, and their progeny differentiates into B-memory cells and effector cells called "plasma cells". In-memory cells have a longer lifetime, etc�continue to Express membrane-bound antibody, which has the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody, but instead they produce the antibody in a form that can secretariats. Sekretiruemyi antibodies are the main effector molecules of the humoral immune response.

T-cells Mature in the thymus and create conditions for the proliferation and differentiation of immature T cells. In the process of maturation of T-cells exhibit rearrangeable genes, which leads to the production of the T-cell receptor and undergo positive and negative selection, which facilitates the determination of the cell surface phenotype of Mature T cells. Characteristic cell surface markers of Mature T cells are the complexes of the CD3:T-cell receptor and one of coreceptors, CD4 or CD8.

In trying to identify effective cellular targets for cancer therapy studies have been conducted to identify transmembrane or other membrane-bound polypeptides that are specifically expressed on the surface of cancer cells of one or more specific types compared with one or more normal non-cancer cells. In most cases, these membrane-bound polypeptides in a large number of them are expressed at the surface�T. cancer cells but not on the surface of cancer cells. Identification of such associate with tumor polypeptides of antigens on the cell surface provides the opportunity to specifically destroy cancer target cells by therapy with the use of antibodies. In this regard, it should be noted that treatment based on antibodies proved to be very effective for the treatment of certain cancers. So, for example, Herceptin (HERCEPTIN®) and Rituxan (RITUXAN®) (both antibodies supplied by Genentech Inc., South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a humanized monoclonal antibody, which was obtained by the methods of recombinant DNA and which selectively binds to the extracellular domain of the proto-oncogene receptor human epidermal growth factor 2 (HER2). Overexpression of HER2 protein was observed in 25-30% of cases of primary breast cancer. RITUXAN® is a genetically engineered chimeric monoclonal antibody mouse/man" directed against the CD20 antigen located on the surface of normal and malignant b lymphocytes. Both of these antibodies recombinante are produced in cells SNO.

In other attempts to identify effective �gunning targets for anticancer therapy studies have been conducted to identify (1) polypeptides, are not membrane-bound and which, unlike normal non-cancer cells of one or more specific types, specifically produced by one or more cancer cells of specific types, (2) polypeptides produced by cancer cells on the expression level significantly exceeding the level of expression of the polypeptide by one or more normal non-cancer cells, or (3) polypeptides whose expression, in particular, is limited to the tissues of the same type (or a very limited number of other types of tissues) affected and not affected by cancer (e.g., normal prostate tissue and tumor tissue of the prostate gland). Such polypeptides can be localized inside the cells, or they can secretariats cancer cells. Furthermore, such polypeptides can be expressed is not the tumor cells themselves, the cells that produce and/or secrete the polypeptides, providing a potentiating effect on cancer cells or stimulating the growth of cancer cells. In most cases, such sekretiruemyi polypeptides are proteins that provide the cancer cells but not normal cells, preferential growth, and such polypeptides are, for example, angiogenic factors, cell adhesion factors, facto�s growth, etc. This assumes that the identification of antagonists of these polypeptides, which are not membrane-bound, will identify effective therapeutic agents for treatment of these cancers. In addition, identification of the expression pattern of these polypeptides can be used to diagnose specific cancers in mammals.

Despite the aforementioned progress in anti-cancer therapy in mammals, the need for additional therapeutic agents capable of detecting the presence of tumor in mammals and to effectively inhibit the growth of tumor cells, respectively, remains particularly relevant. Accordingly, the present invention is the identification of polypeptides, membrane-bound, secreted or intracellular polypeptides whose expression, in particular, is limited to the tissues of only one type (or a very limited number of other types of tissues), hematopoietic tissues, affected and not affected by cancer; and the use of such polypeptides and nucleic acids encoding these polypeptides, to obtain compositions according to the invention, which can be used for therapy and/or diagnosis of hematopoietic cancer in mammals.

CD79 is a with�Galiny component b-cell receptor, consisting of covalently bound heterodimer containing CD79a (Igα, mb-1) and CD79b (Igβ, B29). Each of CD79a and CD79b contains extracellular immunoglobulin domain (Ig), transmembrane domain, intracellular signaling domain and activation domain of immunoreceptor having a tyrosine motif (ITAM). CD79 is expressed on B-cells and in cells of non-Hodgkin's lymphoma (NHL) (big head et al., Haematologica, 84:413-418 (1999); D'arena et al., Am. J. Hematol., 64:275-281 (2000); Olejniczak et al., Immunol. Invest., 35:93-114 (2006)). All CD79a and CD79b and sIg required for surface expression of CD79 (Matsuuchi et al., Curr. Opin. Immunol., 13(3):270-7)). The average level of surface expression of CD79b in the NHL is similar to its expression on normal b cells, but in a wider range (Matsuuchi et al., Curr. Opin. Immunol., 13(3):270-7 (2001)).

With regard to the expression of CD79b, we can say that it is more effective in producing therapeutic antibodies against CD79b antigen and has minimal or no antigenicity has the antigenicity when administered to patients, and particularly during prolonged treatment. The present invention meets these and other requirements. The present invention relates to anti-CD79b antibodies, which do not have the disadvantages inherent in the current therapeutic compositions, as well as offer other advantages which will be apparent from the following detailed description.

Using conju�of ATA "antibody-drug" (ADC), that is immunoconjugates, in order for local delivery of cytotoxic or cytostatic agents, e.g., drugs, to kill or suppress the growth of tumor cells in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al., (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) Cancer Cell 3:207-212; Syrigos & Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz & Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. patent No. 4975278) allows targeted delivery of the drug in the tumor and to ensure their accumulation inside the cells, whereas systemic administration of these unconjugated drug agents can lead to the production of levels of toxicity that are unacceptable for normal cells and insufficient for the destruction of tumor cells (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986):603-05; Thorpe, 1985, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antobodies 84:Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). In attempts to increase therapeutic index, i.e. to maximize the efficacy and minimize the toxicity of ADC, all efforts were directed at improving the selectivity of polyclonal antibodies (Rowland et al. (1986), Cancer Immunol. Immunother. 21:183-87) and monoclonal antibodies (mAb), as well as to improve properties such as binding to the medication, and releasing drugs (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drug�public funds used in the conjugate "antibody-drug", are bacterial protein toxins, such as diphtheria toxin, plant protein toxins, such as ricin, small molecule, such as auristatin, geldanamycin (Mandler et al., (2000) J. of the Nat.Cancer Inst. 92 (19):1573-1581; Mandler et al.(2000), Bioorganic &Med. Chem. Letters 10:1025-1028; Mandler et al. (2002), Bioconjugate Chem. 13:786-791), maytansinoid (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al. (1986) supra). These medicines can influence the cytotoxic and cytostatic mechanisms, including binding to tubulin, DNA binding or inhibition of topoisomerase. Some cytotoxic drugs, with their conjugation with large antibodies or protein ligands-receptors, tend to loss or reduction of activity.

Medicines, namely auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), i.e. synthetic analogs of dolastatin (WO 02/088172) were anywherevery: (i) chimeric monoclonal antibodies cBR96 (specific to the antigen Lewis Y on carcinomas); (ii) with SAS, which is specific to CD30, who is present on hematological malignancies (Klussman et al. (2004) Bioconjugate Chemistry 154):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; Francisco et al. (2003) Blood 102(4):1458-1465; the publication of the patent application U.S. 2004/0018194); (iii) anti-CD20 antibodies, such as Rituxan® (WO 04/032828) used for the treatment of CD20-expressing cancers and immune disorders; (iv) with anti-EphB2R antibody N used to treat colorectal cancer and colon cancer (Mao et al. (2004) Cancer Research 64(3):781-788); (v) with an antibody against E-selectin (Bhaskar et al. (2003) Cancer Res. 63:6387-6397); (vi) with trastuzumab (HERCEPTIN®, order USA 2005/0238649), and (vii) anti-CD30 antibodies (WO 03/043583). Options auristatin described in U.S. patents 5767237 and 6124431. Monomethylaniline E, conjugated with monoclonal antibodies is described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published March 28, 2004. Auristatin analogues of MMAE and MMAF were anywhereman with different antibodies (application USA 2005/0238649).

The standard method of attachment, i.e. covalent binding of the molecule of the drug with the antibody, essentially allows to obtain a heterogeneous mixture of molecules in which the drug is attached to various portions of antibody molecules. So, for example, cytotoxic drugs usually kongugiruut with antibodies by a large number of lysine residues of the antibody with obtaining a heterogeneous mixture of conjugate “antibody-drug” depending on the reaction conditions heterogeneous mixture usually contains a certain number of antibodies, to which is annexed from 0 to about 8 or more molecules associated medicines. In addition, each subgroup conjugates, with respect to molecules of the drug to the antibody molecules equal to a specific integer, may be a heterogeneous mixture, in which the molecule of the drug attached to different sites of the antibody. Analytical and preparative methods can be unsuitable for the separation and characterization of molecules of conjugates of the antibody-drug” in a heterogeneous mixture resulting from the reaction of conjugation. Antibodies are large, complex and differing in the structure of biomolecules, which in most cases have a variety of reactive functional groups. The ability of these groups to react with linker reagents and intermediate compounds “drug-linker” depends on factors such as pH, concentration, salt concentration and the presence of cosolvent. In addition, the multistep method of conjugation can be playable, due to the difficulties of regulation of the reaction conditions and characterization of reagents and intermediates.

Unlike most amines that are protonated and less nucleophilic at pH~7, the thiol� cysteines are reactive at neutral pH. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins containing cysteine residues, are often oxidized form and are associated with disulfide oligomers, or they contain internal bridged disulfide groups. Extracellular proteins usually do not contain free thiol groups (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, page 55). The thiol group of cysteine antibodies are typically more reactive, more nucleophilic to electrophilic reagents of conjugation than the amino group or the hydroxyl group of the antibody. Cysteine residues were introduced in proteins by means of genetic engineering with the formation of covalent bonds with ligands or with the formation of new intermolecular disulfide bonds (Better et al. (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al. (1994) Bioconjugate Chem. 5:126-132; Greenwood et al. (1994) Therapeutic Immunology 1:247-255; Tu et al. (1999) Proc. Natl. Acad. Sci USA 96:4862-4867; Kanno et al. (2000) J. of Biotechnology, 76:207-214; Chmura et al. (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. patent 6248564). However, the design of the thiol groups of cysteine by substituting various amino acid residues of the protein cysteine residues can be associated with certain problems, especially if there are unbound (free Cys) residues or residues that are relatively available for R�shares or oxidation. In concentrated solutions of the protein, irrespective of whether they are present in the periplasm of E. coli, the culture supernatants or are partially or completely purified protein, unbound Cys residues on the protein surface may contact and oxidize with the formation of intermolecular disulfides, and hence dimers or multimeric protein. The formation of disulfide dimers tells the new Cys residue inability to form conjugates with a drug, ligand or with a different label. In addition, if the protein in the oxidation forms an intramolecular disulfide bond between the new engineered Cys and Cys residue, both Cys thiol group becomes unavailable for operation in the active site and interactions. In addition, this protein can become inactive or non-specific as a result of improper installation or the loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311:1-9).

Designed on the basis of cysteine antibodies were obtained in the form of FAB-fragments of antibodies (thio-Fab) and expressed as full-size monoclonal IgG antibody (thio-Mab) (Junutula, J. R. et al. (2008) J. Immunol Methods 332:41-52; application USA 2007/0092940, the content of which is introduced into the present description by reference). Antibodies thio-Fab and thio-Mab were anywherevery through the linkers to the provisions of the Novo�introduced cysteine thiols using a thiol-reactive linker reagents and reagents "drug-linker" obtaining conjugates of the antibody-drug" (ist-ADC).

All quotations from this work, including patent applications and publications are fully incorporated into this description by reference.

Description of the invention

The present invention relates to anti-CD79b antibodies or their functional fragments, as well as to method of their use for the treatment of hematopoietic tumors.

In one of its aspects the present invention relates to an antibody which binds, preferably specifically, to any of the above or below described polypeptides. This antibody is, but not necessarily, a monoclonal antibody, a fragment of the antibody, including Fab, Fab', F(ab')2- and Fv-fragment, denticula, single-domain antibody, a chimeric antibody, a humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be but are not necessarily, anywhereman with the growth-inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin, maytansinoid, derived dolastatin or calicheamicin, an antibiotic, a radioactive isotope, nucleotidase enzyme or etc. the Antibody according to the invention can be, but not necessarily, produced in Cho cells or bacterial� cells, and preferably induce death of the cells to which they bind. Antibodies according to the invention is used for detection, can be-detectable labeled, attached to a solid carrier, or etc.

In one of its aspects the present invention relates to humanitariannet anti-CD79b antibody, where the monovalent affinity of the antibody (e.g., affinity of the antibody used as the Fab-fragment against CD79b) or affinity bivalent form antibodies against CD79b (e.g., affinity of the antibody used as an IgG fragment against CD79b) is substantially the same, lower or higher than the monovalent affinity or affinity divalent, respectively, of a murine antibody (e.g. affinity of the murine antibody, used as a Fab fragment or IgG fragment against CD79b) or a chimeric antibody (e.g., affinity of the chimeric antibody, used as a Fab fragment or IgG fragment against CD79b), comprising the sequence of the variable domain of the light and heavy chains, or consisting of or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates humanitariannet anti-CD79b antibody, where the specified affinity of the antibody in its bivalent� form to CD79b (e.g., the affinity of antibodies of the IgG type against CD79b) is 0.4 nm, 0.2 nm or 0.5 nm.

In one of its aspects the present invention relates to an antibody that binds to CD79b, where the specified antibody contains at least one, two, three, four, five or six HVR selected from the group consisting of:

(i) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO: 131)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO: 132)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a QQSNEDPLT (SEQ ID NO: 133)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO: 134)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO: 135), and

(vi) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPVYFDY (SEQ ID NO: 136).

In one of its aspects the present invention relates to an antibody that binds to CD79b, where the specified antibody contains at least one variant HVR, where the specified variant HVR contains a modification of at least one residue of the sequence represented in SEQ ID NO.: 131, 132, 133, 134, 135 or 136.

In one of its aspects the present invention relates to an antibody comprising the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or� HVR3-HC, presented on the figure 15 (SEQ ID NO: 164 to 166).

In one of its aspects the present invention relates to an antibody comprising a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 15 (SEQ ID NO: 156-158).

In one of its aspects the present invention relates to an antibody comprising the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 16 (SEQ ID NO: 183-185).

In one of its aspects the present invention relates to an antibody comprising a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 16 (SEQ ID NO: 175-177).

In one of its aspects the present invention relates to an antibody comprising the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 17 (SEQ ID NO: 202-204).

In one of its aspects the present invention relates to an antibody comprising a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 17 (SEQ ID nos: 194-196).

In one of its aspects the present invention relates to an antibody comprising the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 18 (SEQ ID NO: 221-223).

In �bottom of its aspects the present invention relates to an antibody, comprising a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 18 (SEQ ID NO: 213 to 215).

In one of its aspects the present invention relates to anti-CD79b antibody containing the variable domain of the heavy chain selected from SEQ ID NO: 170, 189, 208 or 227. In another aspect, the present invention relates to anti-CD79b antibody containing the variable domain light chain selected from SEQ ID NO: 169, 188, 207 or 226.

In one of its aspects the present invention relates to constructed on the basis of cysteine anti-CD79b antibody comprising one or more free cysteine amino acids and a sequence selected from SEQ ID NO: 251-298. Constructed of cysteine-based anti-CD79b antibody may contact the CD79b polypeptide. Constructed of cysteine-based anti-CD79b antibody can be obtained by a method comprising replacing one or more amino acid residues of a parent anti-CD79b antibody by cysteine.

In one of its aspects the present invention relates to constructed on the basis of cysteine anti-CD79b antibody that contains one or more free amino acids cysteine, where the specified constructed on the basis of cysteine anti-CD79b antibody binds to a CD79b polypeptide, and where the specified antibody was obtained.�Ohm, includes replacement of one or more amino acid residues of a parent anti-CD79b antibody by cysteine, where the specified parent antibody comprises at least one HVR sequence selected from:

(a) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO: 131) or KASQSVDYEGDSFLN (SEQ ID NO: 137);

(b) HVR-L2 containing a sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO: 132);

(c) HVR-L3, containing the sequence C1-C9, where C1-C9 is a QQSNEDPLT (SEQ ID NO: 133);

(d) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO: 134);

(e) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO: 135), and

(f) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPVYFDY (SEQ ID NO: 136) or TRRVPIRLDY (SEQ ID NO: 138).

Constructed of cysteine-based anti-CD79b antibody may be a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be but are not necessarily, anywhereman with the growth-inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin or m�stanzino. Antibodies according to the invention can be, but not necessarily, produced in Cho cells or bacterial cells and preferably these antibodies inhibit the growth or proliferation of cells to which they bind, or induce the death of these cells. Antibodies according to the invention is used for diagnostic purposes, can be-detectable labeled, attached to a solid carrier, or etc.

In one of its aspects the present invention relates to methods for producing antibodies according to the invention. For example, the present invention relates to a method for producing an anti-CD79b antibody (which, as defined in the present application, includes a full-sized antibody and its fragments), where the specified method comprises the expression in a suitable the host cell a recombinant vector according to the invention, encoding the indicated antibody (or its fragment), and the allocation of the specified antibodies.

In one of its aspects the present invention relates to pharmaceutical compositions comprising the antibody according to the invention or conjugate "antibody-drug" according to the invention and a pharmaceutically acceptable diluent, carrier or excipient.

In one of its aspects the present invention relates to industrial product comprising a container, and to compositions containing�cut in this container, where the specified composition includes one or more anti-CD79b antibodies according to the invention.

In one of its aspects the present invention relates to a kit comprising a first container containing a composition comprising one or more anti-CD79b antibodies according to the invention and a second container containing a buffer.

In one of its aspects the present invention relates to the use of anti-CD79b antibodies according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder.

In one of its aspects the present invention relates to the use of industrial products according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder.

In one of its aspects the present invention relates to the use of the kit according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder.

In one of its aspects the present invention relates to a method of inhibiting growth of cells expressing CD79b, where said method comprises contacting the specified cell with the antibody according to the invention and thus the inhibition of growth of the specified cell. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of therapeutic treatment of a mammal having a cancerous tumor that contains cells expressing CD79b, where the method includes introduction to the specified mammal a therapeutically effective amount of the antibody according to the invention and thus effective treatment of the specified mammal. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of treating or preventing a cell proliferative disorder associated with increased levels of expression of CD79b, where the specified method comprises administering to the individual in need of such treatment, an effective amount of an antibody according to the invention and thus effective treatment or prevention of a decree�x cell-proliferative disorder. In one embodiment of the invention the specified proliferative disorder is cancer. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of inhibiting growth of cells that is at least partially dependent on the growth-potentiating action CD79b, where said method comprises contacting the specified cell with an effective amount of an antibody according to the invention and thereby inhibiting the growth of these cells. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of therapeutic treatment of a tumor in a mammal, the growth of which is at least partially dependent on the growth-potentiating action CD79b, where said method comprises contacting the specified cell with an effective amount of an antibody according to the invention and thus effective treatment of the indicated tumor. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. Water from variants of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of treating cancer, comprising administering to the patient a pharmaceutical composition containing described here immunoconjugate and acceptable diluent, carrier or excipient.

In one of its aspects the present invention relates to a method of inhibiting the proliferation of b cells, comprising treating cells immunoconjugates containing the antibody according to the invention, under conditions conducive to binding immunoconjugate with CD79b.

In one of its aspects the present invention relates to a method of detecting the presence of CD79b in a sample that is assumed to contain CD79b, where the specified method comprises treating a specified specimen, the antibody according to the invention and determining the level of binding of the indicated antibody to CD79b in a specified sample, where the specified binding of the antibody to CD79b in a specified sample is indicative of the presence of the indicated protein in the sample.

In one of its aspects the present invention relates to a method of diagnosing a cellular proliferative disorder associated with increased number of cells, such as b cells expressing CD79b, where said method comprises contacting the test cells in a biological sample with any of the above antibodies; defined�tion antibody, associated with the test cells in the sample by detecting binding of the indicated antibody to CD79b; and comparing levels of antibody bound to cells in a control sample, where the level of bound antibodies normalize by the number of CD79b-expressing cells in the test and control samples, and where a higher level of bound antibody in the sample compared to a control sample, indicates the presence of cell-proliferative disorder associated with cells expressing CD79b.

In one of its aspects the present invention relates to a method for the detection of soluble CD79b in blood or serum, where the method comprises contacting the specified test sample of blood or serum from a mammal who is suspected b-cell-proliferative disorder with an anti-CD79b antibody according to the invention, and the detection of increased level of soluble CD79b in the tested blood sample is compared with its level in the control sample of blood or serum taken from a healthy mammal.

In one of its aspects the present invention relates to a method of binding antibodies according to the invention with a cell that expresses CD79b, where said method comprises contacting the specified cell with the specified antibody according to the invention. � one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

Brief description of graphic material

Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) cDNA PRO36249, where SEQ ID NO: 1 is a clone designated “DNA225786" (also referred to herein as “CD79b"). The nucleotide sequence encodes CD79b with start and stop codons are in bold and underlined.

Figure 2 shows the amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: 1 presented on figure 1.

Figure 3 presents the nucleotide sequence (SEQ ID NO: 3) light chain chimeric murine anti-CD79b antibody (chMA79b) IgG1 (MA79b is a murine monoclonal anti-CD79b antibody). The nucleotide sequence encodes the light chain chMA79b with start and stop codons are in bold and underlined.

Figure 4 presents the amino acid sequence (SEQ ID NO: 4), containing the first 18 amino acid signal sequence and derived from the coding sequence of SEQ ID NO: 3, are presented in figure 3. Variable regions are not underlined.

Figure 5 presents the nucleotide sequence (SEQ ID NO: 5) of the heavy chain of chimeric mouse antibodies (chMA79b) IgG1 (MA79b performance�possessing a murine monoclonal anti-CD79b antibody). The nucleotide sequence encodes a heavy chain chMA79b with start and stop codons are in bold and underlined.

Figure 6 presents the amino acid sequence (SEQ ID NO: 6), containing the first 18 amino acids of the signal sequence and the last of lysine (K) before the stop codon and derived from the coding sequence of SEQ ID NO: 5, as presented in figure 5. Variable regions are not underlined.

In the figures 7A-B show the alignment of sequences of variable light chain: consensus sequences of human light chain Kappa I (labeled as "huKI"; SEQ ID NO: 9) with VL-FR1, VL-FR2, VL-FR3, VL-FR4 (SEQ ID NO: 139-142, respectively), murine anti-CD79b antibody (labeled as "MA79b"; SEQ ID NO: 10), MA79b-associated "humanized" antibody (labeled as "huMA79b-hybrid'; SEQ ID NO: 11), MA79b-associated case 17 "humanized" antibody (labeled as "huMA79b.v17"; SEQ ID NO: 169), MA79b-associated case 18 "humanized" antibody (labeled as "huMA79b.v18"; SEQ ID NO: 188), MA79b-associated case 28 "humanized" antibody (labeled as "huMA79b.v28"; SEQ ID NO: 207) and MA79b-associated case 32 "humanized" antibody (labeled as "huMA79b.v32”; SEQ ID NO: 226). The provisions are numbered in Cabatu, and hypervariable region (HVR) of MA79b-joined the consensus frame region variabel�Noi region light chain Kappa (I, specified in the framework.

In the figures 8A-B illustrate the alignment of sequences of the variable regions of heavy chains: a consensus sequence of human heavy chain subgroup III (marked "humIII"; SEQ ID NO: 13) with VH-FR1, VH-FR2, VH-FR3, and VH-FR4 (SEQ ID nos: 143-146), murine anti-CD79b antibody (labeled as "MA79b"; SEQ ID NO: 14), MA79b-associated "humanized" antibody (labeled as "huMA79b-hybrid'; SEQ ID NO: 15) (containing 71A, 73T and 78A), MA79b-associated case 17 "humanized" antibody (labeled as "huMA79b.v17"; SEQ ID NO: 170) (containing 71A, 73T and 78A), MA79b-associated case 18 "humanized" antibody (labeled as "huMA79b.v18"; SEQ ID NO: 189) (containing 71A, 73T and 78A), MA79b-associated case 28 "humanized" antibody (labeled as "huMA79b.v28"; SEQ ID NO: 208) (containing 71A, 73T and 78A) and MA79b-associated case 32 "humanized" antibody (labeled as "huMA79b.v32”; SEQ ID NO: 227) (containing 71A, 73T and 78A). The provisions are numbered in Cabatu, and hypervariable region (HVR) of MA79b-joined the consensus frame region of the variable region of the heavy chain subgroup III, specified in the framework.

Figure 9 shows various HVR sequences of selected variants of MA79b-associated "humanized" antibody (SEQ ID NO: 17-21), where each variant has a single amino acid substitution in one HVR MA79b-associated "humanised" and�of tetela (HVR-L1 (SEQ ID NO: 131); HVR-L2 (SEQ ID NO: 132); HVR-L3 (SEQ ID NO: 133)). Sequence of variable region light chain and the variable region of the heavy chain that is outside of the specified one amino acid replacement that is identical huMA79b-hybrid and the figure is not shown. Any changes in HVR-H1 (SEQ ID NO: 134), HVR-H2 (SEQ ID NO: 135) or HVR-H3 (SEQ ID NO: 136) MA79b-associated "humanized" antibodies are not observed.

Figure 10 shows various HVR sequences of selected variants of MA79b-associated "humanized" antibody (SEQ ID NO: 22-106), including huMA79b L2-2 (also denoted here "L2") and huMA79b H3-10 (also denoted here "H3"), where each variant has many amino acid substitutions in one area HVR MA79b-associated "humanized" antibody (HVR-L2 (SEQ ID NO: 132); HVR-L3 (SEQ ID NO: 133); HVR-H1 (SEQ ID NO: 134); the portion of HVR-H3 (SEQ ID NO: 136) is shown in figure 10 as SEQ ID NO: 107). Sequence of variable region light chain and the variable region of the heavy chain that is outside of the specified amino acid substitutions, identical sequences huMA79b-hybrid and the figure is not shown. Any changes in HVR-L1 (SEQ ID NO: 131) or HVR-H2 (SEQ ID NO: 135) MA79b-associated "humanized" antibodies are not observed.

Figure 11 illustrates Biacore analysis of selected anti-CD79b antibodies, including murine anti-CD79b antibody (labeled as "MA79b"), MA79b-bound "humanized" antibody (convoy�Achensee "huMA79b-hybrid") and variants of MA79b-associated "humanized" antibodies, including huMA79b L2-2 (52R, 53K, 55G, 56R; SEQ ID NO: 22), huMA79b H3-10 (98I, 99R, 100L; SEQ ID NO: 94), huMA79b H1-6 (28P, 30T, 31R, 35N; SEQ ID NO: 57) and huMA79b L2/H3 (mutation L2-2 and H3-10, described below) against these antigens, including the extracellular domain of human CD79b (huCD79becd), the extracellular domain of human CD79b attached to Fc (huCD79becd-Fc) and the peptide of 16 amino acids containing the epitope for MA79b and chMA79b (SEQ ID NO: 16).

Figure 12 illustrates a Biacore analysis of selected anti-CD79b antibodies, including MA79b-bound "humanized" antibody (labeled as "huMA79b-hybrid") and variants of MA79b-associated "humanized" antibody (labeled as 1-34 in the first column or as "full frame" in the first column) against the extracellular domain of human CD79b (antigen huCD79b-ecd). Variants of MA79b-associated "humanized" antibodies include options "all frame areas", in which there are potentially important murine skeleton remains, and options (labeled 1-34) with combinations of mutations in the framework region in the presence or in the absence of mutations HVR variable regions of the heavy chain and variable region light chain. Option 17 MA79b-associated "humanized" antibody (labeled here "huMA79b.v17") specified in the first column as 17, version 18 MA79b-associated "humanized" antibody (labeled here "huMA79b.v18") specified in the first column as 18,�stage configuration 28 MA79b-associated "humanized" antibody (labeled here "huMA79b.v28”) specified in the first column as 28, and version 32 MA79b-associated "humanized" antibody (labeled here "huMA79b.v32”) specified in the first column 32. Styling, characteristic of bivalent binding, represented as Kd specific variants of MA79b-associated "humanized" antibody (labeled "Kdvariant”)/Kd chimeric MA79b antibody (chMA79b) (marked “Kdchimera"); the values under the column entitled "laying typical for divalent linking" represent Kdvariant/Kdchimera. Netdetective binding of labeled in the figure as “NDB”.

In the figures 13A-B (consensus frame variable regions of the heavy chain (VH)) and figure 14 (a consensus of a frame variable region light chain (VL)) indicated a representative consensus of frame acceptor sequence of the human antibody used for the implementation of the present invention, along with the sequence identifiers, such as: (figures 13A-B) consensus frame region of a human VH subgroup I without CDR on Kabuto (SEQ ID NO: 108), the consensus frame region of a human VH subgroup I without extended hypervariable regions (SEQ ID NO: 109-111), the consensus frame region of a human VH subgroup II without CDR on Kabuto (SEQ ID NO: 112), the consensus frame region of a human VH subgroup II without the elongated hypervariable�x regions (SEQ ID NO: 113-115), the consensus frame region of a human VH subgroup III without CDR on Kabuto (SEQ ID NO: 116), the consensus frame region of a human VH subgroup III without extended hypervariable regions (SEQ ID nos: 117-119), the acceptor of a frame region of a human VH without CDR on Kabuto (SEQ ID NO: 120), the acceptor of a frame region of a human VH without extended hypervariable regions (SEQ ID NO: 121-122), the acceptor of a frame region of a human VH 2 without CDR on Kabuto (SEQ ID NO: 123) the acceptor of a frame region of a human VH 2 without extended hypervariable regions (SEQ ID NO: 124-26) and (figure 14) the consensus frame region of a human VL Kappa subgroup I (SEQ ID NO: 127), the consensus frame region of a human VL Kappa subgroup II (SEQ ID NO: 128), a consensus human frame region Kappa subgroup III (SEQ ID NO: 129) and a consensus human frame region Kappa subgroup IV (SEQ ID NO: 130).

In the figures 15A (light chain) and 15B (heavy chain) represented by the amino acid sequence of the antibody according to the invention (huMA79b.v17). Figure 15A (light chain) and 15B (heavy chain) shows the amino acid sequence of a frame region (FR), hypervariable region (HVR), first constant domain (CL or CH1) and Fc region (Fc) of one of the variants of the antibody according to the invention (huMA79b.v17) (SEQ ID NO: 152-159 (figure 15A) and SEQ ID NO: 160-168 (figure 15B)). Also performance�see a full-sized amino acid sequences (variable and constant regions) of the light and heavy chains huMA79b.v17 (SEQ ID NO: 303 (figure 15A) and 304 (figure 15B), accordingly, accentuated with constant domains. Presents the amino acid sequence of the variable domains (SEQ ID NO: 169 (figure 15A for light chain) and SEQ ID NO: 170 (figure 15B for the heavy chain)).

In the figures 16A (light chain) and 16B (heavy chain) represented by the amino acid sequence of the antibody according to the invention (huMA79b.v18). Figure 16A (light chain) and 16B (heavy chain) shows the amino acid sequence of a frame region (FR), hypervariable region (HVR), first constant domain (CL or CH1) and Fc region (Fc) of one of the variants of the antibody according to the invention (huMA79b.v18) (SEQ ID NO: 171-178 (figure 16A) and SEQ ID NO: 179-187 (figure 16B)). Also presents a full-sized amino acid sequences (variable and constant regions) of the light and heavy chains huMA79b.v18 (SEQ ID NO: 305 (figure 16A) and 306 (figure 16B), respectively, accentuated with constant domains. Presents the amino acid sequence of the variable domains (SEQ ID NO: 188 (figure 16A for light chain) and SEQ ID NO: 189 (figure 16B for the heavy chain)).

In figures 17A (light chain) and 17B (heavy chain) represented by the amino acid sequence of the antibody according to the invention (huMA79b.v28). Figure 17A (light chain) and 17B (heavy chain) shows the amino acid sequence of a frame region (FR), hypervariable region (HVR), first constant d�MENA (CL or CH1) and Fc region (Fc) of one of the variants of the antibody according to the invention (huMA79b.v28) (SEQ ID NO: 190-197 (figure 17A) and SEQ ID NO: 198-206 (figure 17B). Also presents a full-sized amino acid sequences (variable and constant regions) of the light and heavy chains huMA79b.v28 (SEQ ID NO: 307 (figure 17A) and 308 (figure 17B), respectively, accentuated with constant domains. Presents the amino acid sequence of the variable domains (SEQ ID NO: 207 (figures 7A-b for light chain) and SEQ ID NO: 208 (figures 8A-B for the heavy chain)).

In figure 18A (light chain) and 18B (heavy chain) represented by the amino acid sequence of the antibody according to the invention (huMA79b.v32). In figure 18A (light chain) and 18B (heavy chain) shows the amino acid sequence of a frame region (FR), hypervariable region (HVR), first constant domain (CL or CH1) and Fc region (Fc) of one of the variants of the antibody according to the invention (huMA79b.v32) (SEQ ID NO: 209-216 (figure 18A) and SEQ ID NO: 217-225 (figure 18B). Also presents a full-sized amino acid sequences (variable and constant regions) of the light and heavy chains huMA79b.v32 (SEQ ID NO: 309 (figure 18A) and 310 (figure 18B), respectively, accentuated with constant domains. Presents the amino acid sequence of the variable domains (SEQ ID NO: 226 (figure 18A for light chain) and SEQ ID NO: 227 (figure 18B for the heavy chain)).

Figure 19 illustrates the alignment of amino acid sequences of human CD79b (SEQ ID NO: 2), dog-like a chimpan�n (cyno) (SEQ ID NO: 7) and mouse (SEQ ID NO: 8). The amino acid sequence CD79b of human and dog-like apes are identical at 85%. Also shown are the signal sequence, the test peptide (epitope consisting of 11 amino acids for MA79b, chMA79b and antibodies against CD79b dog-like monkeys as described in example 1; ARSEDRYRNPK (SEQ ID NO: 12)), transmembrane (TM) domain and the domain of motive activation immunoreceptor-based tyrosine (ITAM). The area shown in the frame that represents a region of CD79b, which is absent in spicerianum embodiment, CD79b (as described in example 1).

Figure 20 is a graph showing inhibition of tumor growth in vivo in a BJAB-luciferase model xenotransplanted where from this graph we see that the introduction of anti-CD79b antibodies ((a) chMA79b-SMCC-DM1, loading of the drug is approximately 2.9 (table 9), and (b) huMA79b L2/H3-SMCC-DM1, loading of the drug is approximately 2.4 (table 9)) to SCID mice having human b-cell tumor, leads to a significant inhibition of tumor growth. Control includes Herceptin® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1).

Figure 21A is a graph showing inhibition of tumor growth in vivo models of xenotransplantation Granta-519 (human lymphoma cells of the cerebral cortex), where the specified carafe can be seen that the introduction of anti-CD79b antibodies ((a) chMA79b-SMCC-DM1, loading of the drug is approx�enforcement of 3.6 (table 10), (b) huMA79b.v17-SMCC-DM1, loading of the drug is approximately 3.4 (table 10), (c) huMA79b.v28-SMCC-DM1, loading of the drug is about 3.3 or 3.4 (table 10), (d), huMA79b.v18-SMCC-DM1, loading of the drug is approximately 3.4 (table 10), and (e) huMA79b.v32-SMCC-DM1, loading of the drug is approximately 2.9 (table 10)) to SCID mice having human b-cell tumor, leads to a significant inhibition of tumor growth. Control includes Herceptin® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1). On the 21V figure presents a graph of the percentage by mass of the studied mice compared to xenograft Granta-519 (figure 21A and table 10), indicating the absence of any significant changes of weight during the first 7 days research. "hu" means a humanized antibody, and "ch" means a chimeric antibody.

Figure 22 presents conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" (ADC), where the molecule of the drug attached to an engineered cysteine group in the light chain (LC-ADC); the heavy chain (HC-ADC); and Fc region (Fc-ADC).

Figure 23 illustrates the steps of: (i) recovery of cysteine adducts of disulfides and Mirzayeva and noticablely disulfides in anti-CD79b antibody, designed on the basis�e cysteine (ThioMab), the reducing agent TCEP (hydrochloride Tris(2-carboxyethyl)phosphine); (ii) partial oxidation, i.e., re-oxidation with the formation of Mirzayeva and noticablely disulfides under the action of dhAA (dehydroascorbic acid); and (iii) conjugation of re-oxidized antibody with intermediate connection "drug-linker" with the formation of the conjugate "cysteine anti-CD79b antibody-drug" (ADC).

Figure 24 shows (A) the light chain sequence (SEQ ID NO: 229) and (B) sequence of the heavy chain (SEQ ID NO: 228) of humanized anti-CD79b antibodies constructed with cysteine (thio-huMA79b.v17-HC-A118C), where alanine is present at position 118 in accordance with the European numbering system (position alanine 118 in accordance with a consecutive numbering system; the provision Cabatu - 114) in the heavy chain was replaced with cysteine. Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 25 show�s (A) the light chain sequence (SEQ ID NO: 231) and (B) sequence of the heavy chain (SEQ ID NO: 230) a humanized anti-CD79b antibodies constructed with cysteine (thio-huMA79b.v18-HC-A118C), where alanine is present at position 118 in accordance with the European numbering system (position alanine 118 in accordance with a consecutive numbering system; the provision Cabatu - 114) in the heavy chain was replaced with cysteine. Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 26 shows (A) the light chain sequence (SEQ ID NO: 233) and (B) sequence of the heavy chain (SEQ ID NO: 232) a humanized anti-CD79b antibodies constructed with cysteine (thio-huMA79b.v28-HC-A118C), where alanine is present at position 118 in accordance with the European numbering system (position alanine 118 in accordance with a consecutive numbering system; the provision Cabatu - 114) in the heavy chain was replaced with cysteine. Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Every figure is a modified amino acid of the show�and bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 27 shows (A) the light chain sequence (SEQ ID NO: 235) and (B) sequence of the heavy chain (SEQ ID NO: 234) anti-CD79b antibodies constructed with cysteine (thio-huMA79b-LC-V205C), where valine at position 205 in Cabatu (position valine 209 in accordance with the sequential numbering system) light chain was replaced with cysteine. Molecule drugs can be attached to the introduced cysteine group in the light chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody.

Figure 28 shows (A) the light chain sequence (SEQ ID NO: 237) and (B) sequence of the heavy chain (SEQ ID NO: 236) anti-CD79b antibodies constructed with cysteine (thio-huMA79b-HC-A118C), where alanine is present at position 118 in accordance with the European numbering system (position alanine 118 in accordance with a consecutive numbering system; the provision Cabatu - 114) in the heavy chain was replaced with cysteine. Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody.

In figures 29A-B presents FACS-graphs, which indicates that the binding of the conjugates anti-CD79b antibody thioMAb-drug" (TDC) according to the invention with CD79b, expressed on the surface of BJAB cells containing luciferase, similar to the binding of the conjugated (A) LC variants(V205C) thio-MAb and (B) options HC(A118C) thio-MAb antibodies chMA79b with MMAF. Detection was performed using mass spectrometry (MS) using PE-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody.

In figures 30A-D presents FACS-graphs, which indicates that the binding of the conjugates anti-CD79b antibody thioMAb-drug" (TDC) according to the invention with CD79b, expressed on the surface of BJAB cells containing luciferase, similar to the binding of (A) "bare" (unconjugated) options HC(AS) thio-MAb huMA79b.v18 and conjugated variants HC(A118C) thio-MAb antibodies huMA79b.v18 you provide during account creation with different�mi conjugates of drugs ((B) MMAF, (C) MMAE and (D) DM1)). Detection was performed using mass spectrometry (MS) using PE-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

In the figures 31A-D presents FACS-graphs, which indicates that the binding of the conjugates anti-CD79b antibody thioMAb-drug" (TDC) according to the invention with CD79b, expressed on the surface of BJAB cells containing luciferase, similar to the binding of (A) "bare" (unconjugated) options HC(AS) thio-MAb huMA79b.v28 and conjugated variants HC(A118C) thio-MAb antibodies huMA79b.v28 with different specified conjugates of drugs ((B) MMAE, (C) DM1 and (D) MMAF)). Detection was performed using mass spectrometry (MS) using PE-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

In the figures 32A-D presents FACS graphs, indicating that the binding of conjugates of an antibody against CD79b dog-like apes thio-MAb-drug" (TDC) according to the invention with CD79b, expressed on the surface of BJAB-cells expressing CD79b dog-like apes, similar to the binding of (A) "bare" (unconjugated) options HC(AS) thio-Mab against CD79b dogs�like monkeys (ch10D10) and conjugated variants HC(A118C) thio-MAb against CD79b dog-like apes (ch10D10) with these different conjugates of drugs ((B) MMAE, (C) DM1 and (D) MMAF)). Detection was performed using mass spectrometry (MS) using PE-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody.

Figure 33A is a graph showing inhibition of tumor growth in vivo models of xenotransplantation Granta-519 (human lymphoma cells of the cerebral cortex), which shows that the introduction of anti-CD79b TDC, which differ in the provisions of the introduced cysteine (LC (V205C) or HC (A118C)) and/or with different doses of the drug, to SCID mice having human b cell tumors, leads to a significant inhibition of tumor growth. Model xenotransplanted treated with thio-chMA79b-HC(A118C)-MC-MMAF with drug loading approximately 1.9 (table 11) or thio-chMA79b-LC(V205C)-MC-MMAF with drug loading approximately 1.8 (table 11), found a significant inhibition of tumor growth during the study. As a control, the hu-anti-HER2-MC-MMAF and thio-hu-anti-HER2-HC(A118C)-MC-MMAF and chMA79b-MC-MMAF. Figure 33B is a graph showing the percent change in mass of the studied mice compared to xenograft Granta-519 (figure 33A and table 11), indicating the absence of any significant changes of weight during the first 14 days of research. "Thio" means constructed on the basis of cysteine antibody, � "hu" means a humanized antibody.

Figure 34A is a graph showing inhibition of tumor growth in vivo models of xenotransplantation BJAB cells containing the luciferase (Burkitt's), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, drug loading of approximately 1,87 (table 12), thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 12), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading of approximately 1,95 (table 12), showed significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE), a control antibody, huMA79b.v28 (huMA79b.v28-SMCC-DM1 and thio-huMA79b.v28-HC(A118C)) and control anti-CD22 antibody (thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF). Figure 34B presents a graph of the percentage of the mass of the investigated mice compared to xenograft BJAB-luciferase (figure 34A and table 12), indicating the absence of any significant changes of weight during the first 7 days research. "Thio" means scans�Rurouni on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 35A is a graph showing inhibition of tumor growth in vivo models of xenotransplantation WSU-DLCL2 (diffuse large cell lymphomas), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, drug loading of approximately 1,87 (table 13), thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 13), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading of approximately 1,95 (table 13), revealed significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE), a control antibody, huMA79b.v28 (huMA79b.v28-SMCC-DM1 and thio-huMA79b.v28-HC(A118C)) and control anti-CD22 antibody (thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF). Figure 35B is a graph showing the percent change of mass of the investigated mice compared to xenograft WSU-DLCL2 (figure 35A and table 13), indicating the absence of any significant changes of weight during the first 7 days of conducting the study�tions. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 36 is a graph showing inhibition of tumor growth in vivo models of xenotransplantation DOHH2 (follicular lymphoma), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-BMPEO-DM1 (with loading of the drug is approximately 1,85 (table 14)), thio-huMA79b.v28-MC-MMAF (with loading of the drug approximately 1,95 (table 14)) or thio-MA-HC(A118C)-MCvcPAB-MMAE (with loading of the drug approximately 1,87 (table 14)), found a significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE), a control antibody, huMA79b.v28 (huMA79b.v28-SMCC-DM1 and thio-huMA79b.v28-HC(A118C)) and control anti-CD22 antibody (thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 37 is a graph showing inhibition of tumor growth in vivo models of xenotransplant�Tata BJAB cells, containing luciferase (Burkitt's), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), and/or in different doses to SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 15), thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, drug loading approximately 1.9 (table 15), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading approximately 1.9 (table 15), revealed significant inhibition of tumor growth during the study. As a control, the media (only buffer), control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE), a control antibody, huMA79b.v28 (thio-huMA79b.v28-HC(A118C)) and control anti-CD22 antibody (thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 38A is a graph showing inhibition of tumor growth in vivo models of xenotransplantation Granta-519 (human lymphoma cells of the cerebral cortex), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules�inter-drug" (BMPEO-DM1 or MC-MMAF) and/or in different doses to SCID mice, having a human b-cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 16) or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading of approximately 1,95 (table 16), revealed significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF). Figure 38B is a graph showing the percent change of mass of the investigated mice compared to xenograft Granta-519 (figure 38A and table 16), indicating the absence of any significant changes of weight during the first 14 days of research. "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 39 is a graph showing inhibition of tumor growth in vivo models of xenotransplantation WSU-DLCL2 (diffuse large cell lymphomas), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE) and/or in different doses to SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 17), thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading approximately 1.9 (table 17) or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE with loading of the drug approximately 1.9 (table 17), revealed significant inhibition of tumor growth during the study. As a control, the media (only buffer) and control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 40 is a graph showing inhibition of tumor growth in vivo models of xenotransplantation Granta-519 (human lymphoma cells of the cerebral cortex), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (BMPEO-DM1 or MCvcPAB-MMAE) and/or in different doses to SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 18) or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE with a drug loading of approximately 1,87 (table 18), found a significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody� (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 41 presents a chart based on the results of the analysis on the proliferation of tumor cells in vitro (A) BJAB, (B) Granta-519 or (C) WSU-DLCL2 treated with various concentrations of 0.001-10000 ng TDC per ml, including: (1) control antibody thio-hu-anti-gD-HC(A118C)-MCvcPAB-MMAE, with a loading of 2.0 MMAE/Ab, (2) the control antibody thio-hu-anti-gD-HC(A118C)-MC-MMAF, c loading of 2.1 MMAF/Ab, (3) control antibody thio-hu-anti-gD-HC(A118C)-BMPEO-DM1, with a loading of 2.1 DM1/Ab, (4) thio-huMA79b.v18-HC(A118C)-MC-MMAF, with a loading of 1.91 MMAF/Ab, (5) thio-huMA79b.v18-HC(A118C)-BMPEO-DM1, with a loading of 1.8 DM1/Ab; and (6) thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, with a loading of 2.0 MMAE/Ab. “Thio” means constructed on the basis of cysteine antibodies, and “hu” means a humanized antibody. “gD” means a glycoprotein D.

Figure 42 presents the nucleotide sequence of cDNA (SEQ ID NO: 238) PRO283627, where SEQ ID NO: 235 is a clone designated “DNA548455” (also referred to herein as “cyno CD79b”). The nucleotide sequence encodes CD79b dog-like monkey with start and stop codons are indicated in bold and underlined.

Figure 43 presents the amino acid sequence (SEQ ID NO: 239), derived from the coding sequence of SEQ ID NO: 235, presented in figure 42.

Figure 44 presented�ena nucleotide sequence (SEQ ID NO: 240) light chain antibodies against CD79b dog-like apes (ch10D10). The nucleotide sequence encodes the light chain of antibodies against CD79b dog-like apes (ch10D10) with start and stop codons are indicated in bold and underlined.

Figure 45 presents the amino acid sequence (SEQ ID NO: 241), containing the first 18 amino acid signal sequence and derived from the coding sequence of SEQ ID NO: 240, presented on figure 44. Variable region (SEQ ID NO: 302) are not underlined.

Figure 46 presents the nucleotide sequence (SEQ ID NO: 242) of the heavy chain of antibodies against CD79b dog-like apes (ch10D10). The nucleotide sequence encodes a heavy chain antibodies against CD79b dog-like apes (ch10D10) with start and stop codons are indicated in bold and underlined.

Figure 47 presents the amino acid sequence (SEQ ID NO: 243), containing the first 18 amino acids of the signal sequence and the last of lysine (K) before the stop codon and derived from the coding sequence of SEQ ID NO: 242, presented in figure 46. Variable region (SEQ ID NO: 301) are not underlined.

Figure 48 shows (A) the light chain sequence (SEQ ID NO: 245) and (B) sequence of the heavy chain (SEQ ID NO: 244) antibodies against CD79b dog-like apes, constructed on the basis of cysteine (thio-anti-cynoCD79b-HC-A118C), where Alan�n, is present at position 118 in accordance with the European numbering system (position alanine 118 in accordance with a consecutive numbering system; the provision Cabatu - 114) in the heavy chain was replaced with cysteine. Amino acid D at position 6 in accordance with the European numbering system (shaded in the figure) in the heavy chain may alternatively be a E. Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody.

Figure 49 shows (A) the light chain sequence (SEQ ID NO: 300) and (B) sequence of the heavy chain (SEQ ID NO: 299) antibodies against CD79b dog-like apes, constructed on the basis of cysteine (thio-anti-cynoCD79b LC-V205C), where valine at position 205 in Cabatu (position valine 209 given in accordance with the sequential numbering system) in the light chain was replaced with cysteine. Amino acid D at position 6 in accordance with the European numbering system (shaded in the figure) in the heavy chain may alternatively represent a Molecule of E. Lekarstvo�th means can be attached to the introduced cysteine group of the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Constant region are underlined with a single slash. Variable regions are not underlined. Fc-region is shown in italics. "Thio" means constructed on the basis of cysteine antibody.

Figure 50 is a graph showing inhibition of tumor growth in vivo models of xenotransplantation BJAB-cynoCD79b (BJAB cells expressing cynoCD79b) (Burkitt's), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules, the linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 19), thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading approximately 1.9 (table 19), or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE with loading of the drug approximately 1.9 (table 19), thio anti-cyno-CD79b (ch10D10)-HC(A118C)-BMPEO-DM1 with a drug loading approximately 1.8 (table 19), thio anti-cyno-CD79b (ch10D10)-HC(A118C)-MC-MMAF with drug loading approximately 1.9 (table 19), thio anti-cyno-CD79b (ch10D10)-HC(A118C)-MCvcPAB-MMAE with a drug loading of approximately 1,86 (table 19), was found to have a significant�th inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE, thio-hu-anti-HER2-HC(A118C)-MC-MMAF). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Figure 51 is a graph showing inhibition of tumor growth in vivo models of xenotransplantation BJAB-cynoCD79b (BJAB cells expressing cynoCD79b) (Burkitt's), where it is shown that the introduction of anti-CD79b TDC, conjugated with various molecules "VMRO-DM1-linker-drug", in various doses to SCID mice having human b cell tumors, has led to significant inhibition of tumor growth. Model xenotransplanted treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 with a drug loading of approximately 1,85 (table 20) or thio anti-cyno(ch10D10)-HC(A118C)-BMPEO-DM1 with a drug loading approximately 1.8 (table 20), found a significant inhibition of tumor growth during the study. As a control, the control anti-HER2 antibody (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1), a control antibody, huMA79b.v28 (thio-huMA79b.v28-HC(A118C) and control antibody anti-cynoCD79b(ch10D10) (thio-anti-cynoCD79b(ch10D10)-HC(A118C)). "Thio" means constructed on the basis of cysteine antibody, and "hu" means a humanized antibody.

Detailed description of preferred Varian�s for carrying out the invention

The present invention relates to methods, compositions, kits, and manufactured products used to identify compositions useful for the treatment of hematopoietic tumor in mammals and to methods of using such compositions according to the invention for these purposes.

A detailed description of these methods, compositions, kits and industrial products is given below.

I.General methods

The present invention can be carried out, unless otherwise agreed, the standard methods used in molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, and known in the art. Such methods are described in detail in the literature, for example, in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and in contemporary periodicals); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001).

II. Definitions

For a better understanding of the present invention, the definitions of terms used in the present description, wherein, unless otherwise agreed, it is understood that the terms used� in the singular, mean nouns in the plural, and Vice versa. If the definition of any term in this description conflicts with the definition given in any document entered into the present description by reference, they can be dealt with in accordance with the description below.

Used here, the term “marker b-cell surface” or “antigen b-cell surface” means an antigen, expressed on the surface of b cells, which can be directed antagonist that binds to that cell, including, but not limited to, antibodies against antigen b-cell surface or soluble forms of the antigen In the cell surface, having the ability to inhibit ligand binding with the natural b-cell antigen. Examples of markers of b-cell surface markers are the surface of leukocytes, such as CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 (description, see The Leukocyte Antigen Facts Book, 2ndEdition, 1997, ed. Barclay et al., Academic Press, Harcourt Brace & Co., New York). Other markers of b-cell surface are RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. Marker b-cell surface that are of particular interest, expressio�tsya predominantly on b-cells, unlike other non-b cell tissues of a mammal and may be expressed as b-cell precursors and Mature b cells.

As used herein, the term "CD79b" means any natural CD79b, derived from any vertebrate, including mammals such as primates (e.g., human, dog-like monkey (cyno)) and rodents (e.g. mice and rats), unless otherwise agreed. Human CD79b is also referred to here as the "PRO36249" (SEQ ID NO: 2) and is encoded by a nucleotide sequence (SEQ ID NO: 1), also referred to here as the "DNA225786". CD79b dog-like apes is also indicated here "cyno-CD79b" or "PRO283627" (SEQ ID NO: 239) and is encoded by a nucleotide sequence (SEQ ID NO: 238), also referred to here as the "DNA548455". The term "CD79b" encompasses "full-size" reprezentirovanii CD79b, as well as any form of CD79b, which is formed by processing in the cell. The term also covers natural options CD79b, for example, spliced variants, allelic variants and isoforms. Described here CD79b polypeptides can be isolated from different sources, e.g., from human tissue or from other sources, or they can be obtained by recombinant methods or by methods of synthesis. A "native sequence CD79b polypeptide" includes a polypeptide having the same amino acid sequence�performance as the corresponding natural CD79b polypeptide. Such CD79b polypeptides with a native sequence can be isolated from a natural source, or they can be obtained by recombinant methods or by methods of synthesis. The term "CD79b polypeptide with native sequence" specifically encompasses truncated natural or synthesized forms specific CD79b polypeptide (for example, the sequence of the extracellular domain), natural options (e.g., alternative spliced forms) and natural allelic variants of the polypeptide. In some embodiments of the invention described here CD79b polypeptides with a native sequence represents the Mature polypeptides or full-sized polypeptides with a native sequence containing a full-sized amino acid sequence presented in the description of graphic material. In the description of graphic material, start - and stop-codons (if specified) are shown in bold and underlined. Remnants of a nucleic acid, designated “N” in the description of graphical material, are any remnants of the nucleic acid. Although CD79b polypeptides referred to in the description of graphic material, start with methioninol residues designated herein as amino acid position 1, however, possible that in Kutch�as the starting amino acid residue for CD79b polypeptides can be used and other methionine residues, located above or below from amino acid position 1, as shown in the description of graphic material.

As used herein, the terms "MA79b" or "murine anti-CD79b antibody" or "murine antibodies against CD79b, in particular, means a murine monoclonal anti-CD79b antibody that contains a variable domain light chain SEQ ID NO: 10 (figures 7A-B) and the variable domain of the heavy chain SEQ ID NO: 14 (figures 8A-B). Murine monoclonal anti-CD79b antibody may be purchased from commercial firms, such as Biomeda (antibody against human CD79b; Foster City, CA), BDbioscience (antibody against human CD79b; San Diego, CA) or Ancell (antibody against human CD79b; Bayport, MN), or it may be selected from hybrid clone 3A2-2E7, deposited in the American type culture collection (ATCC) under Deposit number HB11413 assigned ATCC July 20, 1993

As used herein, the term "chMA79b" or "chimeric MA79b antibody, in particular, means a chimeric antibody against human CD79b (described earlier in the application for U.S. patent No. 11/462336, filed August 3, 2006), where the specified chimeric anti-CD79b antibody contains a light chain SEQ ID NO: 4 (figure 4). Light chain SEQ ID NO: 4 also contains a variable domain SEQ ID NO: 10 (figures 7A-B) and a constant domain of the light chain of human IgG1. Chimeric anti-CD79b antibody contains a heavy chain SEQ ID NO: 6 (figure 6). Heavy chain SEQ ID N: 6 also contains a variable domain SEQ ID NO: 14 (figures 8A-B) and a constant domain of the heavy chain of human IgG1.

As used herein, the term "anti-cynoCD79b" or "antibody against CD79b dog-like apes" means antibodies that bind to CD79b dog-like monkey (SEQ ID NO: 239 figure 43) (as previously described in application for U.S. patent No. 11/462336, filed August 3, 2006). As used herein, the term "anti-cynoCD79b (ch10D10)" or "ch10D10" means a chimeric antibody against CD79b dog-like apes (described earlier in the application for U.S. patent No. 11/462336, filed August 3, 2006) that binds to CD79b dog-like monkey (SEQ ID NO: 239 figure 43). Anti-cynoCD79b(ch10D10) or ch10D10 is a chimeric antibody against CD79b dog-like apes, which contains the light chain is SEQ ID NO: 241 (figure 45). Anti-cynoCD79b(ch10D10) or ch10D10 also contains a heavy chain SEQ ID NO: 243 (figure 47).

As used herein, the term "MA79b-hybrid" or "MA79b-associated humanized antibody" or "huMA79b-hybrid" in particular means a hybrid produced by attaching hypervariable regions derived from murine anti-CD79b antibody (MA79b), the acceptor sequence is a human consensus VL Kappa I (huKI) and human consensus VH subgroup III (huIII) with substitutions R71A, N73T and L78A (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992)) (see example 1A and figure 7 (SEQ ID NO: 11) and 8 (SEQ ID NO: 15)).

Used here, the term "modification" of amino acid residue/position means replacing the primary AMI�kislotno sequence compared to the original amino acid sequence, where the specified substitution is the result of a modification to the sequence incorporating the amino acid residues/positions. For example, typical modifications are the replacement of the remainder (or residue at the position) to other amino acid residue (e.g., conservative or non-conservative substitution), insertion of one or several (usually less than 5 or 3) amino acids adjacent to the specified residue/position, and deletion of the specified residue/position. The term "amino acid substitution" or its variant means replacing the existing amino acid residue in a pre-defined (original) amino acid sequence with other amino acid residue. Generally and preferably, the modification leads to a change in at least one physico-biochemical activity of the polypeptide variant in comparison with the activity of the polypeptide containing the original amino acid sequence (or "wild-type sequence"). For example, in the case of antibodies, modified physico-biochemical activity can be affinity-binding molecule-target, the ability to bind with the molecule-target and/or influence on the binding molecule-target.

As used herein, the term “antibody” is used in the broadest sense and specifically covers otdeleniye anti-CD79b antibodies (including agonist antagonists, neutralizing antibodies, full-size monoclonal antibody or intact monoclonal antibodies), compositions of anti-CD79b antibodies with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bespecifically antibodies, provided that they possess the desired biological activity), obtained from at least two intact antibodies, single-chain anti-CD79b antibodies and fragments of anti-CD79b antibodies (see below), including Fab, Fab', F(ab')2- and Fv - fragments, denticula, single-domain antibodies (sdAb), provided that they possess the desired biological or immunological activity. As used herein, the terms "immunoglobulin (Ig)" and "antibody" are synonyms. The antibody may be human, humanized and/or affinity Mature.

The term "anti-CD79b antibody" or "antibody that binds to CD79b" means an antibody that is able to contact with CD79b affinity sufficient for use of this antibody as a diagnostic and/or therapeutic agents that target CD79b. Preferably, the level of binding of anti-CD79b antibody with an unrelated protein, i.e. a protein, a non-CD79b, is less than about 10% of the binding of an antibody to CD79b, as determined using, for example,radioimmunoassay (RIA). In some embodiments of the invention, the antibody that binds to CD79b, has a dissociation constant (Kd), dimension ≤1 µm ≤100 nm, ≤10 nm, ≤1 nm, or ≤0.1 nm. In some embodiments of the invention, the anti-CD79b antibody binds to the epitope CD79b, which is conservative in CD79b various kinds.

"Isolated" antibody is an antibody that was identified and isolated and/or purified from components of its natural environment. Contaminating components of its natural environment are materials that negatively affect therapeutic efficacy of the antibody, and these components include enzymes, hormones and other protein or non-protein solute. In preferred embodiments of the invention, the specified antibody can be purified: (1) more than 95% by weight of antibody as may be determined by the method of Lowry, and more preferably not more than 99% by weight of antibody, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal part of the amino acid sequence that can be determined using the sequencer, equipped with a centrifuge vessel, or (3) to homogeneity, what can be confirmed using electrophoresis in LTOs-page in reducing or Sevostyanova conditions with staining of Kumasi blue Il�, preferably, silver. The term “selected antibody includes the antibody in situ within recombinant cells, unless there is at least one natural component of this antibody. Usually, however, the selected antibody can be obtained in at least one stage of cleaning.

Basic 4-chain antibody molecule is heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 main heterotetrameric molecules together with an additional polypeptide called J chain, and therefore it contains 10 antigen-binding sites, while secreted IgA antibodies can polymerize with the formation of polyvalent structures comprising 2-5 of the basic 4-chain molecules with J-chain). In the case of IgG 4-chain molecule mainly has a size of approximately 150000 daltons. Each L-chain is associated with the H chain by one covalent disulfide bond, while the two H chains are linked together by one or more disulfide bonds depending on the isotype of the H-chain. Each H and L chain also has regularly spaced noticeplease disulfide bridges. Each H chain has at its N-Terminus a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CHdomain for and�of Osipov μ and ε. Each L chain has at its N-Terminus a variable domain (VL) followed by a constant domain (CL) at its other end. VLlocated on one line with VHand CLlocated on one line with the first constant domain of the heavy chain (CH1). It is obvious that the specific amino acid residues form a boundary region between the variable domains of the light chain and heavy chain. The pairing of VHand VLleads to the formation of a single antigen-binding site. Structure and properties of different classes of antibodies are described, for example, in the publication ofBasic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

L-chain antibodies derived from any vertebrate species can belong to one of two clearly distinguishable types, called Kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins, depending on the amino acid sequence of the constant domain of their heavy chains (CH), can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. Classes γ and α are also divided into subclasses on the basis of relatively minor differences in the sequences and funkcijas Hfor example , the person expressed immunoglobulins of the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The terms "variable region" or "variable domain" of an antibody means the amino-terminal domains of the heavy or light chain of the antibody. Variable domain of the heavy chain may be referred to as “VH”. Variable domain light chain may be referred to as “VL”. These domains are mostly the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to certain segments of the variable domains, which have significant differences in the sequences of different antibodies. Domain V mediates the binding to the antigen and determines the specificity of a particular antibody to a specific antigen. However, the variability is unevenly distributed on all variable domains consisting of 110 amino acids. But usually region V consists of a relatively invariant segments called frame regions (FR), consisting of 15-30 amino acids separated by shorter regions hypervariability called "hypervariable regions", each of which has a length of 9-12 amino acids. Each variable domain of native heavy and light chains contain four FR with mainly β-folded configuration and the United three hypervariability, which form loops connecting, and in some cases forming part of β-folded structure. Hypervariable regions in each chain are held in close proximity to each other through FR, and together with the hypervariable regions of the other chains involved in the formation of the antigen-binding site of antibodies (see Kabat et al.Sequences of Proteins of Immunological Interest, Sth. Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Constant domains are not directly involved in the binding of the antibody to an antigen, but exhibit different effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

“Intact” antibody is an antibody containing the antigen-binding site, and CLand at least the constant domains of the heavy chain, CH1, CH2 and CH3. The constant domains may represent the constant domains of the native sequence (for example, the constant domains of a native human sequence) or variants of their amino acid sequences. Intact antibody preferably has one or more effector functions.

Used here, the term "naked antibody" means an antibody that is not conjugated with a cytotoxic molecule or a radioactive label.

"Fragments of the antibodies contain a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of fragments of antibodies are Fab-, Fab', F(ab')2- and Fv-fragments; dianthicola; linear antibodies (see U.S. patent No. 5641870, example 2; Zapata et al.,Protein Eng.8(10):1057-1062 [1995]); molecules of single-chain antibodies; and multispecific antibodies formed from fragments of antibodies. In one embodiment of the invention, the antibody fragment contains the antigen-binding site of the intact antibody, and therefore it maintains its ability to bind to the antigen.

As a result of hydrolysis of the antibody with papain form two identical antigen-binding fragments, called “Fab”fragments, and one remaining “Fc”fragment, whose name indicates its ability to easily crystallize. Fab-fragment consists of a full-sized L-chain with a domain variable regions of the H-chain (VHand the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent in binding to the antigen, i.e. it has a single antigen-binding site. The processing of the antibody with pepsin is formed a single large F(ab')2-the fragment which roughly corresponds to two disulfide linked Fab communication-fragments with double-shaft�Noi binding activity and ability to cross communicate with the antigen. Fab'fragments differ from Fab fragments by the presence of several additional residues at the carboxy-end of domain CH1, including one or more cysteines, derived from a hinge region of the antibody. Fab'-SH used in the present application, is a Fab' in which the cysteine(C) residue(tki) constant domain has the(s) free thiol group. F(ab')2-fragments of the antibodies were initially obtained in the form of pairs of Fab'-fragments, which are located between the hinge cysteines. Professionals are also known other methods chemical binding fragments of antibodies.

Fc-fragment contains a carboxy-terminal parts of both H-chains connected by disulfide bonds. Effector functions of antibodies are determined by sequences in the Fc-region, which are also the part recognized by Fc receptors (FcR) found on cells of certain types.

"Fv" is the minimum antibody fragment that contains a full-sized antigen-recognizing site and the antigen-binding site. This fragment consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected with each other by non-covalent bond. In single-chain Fv (scFv) single variable domain of the heavy chain and one variable domain light chain can be covalently bound to a flexible peptide linker, bringing light and heavy chains can associate with each other to form a "dimeric" structure analogous to the structure of double-stranded Fv. After laying these two domains formed by six hypervariable loops (3 loops, each of which is derived from the H and L-chains), which provide amino acid residues for binding to the antigen and inform the antibody binding specificity to the antigen. However, even a single variable domain (or half of Fv containing only three CDRs specific for an antigen) has the ability to recognize the antigen and contact them, although with lower affinity than the entire binding site.

"Single-chain Fv-fragments," also known as "sFv" or "scFv", represent fragments of antibodies, which include domains VHand VLantibody, United in a single polypeptide chain. Preferably, the scFv polypeptide also contains between domains VHand VLthe polypeptide linker that provides education scFv with the structure required for binding to the antigen. Description scFv can be found in the work Pluckthun inThe Pharmacology of Monoclonal Antibodiesvol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below.

The term “dentical” means fragments of antibodies with two antigen-binding sites, where these fragments comprise variable domain �agelou chain (VH), connected to the variable domain light chain (VL) in the same polypeptide chain (VH-VL). Small fragments of the antibodies produced by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between domains VHand VLso as to achieve mezzapesa, but not noticeplease, the pairing of the V domains, with the formation of bivalent fragment, i.e., fragment having two antigen-binding site. Dianthicola can be divalent or bespecifically. Bespecifically venticelli are heterodimeric two "cross-linked" scFv fragments in which domains VHand VLthe two antibodies are present on different polypeptide chains. Dianthicola described in more detail, for example, in EP 404097; WO 93/11161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Trinchitella and tyrantical also described in the publication of Hudson et al., Nat. Med. 9:129-134 (2003).

Used herein the term “monoclonal antibody” means an antibody obtained from a population of mostly homogeneous antibodies, i.e. the individual antibodies included in this population and are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and dir�us against one antigenic determinants. In addition, in contrast to the preparations of polyclonal antibodies, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized so that they do not contain impurities of other antibodies. The term “monoclonal antibody” does not mean that the antibody should be produced by any particular method. For example, the monoclonal antibodies according to the invention can be obtained by using hybrid technology, first described by Kohler et al. (1975)Nature256:495, or they can be obtained by methods of recombinant DNA in bacterial cells, eukaryotic organisms or plants (see, e.g., U.S. patent No. 4816567). “Monoclonal antibodies” may also be isolated from phage libraries of antibodies using procedures described, for example, Clackson et al.,Nature, 352:624-628 (1991) and Marks et al.,J. Mol. Biol., 222:581-597 (1991).

Used here, the monoclonal antibodies include, inter alia, a “chimeric” antibodies in which a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of anti�l, and the rest(s) chain(s) is identical to(s) or homologous(s) corresponding sequences of antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they possess the desired biological activity (see U.S. patent No. 4816567 and publication of Morrison et al.Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Of interest is a chimeric antibody according to the invention are "primaryservername" antibody containing the antigen-binding sequences of the variable domains, the sequences derived from the constant region of primates, non-human (e.g., monkeys, apes, etc.), and man.

“Humanized form” non-human antibodies (e.g., antibodies rodents) are chimeric antibodies that contain minimal sequence derived from nonhuman antibody. For the most part, humanized antibodies are human immunoglobulins (antibody-recipient), in which residues originating from the hypervariable region of antibodies of the recipient are replaced by residues originating from the hypervariable region of nonhuman antibody (donor antibody) such as mouse antibody, rat antibody, rabbit antibody or a�titulo primates, not a person, where the antibodies have the desired specificity, affinity and binding capacity. In some cases the remains of the frame region (FR) of a human immunoglobulin are replaced by corresponding residues of the non-human antibody. Furthermore, humanized antibodies may contain residues that are not found in the antibody-recipient or antibody-donor. These modifications introduced to improve the properties of the antibody. In General, a humanized antibody may include substantially all or at least one, and typically two, variable domain, in which all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin, and all or nearly all FR are FR sequence of a human immunoglobulin. A humanized antibody also includes, but not necessarily, at least a portion of constant region of immunoglobulin (Fc), typically a human immunoglobulin. A more detailed description, see Jones et al.Nature, 321:522-525 (1986); Riechmann et al. Nature 332:323-329 (1998) and PrestaCurr. Op. Struct. Biol., 2:593-596 (1992). Cm. also the following review articles and cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

As used herein, the term "thio" relative�tsya to the antibody, constructed with cysteine, as used herein, the term “hu” refers to humanitarianlaw the antibody.

"Human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of the antibody produced in humans and/or received by any of the methods of producing human antibodies described in this application. This definition of a human antibody specifically excludes a humanized antibody containing the antigen-binding residues, non-human antibody. Human antibodies can be obtained using different methods known in the art, including the use of phage libraries of submission. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). To obtain human monoclonal antibodies can also be applied to methods described in publications Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). Cm. also van Dijk &van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be obtained by introducing antigen transgenic animal that has been modified to produce such antibodies in response to antigenic stimulation, but which were blocked endogenous loci, for example, immunized mice compared to xenograft (�, for example, U.S. patents №№ 6075181 and 6150584 relating to XENOMOUSE technologyTM). Cm. also, for example, the publication of Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies obtained using the technology of the human b-cell hybridomas.

As used herein, the terms “hypervariable region”, “HVR” or “HV” means the area variable domain of antibodies, which are in sequence hypervariable and/or form a loop a certain structure. In General, these antibodies contain six hypervariable regions; three regions in the VH (H1, H2, H3) and three regions in the VL (L1, L2, L3). In the present application uses a number of hypervariable regions that fall within the scope of the present invention. Hypervariable region (complementarity-determining region (CDR)) Cabatu have a high degree of sequence variability and are widely used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead of determining hypervariable regions on Kabuto, Cotija proposed to identify hypervariable region of localization of the structural loops (Chothia & Lesk, J. Mol.Biol. 196:901-917 (1987)). The end of the loop of CDR-H1 on Cotia, in the numbering according to the numbering Kabata varies between the provisions of H32 and H34 depending on the length of the loop (e�about is because in accordance with the numbering scheme for Kabuto insertions located at positions H35A and H35B; if not present 35A or 35B, the loop ends at position 32; if it only has 35A, the loop ends at position 33; and if present 35A and 35B, the loop ends at position 34). The definition of the hypervariable regions AbM represents a compromise between the “CDR” Cabatu and “structural loops” on CATIA, and such definitions are used in the computer program for the modeling of antibody Oxford Molecular's AbM. "Contact" hypervariable region is determined based on the analysis of the available complex crystal structures. Remnants of each of these hypervariable regions are provided below.

LoopOn CabatoAbMOn ScotiaThe contact area
L1L24-L34L24-L34L26-L32L30-L36
L2L50-L56 L50-L56L50-L52L46-L55
L3L89-L97L89-L97L91-L96L89-L96
H1N-NWN-NWN-NN-NW
(Numbering according Cabatu)
H1N-NN-NN-NN-N
(The numbering of CATIA)
H2Uw50-NUw50-NN-NN-N
H3N95-NN95-NN-NN-N

The term “hypervariable region” may include “extra long hypervariable�nye region”, namely: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL; and 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. Residues of the variable domains for each of these definitions are numbered in Cabatu, etc., see above.

“Framework” or “FR” residues are residues of the variable domains, except for residues of the hypervariable regions, as defined above.

Used here, the term “variable domain residue numbered according Kabuto” or “amino acid position numbered in Kabuto” and its variants means a system used for numbering the variable domains of the heavy chain or variable light chain domains of the antibody described in the reference antibody Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In accordance with this numbering system, the actual primary amino acid sequence may contain fewer or additional amino acids corresponding to a shortened or lengthened FR or CDR of the variable region domain. For example, the variable domain of the heavy chain may include the insertion of one amino acid (residue 52a according to the numbering Kabat) after residue 52 of H2 and residues (e.g. residues 82A, 82b and C, etc., in accordance with the numbering Kabata) built after residue 82 FR of the heavy chain. Well�erace balances on Kabuto can be carried out after alignment of its sequences in regions of homology with a “standard” sequence, numbered on Kabuto.

The numbering system on Cabatu usually applied to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain)(e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The European numbering system "EU" or "Eu index" is generally applied to residues of the constant region of the heavy chain of immunoglobulin (e.g., the EU index as described in Kabat et al., see above). The term "EU-index Kabuto" means the residue numbering of the human IgG1 antibody in accordance with the European numbering system. If this is not indicated in the present description, the indication of the number of residues in the variable domain of antibodies means residues are numbered according to the numbering system Kabata. If this is not indicated in the present description, the indication of the number of residues in the constant domain of antibodies means residues, numbered in accordance with the European numbering system (see, e.g., provisional patent application U.S. No. 60/640323; in the figures given by the European numbering).

“Affinely” antibody is an antibody having one or more modifications in one or more HVR, where these modifications increase the affinity of the antibody for antigen, compared to a parent antibody that does not have this(or their) option�operators(ies). Preferred affinnative antibodies have mu or even low picomolar affinity to the antigen target. Affinnative antibodies get methods known in the art. The publication Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by permutation of the domains VH and VL. Nonspecific mutagenesis HVR and/or frame of the residues described in the publications Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

"Blocking" antibody or "antibody antagonist" is an antibody, inhibiting or reducing the biological activity of the antigen with which it is associated. Preferred blocking antibodies or antibody antagonist mainly or completely inhibit the biological activity of the antigen.

As used herein, the term "antibody agonist" means an antibody which mimics at least one of the functional activities of interest polypeptide.

"Species-specific antibody, e.g., antibody of a mammal against human IgE is an antibody that has a higher binding affinity to the antigen, derived from a mammal of a first species, in comparison with the homologue of the antigen derived from the mammal verogalid. Usually species-specific antibody "specifically binds" to a human antigen (i.e. has a value of binding affinity (Kd) of no more than about 1×10-7M, preferably not more than about 1×10-8and most preferably no more than about 1×10-9M), whereas the affinity of binding to the homologue of the antigen from a mammal of the second species, not human, at least about 50-fold or at least about 500 fold, or at least about 1000 times lower affinity of binding to human antigen. Species-specific antibody may be any of the antibodies of various types, as defined above, but preferably such an antibody is a humanized or human antibody.

The term “binding affinity” essentially means the strength of the total noncovalent interactions of a single binding site of a molecule (e.g. antibody) to its binding partner (e.g. antigen). Unless specified otherwise, as used herein, the term “binding affinity” means a natural binding affinity for the interaction of 1:1 between members of pairs of binding (e.g., antibody to the antigen). The binding affinity of a molecule X and Y, in General terms, can be called constant� dissociation (Kd). Affinity can be determined by standard methods, known in the art, including methods described herein. Discapline antibodies generally bind antigen slowly and tend to easily dissociate, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound for a longer period of time. Specialists in this field there are various methods of measuring the affinity of binding, and for the implementation of the present invention can be applied any of these methods. Specific representative embodiments of the invention described below.

Used here the definition of "or higher", if it is used in relation to the affinity of binding indicates a higher level of binding of the molecule to its binding partner. Used here the definition of "or higher", if it is used in this application, means a stronger binding defined by a smaller numerical value Kd. For example, an antibody that has a binding affinity to the antigen "of 0.6 nm or higher" means an antibody that has a binding affinity to the antigen <0.6 nm, i.e. of 0.59 nm, of 0.58 nm, 0,57 nm, etc., or any value less than 0.6 nm.

In one embodiment of the invention, the “Kd” or “Velich�well Kd according to the invention is determined by analyzing the binding of radioactively labeled antigen (RIA), carried out using Fab-option of interest antibody and its antigen as described below, where the analysis allows to measure the affinity of binding of the Fab to the antigen in solution by balancing minimal Fab concentration (125(I)-labeled antigen in the presence of a titration of a set of unlabeled antigens, with subsequent immobilization of the bound antigen on the tablet, sensitized by antibody against Fab-fragment (Chen et al. (1999) J. Mol. Biol. 293:865-881). In order to create appropriate conditions for analysis, the microtiter plates (Dynex) sensitize during the night of 5 µg/ml the binding of an anti-Fab antibody (Cappel Labs) in 50 mm sodium carbonate (pH 9,6), and then blocked with 2% (wt./about.) the bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In neabsorbiruemye tablet (Nunc # 269620) of 100 PM or 26 PM [125I]-antigen are mixed with serial dilutions of Fab of interest (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al. (1997) Cancer Res. 57:4593-4599). Then Fab of interest were incubated overnight, however, to guarantee the attainment of equilibrium, the incubation can be carried out over a longer period of time (for example, 65 hours). Thereafter, the mixture was transferred to a tablet for immobilization and incubated at room�a combined temperature (for example, within one hour). The solution was then removed and the plate washed eight times with 0.1% Tween 20 in PBS. After drying, the tablets, add 150 ál/well of scintillation fluid (MicroScintTM-20; Packard), and the plates are counted on a gamma counter Topcount (Packard) for ten minutes. The concentration of each Fab, which account for 20% or less of the maximum binding, selected for their use in the analysis of competitive binding.

In accordance with another variant of the invention, the Kd or Kd value is measured in the analysis by surface plasmon resonance using a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using cm 5 chips with immobilized them by antigen when the value of the response units (RU) of ~10. Briefly, biosensor chips with carboxyethylgermanium the dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in accordance with the instructions of the suppliers. Antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) and then injected at a flow rate of 5 μl/minute to achieve the magnitude of response units (RU) of the associated protein, component of ~10. After injection of the antigen to block unreacted groups injected with 1M ethanolamine. To measure the kinetics of the reaction injections administered twice seronegativity Fab (0,78 nm-500 nm) in PBS, containing 0.05% Tween® 20 (PBST) at 25°C and at a flow rate of approximately 25 μl/min Rate of Association (kon) and the rate of dissociation (koff) is calculated using a simple langourously model binding 1:1 (BIAcore Evaluation Software version 3.2) while building sensogram Association and dissociation. The equilibrium constant of dissociation (KD) is calculated as the ratio of koff/kon. See, for example, Chen, Y., et al. (1999) J. Mol. Biol. 293:865-881. If the velocity of the Association exceed 106M-1with-1as defined above, the method of surface plasmon resonance, such that the rate of Association can be determined by the method of quenching the fluorescence, which allows us to measure the increase or decrease in the intensity of fluorescence emission (excitation = 295 nm; emission = 340 nm, a bandwidth of 16 nm) at 25°C for 20 nm antibodies against the antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a spectrophotometer, equipped with a flow limiter (Aviv Instrumtnts), or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a cuvette for mixing, containing red dye.

"Rate of Association" ("on-rate" or "kon"according to the invention may also be defined as described above by the method of surface plasmon re�of anansa using BIAcore TM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ).

As used herein, the terms “substantially similar” or “substantially the same” means a sufficiently high degree of similarity between two numerical values (usually between one value corresponding to the antibody according to the invention, and another value corresponding to a reference/comparator antibody), namely, a degree of similarity that would allow the person skilled in the art to consider the difference between these two values is insignificant or biologically and/or statistically insignificant from the point of view of their biological properties determined by these values (e.g., Kd values). The differences between these two values is preferably less than about 50%, more preferably less than about 40%, even more preferably less than about 30%, even more preferably less than about 20%, and most preferably less than about 10% compared with the reference values/compare antibodies.

Used herein, the terms "substantially reduced" or "substantially different" means a sufficiently high degree of difference between two numerical values (usually between one value corresponding to the antibody according to the invention, and another value corresponding to the standard�WMD/comparator antibody), namely, such a degree of difference that would allow the specialist in this field is the difference between these two values is statistically significant from the point of view of their biological properties determined by these values (e.g., Kd values, NAMA-answer). The differences between these two values is preferably more than about 10%, more preferably more than about 20%, even more preferably more than about 30%, even more preferably more than about 40%, and most preferably more than about 50% compared to reference values/compare antibodies.

The term "antigen" means a pre-defined antigen, which can be selectively contacted by the antibody. The antigen target can be polypeptide, carbohydrate, nucleic acid, lipid, hapten or other natural or synthetic compound. The preferred antigen is the target is a polypeptide.

As used herein, the term "acceptor human skeleton area" means a frame region containing the amino acid sequence of a frame region VL or VH derived from a human frame region of an immunoglobulin or a human consensus frame region. Acceptor human skeleton region, "derived from" a human�th frame region of an immunoglobulin or a human consensus frame region, may contain the same amino acid sequence or it may contain amino acid sequence with the existing changes. If in amino acid sequence were already changing, it is preferred that the number of such changes does not exceed 5, and preferably 4 or less, or 3 or less. If amino acid changes are present in a VH, preferably those changes were only three, two or one of the provisions 71H, 73H and 78H; for instance, the amino acid residues in these positions may be 71A, 73T and/or 78A. In one embodiment of the invention the acceptor human skeleton VL region identical to the sequence of the human frame VL region of an immunoglobulin or a human consensus frame of the sequence.

"Human consensus frame region is a frame region, which includes the most frequently occurring amino acid residues used in the selection of frame sequences of the VL or VH of a human immunoglobulin. Usually sequences of the VL or VH of a human immunoglobulin chosen from the group of sequences of variable domains. Basically, the subgroup of sequences is determined by Kabuto, etc., In one embodiment of the invention, for the VL, the subgroup I�is subgroup Kappa I on Kabuto, etc. In one embodiment of the invention, for the VH, the subgroup is subgroup III as in Kabuto, etc.

"The consensus frame region VH subgroup III contains a consensus sequence selected from the amino acid sequences of heavy chain subgroup III Cabatu, etc., In one embodiment of the invention a consensus amino acid sequence of a frame region VH subgroup III contains at least a portion of each of the following sequences or all sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 143)-H1-WVRQAPGKGLEWV (SEQ ID NO: 144)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 145)-H3-WGQGTLVTVSS (SEQ ID NO: 146).

"The consensus frame region VL subgroup I contains a consensus sequence selected from the amino acid sequences of variable light chain Kappa subgroup I on Kabuto, etc., In one embodiment of the invention a consensus amino acid sequence of a frame region VL subgroup I contains at least a portion of each of the following sequences or all sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 139)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 140)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 141)-L3-FGQGTKVEIKR (SEQ ID NO: 142).

"Unmodified human frame region is a human frame region having the same amino acid sequence as the acceptor human to�resna region, for example, there are no replacement amino acids of human to non-human amino acid, as in the acceptor human skeleton area.

Used here, the term "modified hypervariable region" means a hypervariable region containing one or more (e.g., from one to about 16) amino acid substitutions.

As used herein, the term "unmodified hypervariable region" means the hypervariable region having the same amino acid sequence as the sequence of the non-human antibody from which it occurs, i.e., a sequence not containing one or more amino acid substitutions.

An antibody that “binds” with interest antigen, e.g., tumor-associated polypeptide antigen target, is antitela that binds to the antigen with an affinity that is sufficient to ensure that this antibody could be used as a therapeutic agent for delivery to a cell or tissue expressing the antigen, and that this antibody had no significant cross-reactivity with other proteins. In these embodiments of the invention, the level of binding of the antibody to the protein, which "is not a target" is less than �eat about 10% of the level of binding of the antibody with its specific protein target, as was determined through analysis conducted using cell sorting with activation of fluorescence (FACS) or radioimmunoprecipitation (RIA). As to the binding of the antibody to the molecule-target, the term "specifically binding" or "specifically binds to" a particular polypeptide or epitope on a particular polypeptide target or "specific for" a particular polypeptide or epitope on a particular polypeptide target means that the binding of an antibody is significantly different from non-specific interactions. Specific binding can be measured, for example, by determining the level of binding of a molecule compared to binding of a control molecule, which essentially is a molecule having a similar structure, but do not have binding activity. For example, specific binding can be determined by evaluating the competitive binding of a control molecule that is similar to the target device, for example, by excess unlabeled target. In this case, specific binding is detected when the binding of the labeled target probe competitive inhibited by excess unlabeled target. As used herein, the term "specifically binding" or "specifically binds to" specific�peptides or epitope on a particular polypeptide target or "specific for" a particular polypeptide or epitope on a particular polypeptide target can for example, mean that this molecule has a Kd against a target of at least about 10-4M, alternatively at least about 10-5M, alternatively at least about 10-6M, alternatively at least about 10-7M, alternatively at least about 10-8M, alternatively at least about 10-9M, alternatively at least about 10-10M, alternatively at least about 10-11M, alternatively at least about 10-12M or more. In one embodiment of the invention, the term "specific binding" means binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide, but mostly not associated with any other polypeptide or polypeptide epitope.

An antibody that "inhibits the growth of tumor cells expressing CD79b polypeptide" or "growth-inhibitory antibody" is an antibody that significantly inhibits the growth of cancer cells expressing or sverkhekspressiya corresponding to a CD79b polypeptide. The CD79b polypeptide may be a transmembrane polypeptide expressed on the surface of cancer cells, or it can be a polypeptide produced and secreted by cancer cells. Preferred growth-ingebyra�ing anti-CD79b antibodies inhibit the growth of CD79b-expressing tumor cells more than 20%, preferably about 20%-50%, and even more preferably more than 50% (about 50%-100%), compared with the corresponding control, which is usually a tumor cell not treated with the test antibody. In one embodiment of the invention, the growth inhibition can be measured at the antibody concentration of approximately 0.1 to 30 μg/ml or about 0.5 to 200 nm in cell culture, where the growth inhibition is determined 1-10 days after treatment of tumor cells with antibody. The inhibition of growth of tumor cells in vivo can be determined by various methods described below in the Experimental section examples". The specified antibody inhibits growth in vivo, if the introduction of anti-CD79b antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or the proliferation of tumor cells during the period of time from about 5 days to 3 months, and preferably about 5 to 30 days after the first administration of the antibody.

An antibody that “induces apoptosis” is an antibody that induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, shrinkage of cells, expansion of endoplasmic reticulum, fragmentation of cells and/or images�of membrane vesicles (called apoptotic bodies). Such a cell is usually the cell, sverkhekspressiya CD79b polypeptide. The preferred cell are tumor cells, e.g., hematopoietic cells such as b cells, T cells, basophils, eosinophils, neutrophils, monocytes, platelets or erythrocytes. To assess the cellular events associated with apoptosis, there are different methods. So, for example, translocation of phosphatidylserine (PS) can be determined by binding with annexin; DNA fragmentation can be evaluated by the formation of DNA-ladder; and condensation of nucleus/chromatin along with DNA fragmentation can be evaluated by any increase hypodiploid cells. Preferably the antibody inducing apoptosis, is an antibody that in the analysis on the binding of annexin responsible for approximately 2-50-fold, preferably about 5-50-fold, and most preferably about 10-50-fold induction by binding to annexin compared to untreated cells.

An antibody which "induces cell death" is an antibody that turns viable cells non-viable cells. Such cells are cells expressing CD79b polypeptide, and cells of a particular type that specifically Express or sverkhekspressiya CD79b polypeptide. These cells can be cancerous Il� normal cells of a particular type. The CD79b polypeptide may be a transmembrane polypeptide expressed on the surface of cancer cells, or it can be a polypeptide produced and secreted by cancer cells. These cells can be cancer cells, e.g. b cells or T cells. Cell death in vitro may be determined in the absence of complement and immune effector cells to identify the cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, the analysis of cell death may be performed using thermoinactivation serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether this antibody to induce cell death, can be estimated a loss of integrity of their membranes by uptake of propidium iodide (PI), trypan blue (see Moore et al.Cytotechnology17:1-11 (1995)) or 7AAD compared to untreated cells. Preferred antibodies which induce cell death, are antibodies, which induce PI uptake in the analysis of PI uptake in cells BT474.

The term "effector functions" of antibodies means the biological activity attributed to the Fc region (Fc-region with the native amino acid sequence or a Fc region�and with a modified amino acid sequence) antibodies and varying depending on the isotype of the antibody. Examples of effector functions of antibodies include: C1q binding and complement-dependent cytotoxicity; binding to Fc-receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; inhibition of the function of cell surface receptors (e.g. b cell receptor); and activation of b-cells.

As used herein, the term "Fc region" means the C-terminal region of the heavy chain of immunoglobulin, including Fc-region with the native sequence and the Fc region with an altered sequence. Although the boundaries of the Fc region of the heavy chain of an immunoglobulin may vary, however, the Fc-region of the heavy chain of human IgG is usually defined as a fragment extending from amino acid residue at position Cys226, or from Pro230, to the carboxy-end. The C-terminal lysine (residue 447 according to the European numbering system) of the Fc region may be removed, for example, in the process of producing or purifying antibodies or by recombinant nucleic acid construct encoding the heavy chain of the antibody. Accordingly, the composition of intact antibodies may include populations of antibodies with all remote K447 residues, populations of antibodies with the remnants C that were not deleted, and populations of antibodies with a mixture of antibodies, which are present or absent the K447 residue.

"Functionality�of obayatelnaya, charming Fc region" possesses "effector function" Fc-region with the native sequence. Representative effector functions are the C1q binding; CDC; the binding of Fc-receptor; ADCC; phagocytosis; inhibition of the function of cell surface receptors (e.g. B cell receptor; BCR), etc. To achieve the effector functions usually requires that the Fc-region has been combined with a binding domain (e.g., variable domain antibodies), and such effector functions can be assessed using various assays as described, for example, in the section "Definitions" herein.

"The Fc-region with the native sequence" includes an amino acid sequence identical to the amino acid sequence of the natural Fc-region. Human Fc-areas with native sequence are the Fc-region of a native sequence human IgG1 (non-A and A-allotypes); Fc-region of a native sequence human IgG2; Fc-region of a native sequence human IgG3; and the Fc-region of a native sequence human IgG4, as well as their natural options.

A "variant Fc region" includes an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc-region has less�her least one amino acid substitution, for example, at least the replacement of about 1-10 amino acids, and preferably about 1-5 amino acids, compared with a native sequence Fc region or Fc region of the parent polypeptide. Variant Fc region described in the present application, preferably at least about 80%, more preferably at least about 90% and most preferably at least about 95% homologous to a native sequence Fc region and/or Fc region of the parent polypeptide.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc-receptors (FcR) present on certain cytotoxic cells (e.g. natural cells killer cells (NK), neutrophils and macrophages) enables these cytotoxic effector cells specific to contact with antigen-bearing target cells and thereby destroy these target cells under the action of cytotoxins. These antibodies "arm" the cytotoxic cells and are absolutely required for such cytolysis. Primary cells mediating ADCC, i.e., NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The data obtained for FcR expression on hematopoietic cells, are systematized in table�e 3 on page 464 of Ravetch & Kinet Annu. Rev. Immunol. (1991) 9:457-92. To assess ADCC activity of interest molecules can be analyzed for ADCC in vitro, such as analysis, described in U.S. patents №№ 5500362 or 5821337. Effector cells suitable for such analyses, are mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additionally, ADCC activity of interest molecules can be assessed in vivo, e.g., in animal models, such as the model described in Clynes et al., Proc. Natl. Acad. Sci., USA, 95:652-656 (1998).

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc-region of antibodies. The preferred FcR is a human FcR with a native sequence. Moreover, a preferred FcR is FcR, which binds to the IgG antibody (a gamma receptor) and receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and alternative spliced forms of these receptors. The FcγRII receptors are FcγRIIA (an“activating receptor”) and FcγRIIB (an“inhibiting receptor”), which have similar amino acid sequences that differ primarily by their cytoplasmic domains. Activating receptor FcγRIIA contains in its cytoplasmic domain activates the motive of immunoreceptor-based tyrosine (ITAM). Inhibits re�eptor FcγRIIB contains in its cytoplasmic domain inhibiting the motive of immunoreceptor-based tyrosine (ITIM) (see browse to Daeron M.,Annu. Rev. Immunol., 15:203-234 (1997)). FcR is described in the publications Ravetch &Kinet,Annu. Rev. Immunol.9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994) and de Haas et al.,J. Lab. Clin. Med., 126:330-41 (1995). As used herein, the term “FcR” also extends to other FcR, including FcR, which will be identified in the future. This term also includes the receptor present in newborns, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al.,J. Immunol., 117:587 (1976) and Kim et al.,J. Immunol., 24:249 (1994)).

Binding to human FcRn in vivo and the half-life of polypeptides binding to human FcRn high affinity binding can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates, which were introduced polypeptides with a variant Fc-region. In WO 2000/42072 (Presta) describes variants of antibodies with enhanced or reduced activity of binding to FcR. See, also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

"Human effector cells" are leukocytes that Express one or more FcR and having effector functions. Preferably, these cells Express at least FcγRIII and have ADCC effector function. Examples of human leukocytes that mediate ADCC, are mononuclear to�etki peripheral blood (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; thus are preferred PBMC and NK cells. Effector cells may be isolated from their natural source, e.g., from the blood.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Classical pathway complement activation is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are associated with them cognatum antigen. To assess activation of complement can be analyzed at CDC, for example, as described in Gazzano-Santoro et al.,J. Immunol. Methods, 202:163 (1996). Polypeptide variants having a modified amino acid sequence of the Fc-region (polypeptides with a variant Fc region) and having enhanced or reduced ability to bind to C1q described in U.S. patent No. 6194551B1 and in WO99/51642. Cm. also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

The term "antibody containing the Fc-region" means an antibody comprising a Fc-region. C-terminal lysine (residue 447 according to the European EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant nucleic acid construct that encodes the antibody. Accordingly, a composition comprising an antibody having an Fc region according to the invention, may contain the antibody with K447 residue; antibody where all K447 removed, or a mixture of antibodies containing and not containing the K447 residue.

"Extracellular domain of CD79b polypeptide or "ECD" is a form of CD79b polypeptide, basically do not contain the transmembrane and cytoplasmic domains. Usually ECD CD79b polypeptide is less than 1% of these transmembrane and/or cytoplasmic domains and preferably less than 0.5% of such domains. It should be noted that any transmembrane domains identified for CD79b polypeptides according to the invention, identified in accordance with the criteria commonly used by analysts to identify hydrophobic domain of this type. The exact boundaries of a transmembrane domain may vary but most likely that they differ by no more than about 5 amino acids at either end of a domain originally identified. Therefore, the extracellular domain of CD79b polypeptide may include, but not necessarily, about 5 or fewer amino acids on either side of the edge region of the transmembrane domain/extracellular domain, identified as described in the examples or in the description of the present application, and such polypeptides, with or without attached signal peptide, and nucleic acid that encodes these polypeptid�s, included in the scope of the present invention.

The presumed localization of the "signal peptides" described here CD79b polypeptide may be specified in the description of the present application and/or in the description of graphic material. However, it should be noted that the C-terminal boundary of the signal peptide may vary, but it is most likely that the difference is not more than about 5 amino acids either side of the C-terminal boundary of the signal peptide, originally identified in the present application, where the specified C-terminal boundary of the signal peptide may be identified in accordance with the criteria commonly used by specialists to identify the item the amino acid sequence of this type (e.g., Nielsen et al.,Prot. Eng.10:1-6 (1997) and von Heinje et al.,Nucl. Acids. Res.14:4683-4690 (1986)). In addition, it is also known that in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in the formation of more than one secreted molecules. These Mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide, identified in the present application, and polynucleotides encoding such polypeptides, are included in this volume�th invention.

The term "variant of the CD79b polypeptide" means a CD79b polypeptide, preferably an active CD79b polypeptide as defined in the present description and having an amino acid sequence that is at least about 80% identical to full-sized native sequence CD79b polypeptide described in this application; sequence CD79b polypeptide that does not contain signal peptide described in this application; the sequence of the extracellular domain of CD79b polypeptide containing or not containing a signal peptide described in this application; or sequence with any other fragment of a full-CD79b polypeptide described in this application (such as a polypeptide encoded by the nucleic acid, which is only part of the full-size sequence that encodes a full-sized CD79b polypeptide). Such options CD79b polypeptide include, for example, CD79b polypeptides in which one or more amino acid residues added or deleterows N - or C-end full-sized native amino acid sequence. Usually a variant of the CD79b polypeptide has an amino acid sequence that is at least about 80%, and alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicon� a full-sized amino acid sequence of native CD79b polypeptide, described in this application; sequence CD79b polypeptide that does not contain signal peptide described in this application; the sequence of the extracellular domain of CD79b polypeptide containing or not containing a signal peptide described in this application; or the sequence of any other specifically defined fragment of the full-sized CD79b polypeptide described in this application. Usually versions of the CD79b polypeptide has a length of at least about 10 amino acids, or alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids or more. Options CD79b polypeptides have, but are not necessarily, no more than one conservative substitution and, alternatively, not more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to the native sequence CD79b polypeptide.

"Percent (%) identity of amino acid sequences of peptides or polypeptides, i.e., sequence CD79b polypeptide identified in the present application, is defined as the percentage of amino acid residues in the sequence of the candidate that are identical with amino acid residues in the specific peptide or polypeptide n�coherence, that is, the sequence CD79b polypeptide, after aligning the sequences and the introduction of “gaps, if necessary, to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the identical sequences. Alignment carried out to determine the percent identity of amino acid sequences may be conducted by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR). The person skilled in the art can determine appropriate parameters of the alignment, including any algorithms needed to achieve optimal alignment over the entire length of the compared sequences. However, for the purposes of the present invention, the % identity of amino acid sequences is calculated using computer program ALIGN-2, used to compare sequences, where the full source code of the program ALIGN-2 is shown in table 1 below. Computer program ALIGN-2, used to compare sequences, was developed by Genentech, Inc. and the source code provided in table 1 below, was listed� in the documentation for users of the registration Office for copyright in the USA, Washington, D. C., 20559, under registration number U. S. Copyright Registration No. TXU510087. The program ALIGN-2 is a public program supplied by the company Genentech, Inc., South San Francisco, California, or it can be compiled from source code provided in table 1 below. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All parameters of comparison sequences were set in the program ALIGN-2 was not changed.

In the case when for comparing amino acid sequences using a program ALIGN-2, the % identity of a given amino acid sequence And with a given amino acid sequence B (which may alternatively be called a given amino acid sequence And having a single or a certain % identity with the amino acid sequence B) is calculated as follows:

100•X/Y

where X represents the number of amino acid residues that have been assessed as fully relevant when aligning sequences using the program ALIGN-2, with which we compared the sequences A and b, and where Y represents the total number of amino acid residues in the sequence of V. it should be noted that if the length of the amino acid�a combined sequence And not equal to the length of amino acid sequence B, the % identity of amino acid sequence And amino acid sequence In not equal the % amino acid sequence identity with the amino acid sequence of A.

The term "variant of the polynucleotide CD79b" or "nucleic acid sequence CD79b variant" means a nucleic acid molecule encoding the CD79b polypeptide, preferably an active CD79b polypeptide as defined in the present description and having a nucleic acid sequence that is at least about 80% identical to the full nucleic acid sequence that encodes a full-sized native CD79b polypeptide described in this application; a full-sized native CD79b polypeptide that does not contain signal peptide described in this application; the extracellular domain of CD79b polypeptide containing or not containing a signal peptide described in the present application; or sequence with any other fragment of a full-CD79b polypeptide described in this application (such as a polypeptide encoded by a nucleic acid that represents only a portion of the full-size sequence that encodes a full-sized CD79b polypeptide). Usually a variant of the polynucleotide CD79b has a nucleic acid sequence that is at least about na%, alternatively, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence that encodes a full-sized native CD79b polypeptide described in this application; full-sized native sequence CD79b polypeptide that does not contain signal peptide described in this application; the extracellular domain of CD79b polypeptide, or not containing a signal sequence described in this application; or sequence with any other fragment of a full-CD79b polypeptide described in this application. These options do not contain a native nucleotide sequence.

Typically, polynucleotide variants CD79b have a length of at least about 5 nucleotides and, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 nucleotides, where in the context of the present description, the term "approximately" indicates the length of the specified nucleotide sequence ±10% of �Lina consider the sequence.

"Percent (%) identity of nucleic acid sequences, i.e. nucleic acid sequences encoding the CD79b identified in this application, is defined as the percentage of nucleotides in the sequence of the candidate that are identical with the nucleotides in interest with a nucleic acid sequence CD79b, after aligning the sequences and the introduction of "gaps, if necessary, to achieve the maximum percent sequence identity. Alignment carried out to determine the percent identity of the nucleic acid sequences may be conducted by various methods, known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR). However, for the purposes of the present invention, % identity nucleic acid sequences is calculated using computer program ALIGN-2, used to compare sequences, where the full source code of the program ALIGN-2 is shown in table 1 below. Computer program ALIGN-2, used to compare sequences, was developed by Genentech, Inc. and the source code is presented below in table 1, was registered in the documentation for users in Ve�amste copyright USA, Washington, D. C., 20559, under registration number U. S. Copyright Registration No. TXU510087. The program ALIGN-2 is a public program supplied by the company Genentech, Inc., South San Francisco, California, or it can be compiled from source code provided in table 1 below. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All parameters of comparison sequences were set in the program ALIGN-2 was not changed.

In the case when to compare sequences of nucleic acid used in the program ALIGN-2, % identity of the sequence of nucleic acids with the nucleic acid sequence D (which may alternatively be named the nucleic acid sequence With, or having a certain component % identity with the nucleic acid sequence D) is calculated as follows:

100•W/Z,

where W represents the number of nucleotides that were assessed as being fully identical with the sequence alignment using the program ALIGN-2, with which we compared sequences C and D, and where Z represents the total number of nucleotides in the sequence D. it should be noted that if the length of posledovatelno�and nucleic acid C is not equal to the length of nucleic acid sequence D, the % identity nucleic acid sequence With a nucleic acid sequence D is not equal to the % identity nucleic acid With a nucleic acid sequence D. Unless specified otherwise, all values used here % identity nucleic acid sequences prepared as described in the previous paragraph using the computer program ALIGN-2.

In other embodiments of the invention, polynucleotide variants CD79b are nucleic acid molecules that encode the CD79b polypeptide and which have the ability to gibridizatsiya preferably under stringent conditions of hybridization and washing, the nucleotide sequences encoding described here full-CD79b polypeptide. Options CD79b polypeptide can be a polypeptide encoded by the polynucleotide variant CD79b.

The term "full-coding region", if it refers to nucleic acid that encodes a CD79b polypeptide, means a nucleotide sequence encoding a full-CD79b polypeptide according to the invention (which is often localized between start - and stop codons, inclusive, as shown in the accompanying graphic material). The term "full-coding region", if it refers to a nucleic acid deposited with the ATCC, means�no part of the cDNA, encoding a CD79b polypeptide and is built into the vector deposited with the ATCC (which is often localized between start - and stop codons, inclusive, as shown in the accompanying graphic material (where the start and stop codons shown in bold and underlined)).

The term "highlighted" when he refers to the various described here CD79b polypeptide, means a polypeptide that has been identified and isolated and/or purified from components of its natural environment. Contaminating components of its natural environment are materials that have a negative influence on therapeutic effectiveness of the polypeptide, and such components can be enzymes, hormones and other protein or non-protein solute. In preferred embodiments of the invention the specified polypeptide can be purified (1) to the extent reasonably necessary for the introduction of at least 15 residues of N-terminal or internal part of the amino acid sequence, using the sequencer, equipped with a centrifuge vessel, or (2) to homogeneity, which can be confirmed by electrophoresis in LTOs-page in reducing or Sevostyanova conditions with staining of Kumasi blue or, preferably, silver. The term "selected polypeptide includes polypeptide in situ within recombinant cells, if there is no p� least one component of the CD79b polypeptide, present in its natural environment. Usually, however, the selected polypeptide may be obtained in at least one stage of cleaning.

"Isolated" nucleic acid that encodes a CD79b polypeptide, or another nucleic acid that encodes the polypeptide, is a nucleic acid molecule that is identified and separated from at least one impurity nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid encoding the polypeptide. The selected nucleic acid molecule that encodes a polypeptide that has a shape or structure that is different from the form or structure of a natural molecule. Therefore, the selected nucleic acid molecule encoding a polypeptide different from the specific nucleic acid molecule that encodes a polypeptide and exists in natural cells. However, the selected nucleic acid molecule that encodes a polypeptide that includes a nucleic acid molecule encoding the polypeptide contained in cells that normally Express the polypeptide where, for example, the nucleic acid molecule is present in the chromosome at the position different from the provisions of this nucleic acid in natural cells.

The term "regulatory sequence" means a DNA sequence, not�required for expression of the functionally attached to the coding sequence in a particular organism, the host. Regulatory sequences that are suitable for prokaryotes, for example, a promoter, optionally an operator sequence and the binding site with the ribosome. Eukaryotic cells are known, utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "functionally connected", if it is in a functional relationship with another nucleic acid molecule. So, for example, DNA for a pre-sequence or secretory leader sequence is functionally connected to the DNA of the polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer functionally joined to a coding sequence if they affect the transcription of the sequence; and the binding site with the ribosome is functionally joined to a coding sequence if it is located in a position conducive to the broadcast. Generally speaking, the term "functionally connected" means that the DNA sequence associated with each other, are adjacent, and in the case of a secretory leader sequence, they are adjacent and are in the same reading frame. However, enhancers need not be contiguous. The binding is established through the� ligation at suitable restriction sites. If such sites are not available, then synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

"The rigidity of the conditions of the hybridization reaction can be easily determined by an average person skilled in the art, and usually it is calculated empirically based on the length of the probe, the temperature of washing and salt concentration. Basically, the longer the probe, the higher should be the temperature of hybridization, and the shorter the probe, the lower should be the temperature. Hybridization generally depends on the ability of denatured DNA to re-anneal, when complementary strands are present in an environment with a temperature below their melting temperature. The higher the degree of desired homology between the probe and hybridizing sequence, the higher must be the relative temperature. From this it follows that higher relative temperatures create more stringent reaction conditions and lower temperatures create less stringent reaction conditions. A more detailed description and explanation of the conditions of stringency hybridization reactions can be found in the manual Ausubel et al.,Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Terms of rigidity" or "conditions of high stringency" as defined in the present application, can be set as conditions, which include (1) prom�internals at low ionic strength and high temperature, for example, rinsing of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) use during hybridization a denaturing agent such as formamide, for example, 50% (vol./about.) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mm nutrifaster buffer, pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) hybridization overnight in a solution containing 50% formamide, 5×SSC (0,75 M NaCl, of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5× solution of Denhardt, treated with ultrasound DNA salmon sperm (50 µg/ml), 0.1% of LTOs and 10% dextran sulfate at 42°C with a 10 minute wash at 42°C in 0.2×SSC (sodium chloride/sodium citrate) followed by a 10-minute washing in the conditions of high hardness, consisting of 0.1 x SSC containing EDTA at 55°C.

"Conditions of moderate stringency" may be identified as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of less stringent conditions of washing and hybridization (such as, for example, temperature, ionic strength and % LTOs) than the conditions described above. An example of conditions of moderate stringency is incubation over night at 37°C in a solution containing 20% formamide, 5×SSC (150 mm NaCl, 15 mm trisodium citrate), 50 mm sodium phosphate (pH 7.6), 5× solution of Denhardt, 10% Sul�fat dextran and 20 mg/ml denatured fragmented DNA salmon sperm, followed by washing the filters in 1×SSC at about 37-50°C. If it is necessary to adapt these conditions to the relevant parameters, such as probe length, etc., temperature, ionic strength, etc. can be adjusted by methods known in the art.

As used herein, the term "labeled epitope" means a chimeric polypeptide containing a polypeptide or anti CD79b-CD79b antibody attached to the polypeptide-label". Polypeptide-tag must have a certain number of residues, sufficient to create an epitope against which may be sent to the antibody, and must be short enough, i.e. such that it did not affect the activity of the polypeptide to which it is attached. In addition, preferably, such a polypeptide tag was quite unique, i.e. such that the antibody did not possess significant cross-reactivity with other epitopes. Suitable polypeptides tags usually have at least six amino acid residues, and mostly about 8-50 amino acid residues (preferably about 10-20 amino acid residues).

As used herein, the terms "active" or "activity" refers to form(s) of the CD79b polypeptide which retains the biological or immunological activity of natural or native CD79b, where the term "biological�th activity" means a biological function (either inhibitory or stimulatory), reported native or natural CD79b, except for the ability to induce the production of antibodies against an antigenic epitope located on native or natural CD79b, and the term "immunological activity" means the ability to induce the production of antibodies against an antigenic epitope located on native or natural CD79b.

The term "antagonist" is used here in its broadest sense and includes any molecule that partially or fully blocks, inhibits or neutralizes a biological activity of native CD79b polypeptide. Similarly, the term "agonist" is used here in its broadest sense and includes any molecule that mimics a biological activity of native CD79b polypeptide. Suitable molecules are agonists or antagonists, in particular, antibodies are agonists or antagonists or fragments of antibodies, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules CD79b, etc., Methods of identifying agonists or antagonists of the CD79b polypeptide may include contacting CD79b polypeptide molecule is an agonist or antagonist that is used as a candidate, and determining the detected changes in one or more biological�such activities, usually associated with a CD79b polypeptide.

The term "purified" refers to a molecule present in the sample at a concentration of at least 95 wt.% or at least 98 wt.% the sample in which it is contained.

"Allocated" nucleic acid molecule is a nucleic acid molecule that is separated from at least one other nucleic acid molecule with which it is normally associated, for example, in natural surroundings. The selected nucleic acid molecule includes a nucleic acid molecule contained in cells that normally Express this molecule of nucleic acid, but wherein said nucleic acid molecule is present outside of a chromosome or chromosomes that is different from its natural position in the chromosome.

Used here, the term “vector” means a nucleic acid molecule capable of another nucleic acid to which it is attached. One type of vector is a “plasmid”, a circular double stranded DNA loop which can be legirovanyh additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be legirovanyh with the viral genome. Some vectors�ry can autonomously replicate in the host cell, in which they were introduced (e.g., bacterial vectors having a bacterial origin of replication, and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of the host cell after its introduction in a specified host cell, with the result that they can be replicated along with the host genome. Moreover, certain vectors are capable of regulating the expression of genes to which they were functionally connected. Such vectors are referred to here as “recombinant expression vectors” (or simply “recombinant vectors”). In General, expression vectors commonly used in recombinant DNA methods, often have the form of plasmids. In the present description, the terms “plasmid” and “vector” can be used interchangeably as the plasmid is the most common form of the vector.

As used herein, the terms “polynucleotide” or “nucleic acid” are used interchangeably and refer to polymers of any length, consisting of nucleotides, and such polymers are DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or the pic�edstam synthesis reaction. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If necessary, modification to the nucleotide structure may be performed before or after Assembly of the polymer. The nucleotide sequence may be interrupted dinucleotide components. The polynucleotide may be further modified after synthesis, e.g., by conjugation with a label. The other types of modifications include, for example, “cap”; to replace one or more natural nucleotide analogs; magnolioideae modifications such as, for example, modification by introduction of uncharged linkages (e.g., methylphosphonate, phosphotriesters, phosphoamide, carbamates, etc.) and with charged linkages (e.g., fosforito, phosphorodithioates, etc.); modification containing side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications contain intercalating agents (e.g., acridine, psoralen, etc.); modification containing hepatoblastoma agents (e.g., metals, radioactive metals, boron, metals, oxidizers, etc.); modification containing alkylating agents; modification containing modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as nomodifier�EN forms of the polynucleotide(s). In addition, any hydroxyl group normally present in the sugars may be replaced, for example, phosphonate groups, and phosphate groups, protected by standard protective groups; or activated with the formation of additional linkages to additional nucleotides, or they can be anywhereman with solid or semi-solid media. 5'- and 3'-terminal IT can be fosfaurilirovanny or substituted with amines or organic “kairouseki” groups comprising from 1-20 carbon atoms. Other hydroxyls may also be derivationally standard protective groups. Polynucleotides can also contain analogous forms of sugars, such as ribose or deoxyribose, known in the art, including, for example, 2'-O-methylribose, 2'-O-allelism; 2'-fluoro - or 2'-isidoros; carbocyclic analogues of sugars; alpha-anomeric sugars; epimeria sugars, such as arabinose, xylose or lyxose; the sugar pyranose, the sugar furanose; sedoheptulose; acyclic analogs and non-nucleoside analogues, such as methylribose. One or more fosfolipidnyh ties can be replaced by alternative linker groups. Such alternative linker groups include, but are not limited to, cases in which the phosphate is replaced by P(O)S (“tiat”), R(S)S (“ditial”), (O)NR2(“amidate”), RHO)R, P(O)OR', CO or CH2(“Formatul”), where each R or R' independently represents H or substituted or unsubstituted alkyl (C1-20), optionally containing ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not all linkages in the polynucleotide must be identical. The foregoing description applies to all used here, the polynucleotides, including RNA and DNA.

As used herein, the term “oligonucleotide”, in General terms, means a short, mostly single stranded, generally synthetic polynucleotides, the length of which is mainly, but not necessarily, less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The foregoing description of polynucleotides can equally and fully to relate to oligonucleotides.

As used herein, the terms “cancer” and “cancerous” refer to the physiological condition of mammals that is typically characterized by unregulated growth of cells. Examples of cancer include, but are not limited to, cancer of the hematopoietic system or blood cancer, such as lymphoma, leukemia, myeloma or lymphoid malignant disease, and also cancer of the spleen, cancer of the lymph nodes, carcinoma, blastoma and sarcoma. More specific examples of such cancer Zabol�vany are In-cell carcinoma, including, for example, vysokokachestvennyy, srednekagesstroy and nizkolegirovannuju lymphoma (including b-cell lymphomas, such as, for example, b-cell lymphoma of lymphoid tissue mucosa, and non-Hodgkin's lymphoma (NHL), lymphoma cells of the cortex of the brain, Burkitt's lymphoma, small cell lymphocytic lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, Hodgkin lymphoma and T cell lymphomas) and leukemia (including secondary leukemia, chronic lymphocytic leukemia (CLL), such as b cell leukemia (CD5+-B-lymphocytes), myeloid leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia (ALL) and myelodysplasia), and other hematological and/or b-cell or T-cell cancers. In the present invention also includes other cancers of hematopoietic cells, including polymorphically leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes and natural killer cells. In the present invention also includes a cancerous b-cell proliferative disorder selected from the following diseases, such as lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed�tion of asymptomatic NHL untreatable the NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. Areas of education b-cell cancer are the following areas, for example, b-cell lymphoma marginal zone develops in the b-memory cells in the marginal zone, follicular lymphoma and diffuse large b-cell lymphoma develops in centrocytic in the light zone of germinal centers, chronic lymphocytic leukemia and small-cell lymphocytic leukemia develops in cells B1 (CD5+); lymphoma cells of the cerebral cortex develops in the "untrained" b cells In the cortex of the brain, Burkitt lymphoma develops in the like centroblasts in the dark zone of the germinal centers. Tissues, including hematopoietic cells and which are referred to here as "tissues of hematopoietic cells", are the thymus and bone marrow and peripheral lymphoid tissues such as spleen and lymph nodes; the lymphoid tissue associated with mucosa, such as lymphoid tissue of the intestine, tonsils, Peyer's patches and the Appendix, lymphoid tissue associated with other mucosal sites, for example, the lining of the bronchi. Other nodules�governmental examples of such cancers are squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, cancer of the gastrointestinal tract, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer and colon cancer, carcinoma of the endometrium or uterine carcinoma of the salivary glands, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders and various types of head and neck cancer.

As used herein, the term "b-cell malignant tumor" encompasses nahodkinskuju lymphoma (NHL), including nizkolegirovannuju/follicular NHL, small lymphocytic (ML) NHL srednekagesstroy/follicular NHL, srednekagesstroy diffuse NHL, vysokokachestvennyy immunoblastic NHL, vysokokachestvennyy lymphoblastic NHL, vysokokachestvennyy small cell undifferentiated NHL, generalized NHL, lymphoma cells of the cerebral cortex, lymphoma associated with AIDS and waldenstrom's macroglobulinemia; nahodkinskuju lymphoma (NHL), lymphocyte predominantly Hodgkin's disease (LPBH), small cell lymphocytic lymphoma (MLL), chronic lymphatic�itary leukemia (CLL), asymptomatic NHL, including recurrent asymptomatic NHL and asymptomatic NHL, not amenable to treatment with rituximab; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), reticuloendotheliosis, chronic myeloblastic leukemia; lymphoma cells of the cerebral cortex and other hematological malignant tumors. Such malignant disease may be subjected to treatment with antibodies against markers of b-cell surfaces, such as CD79b. Such diseases are considered diseases that can be treated by injecting antibodies against the marker of b-cell surfaces, such as CD79b, where said treatment includes the introduction of an unconjugated ("naked") antibody or an antibody conjugated with a cytotoxic agent, as described in the present application. These diseases are also referred to here as diseases that can be treated by the methods of combination therapy, including the introduction of anti-CD79b antibody or conjugate is anti-CD79b antibody-drug" according to the invention in combination with the introduction of another antibody or other conjugate "antibody-drug", or other cytotoxic agents with radiation therapy or other therapy conducted synchronous� or sequentially. In a representative method of treatment according to the invention, an anti-CD79b antibody according to the invention is administered in combination with anti-CD20 antibody immunoglobulin or CD20-binding fragment, and such treatment may be carried out simultaneously or sequentially. Anti-CD20 antibody may be "naked" antibody or conjugate "antibody-drug". In an embodiment, the combination therapy of anti-CD79b antibody is an antibody according to the invention, and anti-CD20 antibody is Rituxan® (rituximab).

As used herein, the term "non-Hodgkin's lymphoma" or "NHL" means a cancer of the lymphatic system, with the exception of Hodgkin's lymphoma. In General, Hodgkin's lymphoma and non-Hodgkin's lymphoma can vary the fact that the Hodgkin's lymphoma cells are reed-Sternberg and non-Hodgkin's lymphoma, these cells are absent. Examples of non-Hodgkin's lymphomas covered used herein the term, are all lymphomas, which can be identified by a person skilled in the art (e.g., an oncologist or pathologist) in accordance with known classification schemes, such as the Revised Euro-American classication of lymphomas (REAL), described in “Color Atlas of clinical Hematology (3rd edition) (Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.)(Harcourt Publishers Ltd., 2000). See, an hour�ness, the lists shown in Fig.11.57, 11.58 and 11.59. More specific examples of lymphomas include, but are not limited to, recurrent or not amenable to treatment of NHL; border nizkozameshhennoj NHL first line; NHL stage III/IV; NHL that are resistant to chemotherapy; lymphoblastic leukemia and/or lymphoma, containing b-cell precursor; small cell lymphocytic lymphoma; b-cell chronic lymphocytic leukemia and/or prolimfocitarnoj leukemia and/or small cell lymphocytic lymphoma; b-cell prolimfocitarnaâ lymphoma; immunocytoma and/or lymphoplasmacytic lymphoma; lymphoplasmacytic lymphoma; b-cell lymphoma marginal zone; lymphoma marginal zone of the spleen; lymphoma extranodal marginal zone - MALT; nodal marginal zone lymphoma; reticuloendotheliosis; plasmacytoma and/or plasmacytoma myeloma; nizkozameshhennoj/follicular lymphoma; srednestaticheskaya/follicular NHL; lymphoma of the cerebral cortex; follicular-centralita lymphoma (follicular); srednestaticheskaya diffuse NHL; diffuse large b-cell lymphoma; aggressive NHL (including aggressive edge and recurrent aggressive NHL), NHL relapsing after autologous transplantation of stem cells, or the NHL that are resistant to such transpla�orientation; primary mediastinal large cell b-cell lymphoma; primary effusion lymphoma; vysokokachestvennaya immunoblastic NHL; vysokokachestvennaya lymphoblastic NHL; vysokokachestvennaya small cell undifferentiated NHL; generalized NHL; Burkitt lymphoma; large cell granulocytic leukemia progenitor cells (peripheral); mushroom mycosis fungoides and/or Sezary syndrome; lymphoma of the skin (cutaneous lymphoma); anaplastic large cell lymphoma and angiocentric lymphoma.

The term "disorder" means any condition that is curable by a substance/molecule or method according to the invention. These disorders are chronic and acute disorders or diseases including pathological conditions that create this predisposition of a mammal to the disorder. Non-limiting examples of disorders described herein, exposed to treatment are cancers such as malignant and benign tumors; non-leukemias and lymphoid malignant tumor; lesion of neurons, glial cells, astrocytes, hypothalamic and other disorders of the endocrine system, macrophages, epithelium, stroma and blastocele; inflammatory and immune disorders and other disorders of the AU�oneiromancy with angiogenesis. In addition, these disorders are also cancer such as b-cell proliferative disorder and/or b-cell tumors such as lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment of the invention the specified cell-proliferative disorder is cancer.

Used here, the term “tumor” refers to all neoplastic cells undergoing growth and proliferation, regardless of whether they are benign or malignant, and all precancerous and cancerous cells and tissues.

As used herein, the term “autoimmune disease” means a disease or disorder caused by a reaction produced by the body against its own tissues or organs or a co-segregate or manifestation of these disorders and�and their associated status. In many of these autoimmune and inflammatory disorders can be present a certain number clinical laboratory markers, including, but not limited to, hand hypergammaglobulinemia, high levels of autoantibodies, deposition of complex "antigen-antibody" in the tissues, the favorable effect of treatment with corticosteroids or immunosuppressive agents and aggregates of lymphoid cells in the affected tissues. Not limited to any particular theory regarding b-cell autoimmune disease, we can say that in human autoimmune diseases b cells demonstrate its pathogenic action on a variety of mechanisms, including production of autoantibodies, formation of immune complexes, activation of dendritic cells and T cells, synthesis of cytokines, which direct the release of chemokines and the emergence of foci of ectopic politogenesis. Each of these mechanisms in varying degrees may participate in the development of autoimmune diseases.

"Autoimmune disease" can be an organ-specific disease (i.e., an immune response specifically directed at the system of organs such as the endocrine system, hematopoietic system, the skin, the cardiopulmonary system, gastrointestinal tract and liver and renal system, the thyroid, the ears, the neuromuscular system, Central �ervna system, etc.) or a systemic disease, that can affect system of many organs (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, etc.). Preferably such diseases are autoimmune rheumatologic disorders (such as, for example, rheumatoid arthritis, syndrome Segren, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid syndrome antibodies and psoriatic arthritis), autoimmune diseases gastrointestinal and liver disease (such as inflammatory bowel disease (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including vasculitis charge-Strauss, Wegener's granulomatosis and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, syndrome dancing gdas, myasthenia gravis, neuromyelitis optic nerve disease, Parkinson's disease, Alzheimer's and autoimmune polyneuropathy), kidney disease (such as, for example, glomerulonephritis, goodpasture's syndrome, and Berger's disease), autoimmune skin diseases (such as, e.g.�measures psoriasis, urticaria, rash, vulgar pemphigus, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, purpura occurring after transfusion of blood, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune disease of the auditory pathways (such as, for example, disease of the inner ear and hearing loss), Behcet's disease, Raynaud's syndrome, a disease associated with organ transplantation, and autoimmune endocrine diseases (such as, for example, autoimmune diseases associated with diabetes, such as insulin-dependent diabetes mellitus (IDDM), Addison's disease and autoimmune thyroid disease (e.g., graves ' disease and thyroiditis)). From these diseases more preferred are, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, syndrome Segren, graves ' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Specific examples of other autoimmune diseases mentioned in this application, which in some cases encompass those listed above diseases are, but not limited to, arthritis (acute and chronic arthritis, rim�todny arthritis, including juvenile rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, arthritis induced by collagen type II, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, still's disease, arthritis of the vertebrae, osteoarthritis, progredient chronic arthritis, deforming arthritis, chronic primary arthritis, reactive arthritis, menopausal arthritis, arthritis caused by depletion of estrogen and ankylosing spondylitis/rheumatoid spondylitis), autoimmune lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis, such as blaskovic psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis; atopy including atopic diseases such as hay fever and syndrome Jobe; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, urticaria, dermatitis herpetiformis, coin-like dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis and atopic dermatitis; coupled with X-linked Hyper IgM syndrome, allergic intraocular will vocalite� - to-date diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as DCS, accompanied by dysfunction of the spinal cord and the organs of vision, primary progressive MS (APP) and relapsing-remitting clinical RS (RRRS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated (multiple) sclerosis, ataxic sclerosis, neuromyelitis optic nerve (NZN), inflammatory bowel disease (IBD)(e.g., Crohn's disease, autoimmune gastrointestinal disorders, inflammation of the gastrointestinal tract, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenosis colitis, polypoid colitis, Nekrosius enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease); inflammatory bowel disease, gangrenous pyoderma, nodular erythema, primary sclerosing cholangitis, respiratory distress syndrome, including respiratory distress syndrome, adult (rdsw) or acute respiratory distress syndrome, mining�t, inflammation of the entire uveal tract, or part thereof, iritis, hareidit, an autoimmune hematological disorder, graft-versus-host, angioedema, such as hereditary angioedema, cranial nerve, as in meningitis, herpes pregnancy, pemphigoid pregnant, itching in the scrotum, autoimmune premature decline of ovarian function, sudden hearing loss caused by an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and encephalitis with lesions of the extremities and/or brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, agranulocytosis uveitis, valanchery uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with nephrotic syndrome with or without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immunopositivity GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranosa-proliferative GN (MPGN), including glomerulonephritis type I and type II, and rapidly progressive GN (MPGN), proliferative nephritis, autoimmune plyuriglandulyarnaya endocrine insufficiency, �Alanic, including plasmacytosis enveloping balanitis, balanoposthitis, annular centrifugal erythema, ashy dermatosis, mnogoformnaya erythema, granuloma annulare, shiny ringworm, sclerotic atrophic lichen, simple chronic zoster, thorn zoster, oral lichen planus, lamellar ichthyosis, epidermolitichesky hyperkeratosis, premalignant keratosis, gangrenous pyoderma, allergic conditions and responses, food allergies, allergies to medications, allergies to insects, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, aleatoricism eczema, dyshidrotic eczema, and vesicular palmoplantar eczema; asthma, such as asthma and autoimmune asthma; condition caused by the infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as blood group antigens A-b-O fruit produced during pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, insufficient adhesion of leukocytes; lupus, including lupus nephritis, lupus encephalitis, childhood lupus, depositnow lupus, extrarenal lupus, discoid lupus, discoid lupus erythematosus and lupus alopecia; systemic lupus erythematosus (�KV), such as cutaneous SLE or subacute cutaneous SLE, lupus syndrome of newborns (HRV), and disseminated lupus erythematosus; juvenile diabetes (type I), including pediatric insulin-dependent diabetes mellitus (IDDM), diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic lesions of large arteries; immune responses associated with acute delayed-type hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, agranulocytosis; vasculitides (including vasculitis, large blood vessels, such as polymyalgia rheumatica and giant cell arteritis (Takayasu), vasculitis medium-sized blood vessels, such as disease Kavasaki and polyarteritis nodosa/periarteritis nodosa, immunovaccine, CNS vasculitis, cutaneous vasculitis, allergic vasculitis, Nekrosius vasculitis such as fibrinoid Nekrosius vasculitis and systemic Nekrosius vasculitis, ANCA-negative vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss (SCS), Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, �sitina anemia Coombs, anemia diamond-I am, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (pernicious anemia), Addison's disease, pure red cell anemia or aplasia (PRCA), a deficiency of factor VIII, hemophilia a, autoimmune neutropenia, cytopenia, such as pancytopenia, leukopenia, diseases that lead to diabetes of leukocytes, inflammatory disorders of the Central nervous system, Alzheimer's disease, Parkinson's disease, syndrome of defeat of many organs, such as a secondary syndrome associated with sepsis, trauma, or hemorrhage; diseases, it is mediated by the formation of complex antigen-antibody, disease glomerular basal membranes catalyzed reaction of antibody-antigen, antiphospholipid syndrome, narit motor nerve, allergic narit, disease/Behcet's, syndrome Kalman, goodpasture's syndrome, Raynaud's syndrome, syndrome of Segren, syndrome Stevens-Johnson, pemphigoid or pemphigus such as bullous pemphigoid, treatment-resistant pemphigoid (mucous membranes), skin pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceous, pemphigoid mucous membranes-membranous pemphigoid and pemphigus erythematous, acquired epidermolysis bullosa, eye inflammation, the preferred�of allergic inflammation of the eye, such as allergic conjunctivitis, bullous disease associated with linear IgA, inflammation of the conjunctiva induced autoimmune disease, autoimmune polyendocrinopathy, disease or Reiter's syndrome, burn injury, caused by autoimmune disease, preeclampsia, a disease of immune complexes, such as immune complex nephritis, antibody-mediated nephritis, neuro-inflammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (e.g., developing in a patient with myocardial infarction), including thrombotic thrombocytopenic purple (TTP), posttransfusion purple (PTP), heparin-induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia including, for example, idiopathic thrombocytopenic purple (ITP) including chronic or acute ITP; scleritis such as idiopathic keratoconic, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidic, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis is very, such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune disease setuid�Oh cancer, idiopathic hypothyroidic, graves 'disease, ophthalmic graves' disease (ophthalmopathy or ophthalmopathy associated with lesions of the thyroid gland), plyuriglandulyarnaya syndromes, such as autoimmune plyuriglandulyarnaya syndromes, for example, type I (or syndromes plyuriglandulyarnaya endocrinopathy), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as myasthenic syndrome Lambert-Eaton syndrome or Eaton-Lambert syndrome, “stiff person”, encephalomyelitis such as allergic encephalomyelitis (or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as myasthenia gravis associated with thymoma, degeneration of the cerebellum, neuromyotonia, opsoclonus syndrome or “dancing eyes” (LNG) and neuropathy senses, multifocal motor neuropathy system, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (not transmissible, unlike NSIP); Guillain-Barre syndrome, Berger's disease (IgA-nephropathy), idiopathic IgA-nephropathy, linear IgA-dermatosis, acute febrile �atropellis dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumocytes syndrome, autoimmune enteropathy, bowel disease or celiac disease, intestinal sprue (gluten enteropathy), not treatable sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amyotrophique lateral sclerosis (ABS; disease Louis Gehrig), coronary heart disease; autoimmune ear disease such as autoimmune disease of the inner ear (CBA); autoimmune hearing loss; polyhedra, such as untreatable or recurrent polyhedric; pulmonary alveolar proteins, keratitis, such as syndrome Kogan/nezirroticski interstitial keratitis, bell's palsy, a disease/sweet syndrome, rosacea autoimmune, pain associated with shingles, amyloidosis, non-cancerous lymphocytosis, a primary lymphocytosis, including monoclonal b-cell lymphocytosis (e.g., benign a monoclonal gammopathy and a monoclonal gammopathy of unknown etiology, MGUS), peripheral neuropathy, paraneoplastic syndrome; “channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies” of the CNS, autism, inflammatory myopathy and Otago�th or segmental, either focal segmental glomerulosclerosis (USGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalin, atrophy of the stomach, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia areata, General alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility, sclerodactyly and telangiectasia), autoimmune infertility in men and women, for example, caused by antibodies against sperm, mixed connective tissue disease, Chagas ' disease, rheumatic fever, recurrent miscarriage, lung disease, farmers, mnogoformnaya erythema, second syndrome, Cushing's syndrome, lung disease, bird lovers, allergic granulomatous vasculitis, benign lymphocytic vasculitis, alport syndrome, alveolitis such as allergic alveolitis and fibrosis alveolitis, interstitial lung disease, transfusion disease, leprosy, malaria, parasitic diseases such as leishmaniasis, cyanosoma, schistosomiasis, Ascaris, aspergillosis, syndrome Santera syndrome Kaplan, dengue, endocarditis, engomi�cardiac fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, fibrous mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophallic, erythema elevated persistent, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, felty's syndrome, flares, cycle, such as chronic cycle, heterochronies cycle, iridocyclitis (acute or chronic) or cycle Fuchs, purpura Schonlein purpura-Henoch, infection caused by the human immunodeficiency virus (HIV), severe combined immunodeficiency (TCID), acquired immunodeficiency syndrome (AIDS), infections caused by Echovirus; sepsis (a systemic syndrome associated with inflammatory response)); endotoxemia; pancreatitis; Tiresias; infections caused by parvovirus; infection caused by a virus measles rubella; syndrome that develops after vaccination; hereditary infection caused by a virus measles rubella; infections caused by Epstein-Barr; parotitis, Evans syndrome, autoimmune gonadal failure, Sydenham chorea, post-streptococcal nephritis, obliterating thromboangiitis, thyrotoxicosis, tabes, horida, giant-cell polymyalgia, chronic allergic pneumonitis, conjunctivitis, such as vernal allergic conjunctivitis, dry keratoconus�krivit, epidermal keratoconjunctivitis syndrome, idiopathic nephritis, nephropathy, characterized by minimal changes of the renal tissue, benign hereditary and caused by ischemia reperfusion disorders, reperfusion injury in organ transplantation, autoimmune retinal disease, joint inflammation, bronchitis, chronic obstructive disease of the Airways/lungs, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebrovascular insufficiency) such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, spermiogenesis, autoimmune hemolysis, Beck disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylaxis endoftheline, allergic enteritis, nodosa lepromatous erythema, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever syndrome, Hamman-rich; sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia (sympathetic ophthalmic), ophthalmic newborns, narit optic nerve, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, tireo�it Curwen, acquired atrophy of the spinal cord, non-malignant thymoma, lymphovascular limit, vitiligo, toxic shock syndrome, food poisoning, a condition caused by the infiltration of T cells, insufficient adhesion of leukocytes, immune responses associated with acute hypersensitivity and delayed-type hypersensitivity mediated by cytokines and T-lymphocytes, diseases associated with diapedesis leukocyte syndrome lesions of many organs, diseases mediated by the formation of complex antigen-antibody, disease glomerular basal membranes catalyzed reaction “antigen-antibody”, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insult, polyendocrine failure, autoimmune plyuriglandulyarnaya syndromes, including plyuriglandulyarnaya syndrome type I idiopathic hypoparathyroidism adults (VVM), cardiomyopathy such as congestive (dilated) cardiomyopathy, such as congestive cardiomyopathy, acquired epidermolysis bullosa (PBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic�th sinusitis; ethmoid sinusitis, frontal sinusitis, maxillary sinusitis or sphenoidal; allergic sinusitis, eosinophilic disorders such as eosinophilia, pulmonary infiltrates, eosinophilia syndrome, eosinophilia-myalgia syndrome, Lefler, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma or granulomas containing eosinophils; anaphylaxis, spondyloarthropathies, seronegative spondyloarthropathies, polyendocrine autoimmune disease, sclerosing cholangitis, scleritis, episcleritis, chronic mucocutaneous candidiasis syndrome, Bruton, transient hypogammaglobulinemia in children, Wiskott-Aldrich syndrome, ataxia-telangiectasia, angiectasia, autoimmune disorders, associated with collagen disease, rheumatism such as chronic astronautalis, lymphadenitis, in response to reduction of blood pressure, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic disorders associated with hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of myocardial or other tissues, lymphomatosis tracheobron�it inflammatory dermatoses, dermatoses with components of acute inflammation, failure of many organs, bullous diseases, necrosis of the cortical substance of the kidney, acute purulent meningitis or other inflammatory disorders of the Central nervous system, inflammatory diseases of the eye and orbit; syndromes associated with transfusion of granulocytes; toxicity induced by cytokines, narcolepsy, acute serous inflammation, chronic intractable inflammation, pyelitis, hyperplasia of the inner lining of the artery, peptic ulcer, valvewith and endometriosis. Such diseases are considered diseases that can be treated by administering an antibody that binds to a marker of b-cell surfaces, such as CD79b, and such treatment includes administration of an unconjugated ("naked") antibody or an antibody conjugated with a cytotoxic agent, as described in the present application. These diseases are also referred to here as diseases that can be treated by combination therapy, comprising administering an anti-CD79b antibody or conjugate is anti-CD79b antibody-drug" according to the invention in combination with another antibody or conjugate "antibody-drug", with other cytotoxic� means, as well as with radiation therapy or another method of treatment carried out simultaneously or sequentially.

The terms "treatment", "therapy" or "weakening symptoms" refer to therapeutic treatment and prophylactic or preventative measures, which are aimed at preventing or slowing down (weakening) of undesired physiological change or disorder in an individual. The individual in need of treatment is individual, that is already a specified disorder, and the individual who has a predisposition to develop such disorders, or an individual who needs preventive measures to prevent such disorders. Cancer treatment in an individual or mammal, in tumors which Express CD79b polypeptide, is considered successful if after the introduction of a therapeutic amount of an anti-CD79b antibody by methods according to the invention, the patient has a visible and/or measurable reduction in the number of cancer cells or lack of them; reduce the tumor size; inhibition (i.e., slowing to some extent and preferably termination) infiltration of cancer cells into peripheral organs including the spread of cancer cells in soft tissue and bone; inhibition (i.e., slowing to some extent and preferably prekrasan�) tumor metastasis; the inhibition, to some extent, of tumor growth; and/or reducing, to some extent, of one or more symptoms associated with the specific cancer; reduced morbidity and mortality, and improving the quality of life of these individuals. Anti-CD79b antibody, depending on the degree of its ability to prevent the growth of cancer cells and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The weakening of these signs or symptoms may also be felt by the patient.

The above parameters for assessing the effectiveness of treatment and positive dynamics of the disease can be easily determined using routine procedures known to the doctor. As for the treatment of cancer, the treatment efficiency can be evaluated, for example, by determining the elapsed time to disease progression (TTP), and/or determine the speed of the response (RR). Metastases can be detected by tests at the stage of tumor development and by scanning the bone, and also from the analysis of the levels of calcium and enzymes to determine the spread of the tumor into the bone. May also be undertaken for scanning by means of computer tomography (CT) scans in order to identify the spread of the tumor in the pelvis and lymph nodes. To detect metastases of tumors in Le�the cue and the liver, respectively, can be made by chest x-ray and measured levels of enzymes in the liver known methods. Other routine methods of monitoring disease are transrectal ultrasonography (TUE) and transrectal biopsy (TB).

With regard to bladder cancer, which is more clearly localized cancer, the methods of determining the progression of this disease include cytological analysis of urine under the cystoscope, the monitoring of the presence of blood in the urine, visualization tract mucosa was found by conducting ultrasound or intravenous pyelography, computed tomography (CT) and imaging method, magnetic resonance imaging (MRI). The presence of peripheral metastases can be estimated using abdominal CT scan, chest x-ray or radionuclide imaging of the skeleton.

"Permanent" introduction, unlike a single injection, means the introduction of money (funds) in a continuous mode, which allows to maintain constant therapeutic effect (activity) for a long period of time. "Periodic" introduction refers to the introduction, which is not continuous and is conducted in cycles.

The term "individual" means a vertebrate. In some embodiments of the invention oksanamissbeauty is a mammal. Mammals include, but are not limited to, farm animals (such as cows), dogs that participate in sports, Pets (such as cats, dogs and horses), primates, mice and rats. In some embodiments of the invention the specified mammal is man.

"Mammal", which may be subjected to treatment or lessening of the symptoms of cancer, refers to any animal classified as a mammal, including humans, domestic animals, farm animals, animals kept in zoos, animals, participating in sports, or companion animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc., the Preferred mammal is man.

The term "introduction in combination with one or more other therapeutic agents includes simultaneous (competitive) and sequential introduction in any order.

As used herein, the term "media" includes pharmaceutically acceptable carriers, excipients or stabilizers which are nontoxic to cells in which they are administered, or for mammals, which administered these carriers, excipients or stabilizers in the dosages and concentrations. In most cases, physiologically acceptable �setelem is an aqueous pH buffered solution. Examples of physiologically acceptable carriers are buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; a polypeptide of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; hepatoblastoma agents such as EDTA; alcohols of a range of sugars, such as mannitol or sorbitol; soleobrazutaya counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

The term "solid phase" or "solid carrier" means an anhydrous matrix, which can be applied, or to which it may be attached to the antibody according to the invention. Examples of solid phases considered in the present application, are a solid phase obtained by partially or entirely of glass (such as glass with controlled pore size), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In some embodiments of the invention, depending on the context, the solid phase can start� a hole analytical tablet in other embodiments of the invention, such a solid phase is a column for purification (for example, a column for affinity chromatography). This term also includes the heterogeneous solid phase consisting of discrete particles, for example, the phase described in U.S. patent No. 4275149.

"Liposome" is a small vesicles composed of different types of lipids, phospholipids and/or surfactant that can be used to deliver drugs (such as anti-CD79b antibody) to a mammal. Components of liposomes are usually located so that they form a bilayer, similar to the lipid structure of biological membranes.

"Small" molecule or "small" organic molecule is defined here as a molecule having a molecular weight below about 500 daltons.

The term "individual", "individual" or "patient" means a vertebrate. In some embodiments of the invention specified vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), dogs that participate in sports, Pets (such as cats, dogs and horses), primates, mice and rats. In some embodiments of the invention the specified mammal is man.

The term "pharmaceutical composition" means�em a preparation in this form, which provides effective biological action of the active ingredient and does not contain other components, which can be excessively toxic to the individual, to whom said composition is administered. Such a composition may be sterile.

"Sterile" composition is avascular, meaning it does not contain living microorganisms and their spores.

As used herein, the term "effective amount" of an antibody means a quantity sufficient to achieve specific goals. "Effective amount", depending on a particular purpose may be determined empirically and in a routine way.

The term "therapeutically effective amount" means an amount of antibody or other drug effective to "treat" a disease or disorder in an individual or mammal. In the case of such a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) infiltration of cancer cells into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) the development of tumor metastasis; inhibit, to some extent, tumor growth; and/or weaken, to some extent, one or�how many symptoms associated with cancer. Cm. the definition of "treatment". The drug, to some extent, can prevent the growth of cancer cells and/or destroy cancer cells, and this drug may be cytostatic and/or cytotoxic effect. The term "prophylactically effective amount" means an amount which, when administered in the right dose or within the required time period that is effective to achieve the desired prophylactic result. Since a prophylactic dose is administered to the individual prior to the development of the disease or in an early stage of development, it is usually, but not necessarily, a prophylactically effective amount should be less than therapeutically effective amount.

"Growth inhibitory amount" of an anti-CD79b antibody is an amount capable of inhibiting the growth of cells, and especially tumor, e.g., cancer cell, either in vitro or in vivo. "Growth inhibitory amount" of an anti-CD79b antibody used for the inhibition of tumor cell growth may be determined empirically and in a routine way.

"Cytotoxic amount" of an anti-CD79b antibody is an amount capable of causing the destruction of cells, and especially tumor, e.g., cancer cell, either in vitro or in vivo. "CIT�toxic amount of" anti-CD79b antibodies used for the inhibition of tumor cell growth may be determined empirically and in a routine way.

"CD79b-expressing cell" is a cell that expresses endogenous or transfetsirovannyh CD79b polypeptide either on the cell surface or in a secreted form. "A cancerous tumor that expresses CD79b," is a cancerous tumor that contains cells, which have on their surface a CD79b polypeptide or produce and secrete a CD79b polypeptide. "Cancer expressing CD79b" produces, but not necessarily, sufficient levels of CD79b polypeptide on the cell surface, which allows anti-CD79b antibody to contact the polypeptide to exert its therapeutic effect on cancer. In another embodiment, the invention is a cancer expressing CD79b" produces and secretes, but not necessarily, sufficient levels of CD79b polypeptide that allows an anti-CD79b antibody-antagonist of contact with the polypeptide to exert its therapeutic effect on cancer. In the latter case, the specified antagonist may be antimicrobal the oligonucleotide which reduces, inhibits or prevents production and secretion of secreted CD79b polypeptide by tumor cells. A cancer which "sverkhekspressiya" poly�eptid CD79b, is the tumor with significantly higher levels of CD79b polypeptide on the cell surface, or produces and secretes these higher levels compared to non-cancer cells of the tissues of the same type. Such overexpression may be caused by gene amplification or by increased levels of transcription or translation. Overexpression of CD79b polypeptide can be determined by detecting or predictive Analytics by evaluating increased levels of the protein CD79b, which is present on the cell surface, or secreted by the cell (for example, using immunohistochemical analysis using anti-CD79b antibodies produced against selected CD79b polypeptide, which may be produced by methods of recombinant DNA from isolated nucleic acid that encodes a CD79b polypeptide; FACS analysis, etc.). Alternative or additionally, the levels of nucleic acid or mRNA that encodes a CD79b polypeptide, in cells can be measured by means of fluorescence in situ hybridization using nukleinovokisly probe corresponding to CD79b-encoding nucleic acid or its complement (FISH; see application WO98/45479 published October, 1998), using southern blot analysis, Northern blot analysis, or methods based on the polymerase chain reaction (PCR), such as quantitative PCR in the district�real-time (RT-PCR). Can also be analyzed at sverkhekspressiya CD79b polypeptide by measuring the level of "peeling" of the antigen in a biological fluid such as serum, e.g., using assays based on antibodies (see also U.S. patent No. 4933294, issued June 12, 1990; the application WO91/05264 published April 18, 1991; U.S. patent No. 5401638, issued March 28, 1995; and Sias et al.,J. Immunol. Methods132:73-80 (1990)). In addition to the above analyses, the experts in this field can be carried out various tests in vivo. For example, cells in the patient's body can be subjected to contacting with the antibody, which is labeled, but not necessarily, apparently detected by a label, e.g. a radioactive isotope, and binding of an antibody to a cell of a given patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing biopsy taken from a patient, which was introduced this antibody.

As used herein, the term "immunoadhesin" means an antibody-like molecules which have binding specificity of a heterologous protein (an"adhesin") in combination with the effector functions of the constant domain of immunoglobulin. In its structure, immunoadhesin contain a hybrid amino acid sequence with the desired binding specificity, but which are not antigen-recognizing sequence�activities and antigen-binding site of an antibody (i.e. is "heterologous"), and the sequence of the constant domain of immunoglobulin. Adhesiva part of a molecule immunoadhesin typically is a contiguous amino acid sequence containing at least the binding site of the receptor or ligand. The sequence of the constant domain of immunoglobulin in immunoadhesin can occur from any immunoglobulin, such as an immunoglobulin subtypes of IgG-1, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

As used herein, the term "label" means detected compound or composition that directly or indirectly anywhereman with the antibody, so that they form a "labeled" antibody. This mark, in itself, can be apparently detected (for example, label-radioisotopes or fluorescent labels) or, in the case of an enzymatic label, it can catalyze the chemical modification of the connection substrate or composition which is apparently detected.

As used herein, the term “cytotoxic agent” means a substance that inhibits or prevents the functioning of cells and/or causes destruction of cells. The term includes radioactive isotopes (e.g.,211At,131I,125I,90Y186Re,188Re,153Sm212Bi32P and radioactive isotopes of Lu), chemotherapeutic agents, nab�emer, methotrexate, adriamycin, vinylchloride (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleotidase enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, including fragments and/or variants, and the various antitumor or anticancer agents are described below. Other cytotoxic agents are described below. Ofwholesale means, causes destruction of tumor cells.

"Toxin" is any substance capable of exerting an inhibitory effect on the growth or proliferation of the cell.

"Chemotherapeutic agent" is a chemical compound that, regardless of mechanism of action, can be used to treat cancer. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, alkaloids spindle poisonous plants, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photodynamic agents and kinase inhibitors. Chemotherapeutic agents are compounds used in "therapy directed action" and in standard�a combined chemotherapy. Examples of chemotherapeutic agents include erlotinib (TARCEVA®; Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (CIS-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N. J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentatonica-[4.3.0] Nona-2,7,9-trien-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-differbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, rapamycin.

Other examples of chemotherapeutic agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), of imatinib mesilate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (not containing cremophor), designed on the basis of albumin composition n�of nanoparticles of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478 effect, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), infospeed (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); acesulphame such as busulfan, improsulfan and piposulfan; aziridine, such as bestop, carboquone, maturetube and uredia; ethylenimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylaniline; acetogenin (in particular, bullatacin and bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen analogues of mustard gas, such as chlorambucil, chlornaphazine, chloroformed, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy analogue of mustard gas; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, and in particular, Kalyani�in gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., (1994) 33:183-186); dynemicin, including dynemicin a; bisphosphonates, such as clodronate; spiramycin and neocarzinostatin chromophore and related chromoprotein enediyne the antibiotic chromophores), aclacinomycin, actinomycin, astromicin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, morpholinopropan, cyanomethaemoglobin, 2-pyrroline-doxorubicin and desoxidation), epirubicin, zorubicin, idarubicin, marsellaise, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, clamycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, timipre and thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane and testolactone; antiadrenergic agents such �AK aminoglutethimide, mitten and trilostane; a means of compensating the lack of folic acid, such as prolinnova acid; aceglatone; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diaziquone; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; PSK polysaccharide complex® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenotomy acid; triaziquone; 2,2',2”-trihlortrietilamin; trichothecenes (in particular, toxin T-2, verrucarin And, roridin and unguided); urethane; vindesine; dacarbazine; minomycin; Metaponto; mitolactol; pipobroman; Galitsin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine, etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin; to produce remissions in childhood; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; and farm�citiesi acceptable salt, acid and derivatives of all the above compounds.

In the definition of "chemotherapeutic agent" also includes: (i) anti-hormonal measures to regulate or inhibit hormone action on tumors such as antiestrogens and selective modulators of estrogen receptor (SERM), including, for example, tamoxifen (including NOLVADEX® tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and FARESTON® (toremifene citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase and regulate the production of estrogen in the adrenal cortex, such as, for example, 4(5)-imidazolov, aminoglutetimid, MEGASE® (megestrol acetate), AROMASIN® (exemestane, Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole, AstraZeneca); (iii) antiandrogens such as flutamide, nilutamid, bikalutamid, leuprolide and goserelin; as well as troxacitabine (nucleoside casinoby analogue of 1,3-dioxolane); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) inhibitors of the lipid kinase; (vi) antisense oligonucleotides, particularly oligonucleotides that inhibit gene expression pathways of signal transmission involved in the proliferation of undesirable cells, such as, for example, PKC-alpha, Ralf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as inhibition tion�ora of expression of VEGF (e.g., ANGIOZYME®) and inhibitors of HER2 expression; (viii) vaccines such as vaccines for gene therapy, for example, the vaccine ALLOVECTIN® vaccine, LEUVECTIN® vaccine VAXID®; rIL-2 PROLEUKIN®; topoisomerase inhibitors 1, such as LURTOTECAN®; rmRH ABARELIX®; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech) and pharmaceutically acceptable salts, acids and derivatives of all these compounds.

In the definition of "chemotherapeutic agent" also includes therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia) and conjugate "antibody-drug", gemtuzumab, ozogamicin (MYLOTARG®, Wyeth).

Used here, the term “growth-inhibitory agent” means a compound or composition, inhibiting the growth of cells, and in particular, cancer cells expressing CD79b, either in vitro or in vivo. Thus, by means of inhibiting the growth of cells, may be a means of significantly reducing the percentage of cells expressing CD79b, in phase S. Examples of funds that inhibit the growth of cells, are means of blocking the passage of the cell cycle (in the other phase, in addition to S phase), such as funds, inducing blocking the G1 phase, and phase M Classic means of a blocking phase M, are vinylchloride (vincristine and vinblastine), taxanes and topoisomerase inhibitors II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Means that blocks the G1 phase, as well as blocking the transition to the S phase are, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in the publicationThe Molecular Basis of Cancer, Mendelsohn & Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs”, Murakami et al. (WB Saunders: Philadelphia, 1995), and in particular, on page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel induce microtubule Assembly from tubulinea dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of mitosis.

“Doxorubicin” is an anthracycline antibiotic. Doxorubicin has the full chemical name (8S-CIS)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetic)-1-methoxy-5,12-naphthacenedione.

The term “cytokine�� is a General term related to proteins, which are released by one cell population and which act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and standard of polypeptide hormones. In addition to the above definitions, the term “cytokine” includes growth hormones such as human growth hormone, N-nationally human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH); a growth factor for hepatocytes, fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and-β; Mueller inhibitory factor; peptide, associated with murine gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors tissue such as NGF-β; platelet-growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); factors inducing osteogenesis; interferons, such as interferon-α, -β and-γ; colony stimulating factors (CSF), such as macrophage CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte CSF(G-CSF); interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). Used herein, the term cytokine includes proteins derived from natural sources or from recombinant cell culture and biologically active equivalents of cytokines with a native sequence.

As used herein, the term “liner in the package” means the instructions, which are usually invested in available-for-sale packaging of therapeutic products and in which the information concerning the indications, method of use, dosage, method of administration, contraindications and/or warnings concerning the use of such therapeutic products.

The term "intracellular metabolite" means a compound resulting from a metabolic process or metabolic reactions conjugate "antibody-drug" (ADC) inside the cell. Such a metabolic process or reaction may be an enzymatic reaction, such as proteolytic cleavage of the peptide linker of the ADC, or the hydrolysis of functional groups, such as hydrazon, ester or amide. Intracellular metabolites include, but are not limited to, antibodies and free drug, which was subjected�are intracellular cleavage after entering the cell, diffusion or absorption in the cell, or transport into the cell.

The terms "biologically degradable within cells" and "intracellular cleavage" refer to a metabolic process or reaction conjugate "antibody-drug" (ADC) inside the cell, where under the influence of such processes or reactions cleavage of the covalent bond, that is, the linker between the molecule of the drug (D) and the antibody (Ab), which leads to the formation of free medicines, dissociating from the antibody inside the cells. Thus, contain no cleavable groups of the ADC are intracellular metabolites.

The term “bioavailability” means the systemic bioavailability (i.e. the levels of the drug in blood/plasma) for a given amount of the drug, administered to the patient. “Bioavailability” is an absolute term that defines parameters such as the duration (speed) and the total quantity (level) of the drug released from the dosage form and enters the General circulation.

The term "cytotoxic activity" refers cytotoxic, cytotoxic or growth-inhibitory effect of the ADC, or an intracellular metabolite of the ADC. The cytotoxic activity may be expressed as the value of IC50t�dstanley a concentration (molar or mass per unit volume), which survives 50% of the cells.

As used herein, the term "alkyl" means a saturated straight or branched monovalent hydrocarbon radical comprising 1 to 12 carbon atoms (C1-C12), where the specified alkyl radical can be, but is optionally independently substituted by one or more substituents described below. In another embodiment of the invention the alkyl radical has 1 to 8 carbon atoms (C1-C8) or 1-6 carbon atoms (C1-C6). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, CH2(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, (CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2(CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH 3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C((CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH((CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2(CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)With(CH3)3), 1-heptyl, 1-octyl, etc.

The term "alkenyl" means a straight or branched monovalent hydrocarbon radical of 2 to 8 carbon atoms (C2-C8) at least one unsaturated bond, i.e. a carbon-carbon bond and sp2-double bonds, where the alkenyl radical may be, but is optionally independently substituted by one or more substituents described in this application, and includes radicals having "CIS" and "TRANS"orientations, or alternatively, "E" and "Z"orientation. Examples include, but are not limited to, ethylenic or vinyl (-CH=CH2), allyl (-CH2CH=CH2) and etc.

The term "alkynyl" means a straight or branched�th monovalent hydrocarbon radical, consisting of 2-8 carbon atoms (C2-C8) at least one unsaturated bond, i.e. a carbon-carbon bond and sp-a triple bond, where alkynylaryl radical can be, but is optionally independently substituted by one or more substituents described in this application. Examples include, but are not limited to, ethinyl (-C≡CH), PROPYNYL (propargyl, -CH2C≡CH), etc.

The terms "carbocycle", "carbocyclic", "carbocyclic ring" and "cycloalkyl" mean a monovalent non-aromatic saturated or partially unsaturated ring having 3-12 carbon atoms (C3-C12) as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can be arranged, for example, in the form of a bicyclo-[4,5]-, -[5,5]-, -[5,6]- or[6,6] system, and bicyclic carbocycles having 9 or 10 carbon atoms on the ring may be positioned, for example, in the form of bicyclo-[5,6]- or [6,6] system, or as bridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples of monocyclic carbocycles are, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexane�Neal, cycloheptyl, cyclooctyl, cycloneii, cyclodecyl, cyclodecyl, cyclododecyl, etc.

The term "aryl" means a monovalent aromatic hydrocarbon radical comprising 6 to 20 atoms of carbon (C6-C20and resulting from removal of one hydrogen atom from a single carbon atom of the original aromatic system. Some of aryl groups in representative structures designated as “Ar”. Typical Allami are bicyclic radicals containing an aromatic ring condensed with a saturated, partially unsaturated or aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, radicals derived from benzene (phenyl), substituted benzene, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthalene etc Aryl group is independently substituted, but not necessarily, by one or more substituents described in this application.

The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably and refer to a saturated or partially unsaturated (i.e., having one or two double and/or triple bond in the ring) carbocyclic radical having from 3 to 20 atoms in the ring, where at least one atom on the ring is a heteroatom, chosen�tion of nitrogen atoms, oxygen, phosphorus and sulfur and the other atoms on the ring atoms are C, where one or more atoms on the ring of nezavisna replaced, but not necessarily, by one or more substituents described below. Heterocycle may be a monocycle having 3 to 7 members in the ring (2-6 carbon atoms and 1-4 of heteroatom selected from N, O, P and S), or Bicycle, having from 7 to 10 members in the ring (4-9 carbon atoms and 1-6 heteroatoms selected from N, O, P and S), for example, bicyclo-[4,5]-, -[5,5]-, -[5,6]- or[6,6] system. The heterocycles described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly chapters 1, 3, 4, 6, 7 and 9; in the publication “The Chemistry of Heterocyclic Compounds, A series of Monographs” (submitted by John Wiley & Sons, New York, 1950), and in particular volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The term "heterocyclyl" also includes radicals, namely heterocyclic radicals are fused with a saturated, partially unsaturated or aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuran, tetrahydrothieno, tetrahydropyranyl, dihydropyran, tetrahydrothiopyran, piperidine, morpholino, thiomorpholine, dioxane, piperazinyl, homopiperazine, azetidine, oxetane, titanyl, homopiperazine, oxiranyl, tap�wounded, oxazepines, diazepines, thiazepines, 2-pyrrolidyl, 3-pyrrolidyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-DIOXOLANYL, pyrazolyl, dithienyl, dithiolane, dihydropyran, dihydrothieno, dihydrofuran, pyrazolopyrimidines, imidazolidinyl ureido, 3-azabicyclo[4.1.0]hexenyl, 3-azabicyclo[4.1.0]heptenyl, azabicyclo[2.2.2]hexanal, 3H-indole, finalizing and N-pyridylacetic. In the scope of this definition also includes spironolactone. Examples of heterocyclic groups containing 2 carbon atoms in the ring substituted with oxo(=O)-groups, are of pyrimidinones and 1,1-diokso-thiomorpholine. Described here, the heterocyclic group is independently substituted, but not necessarily, by one or more substituents described in this application.

The term "heteroaryl" means a monovalent aromatic radical consisting of 5-, 6 - or 7-membered rings, and includes a condensed cyclic system (at least one of which is aromatic), consisting of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen atoms, oxygen and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridine), imidazolyl, imidazopyridines, pyrimidinyl (including, for example, 4-hydroxypyrimidine), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, itxaso�Il, thiazolyl, oxazolyl, isothiazolin, pyrrolyl, chinoline, ethenolysis, indole, benzimidazole, benzofuranyl, indolinyl, indazoles, indolizinyl, phthalazine, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinol, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furutani, benzofurazanyl, benzothiophenes, benzothiazolyl, benzoxazolyl, chinazoline, chinoxalin, naphthyridines and Phenoperidine. Heteroaryl groups are independently substituted, but not necessarily, by one or more substituents described in this application.

Heterocyclic or heteroaryl groups can be linked, where possible, through the carbon atom linked to a carbon atom or through a nitrogen atom linked to the nitrogen atom). Non-limiting examples linked through carbon heterocycles or heteroaryl are heterocycles or heteroaryl linked in position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5 or 6 pyridazine, in position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, in position 2, 4 or 5 oxazole, imidazole or thiazole, position 3, 4 or 5 isoxazol, pyrazole or isothiazole, in position 2 or 3 of aziridine, in position 2, 3 or 4 azetidine, in position 2, 3, 4, 5, 6, 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 Sochi�oline.

Non-limiting examples linked through nitrogen heterocycles or heteroaryl are heterocycles or heteroaryl connected in position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole; in position 2 of isoindole or isoindoline, in position 4 of a morpholine, and position 9 of a carbazole, or β-carboline.

“Alkylene” represents a saturated, branched, single-chain, or cyclic hydrocarbon radical comprising from 1 to 18 carbon atoms, and having two monovalent radical center formed by removal of two hydrogen atoms to two same or different carbon atoms of the original alkane. Typical alkilirovanny radicals include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-) and so on.

"C1-C10alkylene" represents a straight saturated hydrocarbon group of the formula -(CH2)1-10-. Examples C1-C10alkylene are methylene, ethylene, propylene, butylene, pentile, exile, reptile, octile, Nonlin and decalin.

“Albaniles” performance�possessing an unsaturated, an extensive, single-chain, or cyclic hydrocarbon radical comprising 2-18 carbon atoms and having two monovalent radical center formed by removal of two hydrogen atoms to two same or different carbon atoms of the original alkene. Typical alkenylamine radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

“Akinyan” is an unsaturated, branched, single-chain, or cyclic hydrocarbon radical comprising 2-18 carbon atoms and having two monovalent radical center formed by removal of two hydrogen atoms to two same or different carbon atoms of the original alkyne. Typical alkenylamine radicals include, but are not limited to, acetylene (-C≡C-), propargyl (-CH2With≡C-) and 4-pentenyl (-CH2CH2CH2WITH≡CH-).

"Allen" represents an aryl group which has two covalent bonds and can be present in ortho-, meta - or para-configurations, as shown in the following structures:

where the phenyl group can be unsubstituted, or it can be substituted with 1-4 groups, including, but not limited to, -C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R') -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Arylalkyl" is an acyclic alkyl radical in which one of the hydrogen atoms associated with carbon atom, typically, the terminal carbon atom or sp3-carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-Penilaian-1-yl, 2-Penilaian-1-yl, naphthylmethyl, 2-Nettleton-1-yl, 2-naphthalate-1-yl, naphthalenyl, 2-naphthenate-1-yl, etc. Arylalkyl group contains from 6 to 20 carbon atoms; for example, the alkyl part, including albanello, alkenyl or alkylamino part arylalkyl group has 1-6 carbon atoms and the aryl part has from 5 to 14 carbon atoms.

"Heteroaromatic" is an acyclic alkyl radical in which one of the hydrogen atoms associated with carbon atom, typically, the terminal carbon atom or sp3-a carbon atom replaced with a heteroaryl radical. Typical heteroarylboronic groups include, but are not limited to, 2-benzimidazolylthio, 2-purolater, etc. Heteroallyl group has from 6 to 20 carbon atoms; for example, the alkyl part, including albanello, alkenyl or alkylamino part of heteroaryl�your group has 1-6 carbon atoms, and the heteroaryl portion has 5 to 14 carbon atoms and 1 to 3 heteroatom selected from N, O, P and S. the Heteroaryl portion heteroallyl group may be a monocycle having 3 to 7 members in the ring (2-6 carbon atoms), or Bicycle, having from 7 to 10 members in the ring (4-9 carbon atoms and 1-3 heteroatom selected from N, O, P and S), for example, bicyclo-[4,5]-, -[5,5]-, -[5,6]- or[6,6] system.

Used in this application, the term "prodrug" means a precursor or derivative compounds according to the invention that, compared with the parent compound or drug, are less cytotoxic against tumor cells and is capable of enzymatically or hydrolytically activated or to become more active Mature form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al. “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). Prodrugs according to the invention are, but are not limited to, phosphate-containing prodrugs; thiophosphate-containing prodrug; sulfate-containing prodrugs; peptide-containing prodrugs; a prodrug, a modified D-amino acid; a glycosylated prodrugs; β-lactam-containing prodrugs; prodrug containing neo�Astelin substituted phenoxyacetamide; or prodrug containing optionally substituted phenylacetamide; 5-fortitudine and other 5-ptoluidine prodrug that can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatization with obtaining a Pro-drug forms for use in the present invention include, but are not limited to, compounds according to the invention and chemotherapeutic agents described above.

The term "metabolite" means a product produced as a result of the metabolism of a specific compound or its salts in the body. Metabolites of compounds can be identified by routine methods known in the art, and their activity can be determined using the assays described in the present application. Such products can be formed, for example, oxidation, recovery, hydrolysis, amidation, desametasone, esterification, deesterification, enzymatic degradation of the compound introduced, etc. In accordance with this, the present invention encompasses metabolites of the compounds according to the invention, including compounds produced by a method comprising contacting the compounds according to the invention with the body of a mammal during the period in�Emini, sufficient to produce a product of metabolism.

"Liposome" is a small vesicles composed of different types of lipids, phospholipids and/or surfactant that can be used to deliver drugs to the mammal. Components of liposomes are usually located so that they form a bilayer, similar to the lipid bilayer in biological membranes.

The term "linker" means a chemical group that contains a covalent bond or a chain of atoms that covalently bind the antibody to the molecule of the drug. In various embodiments of the invention the linker is a divalent radical, such as alkerdeel, areldil, heteroaryl, groups such as -(CR2)nO(CR2)n-, the repeating unit of alkyloxy (e.g., polietilene, PEG, polymethylenes) and alkylamino (e.g., polyethylenimine, JeffamineTM), and dibasic ester and amides including succinate, succinamide, diglycolate, malonate and caproamide.

The term “chiral” refers to molecules which are not aligned with their mirrored counterpart, while the term “achiral” refers to molecules that line up with their mirrored counterpart.

The term “stereoisomer” means compounds that have identical chemical structure�, but differ in the spatial arrangement of atoms or groups.

The term “diastereoisomer” refers to a stereoisomer with two or more chiral centers, whose molecules are not mirror images of each other. The diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. Mixtures of diastereoisomers can be separated by high-resolution analytical techniques such as electrophoresis and chromatography.

The term “enantiomers” refers to two stereoisomers of the compounds, mirroring which are not aligned with each other.

Denitions and notation used here stereochemical terms in General are given in S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they are able to rotate the plane pleapositive light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its (their) chiral(s) centre(s). The prefixes d and l or (+) and (-) are used to designate the sign of rotation pleapositive light data connection and the (-) or l OZNA�AET, that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A mixture of 50:50 of the enantiomers is called a racemic mixture or a racemate, which may occur in the event that if in a chemical reaction or chemical process, there is no stereoselectivity or stereospecificity. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric molecules that do not possess optical activity.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which can be transformed into each other over a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) are tautomers, which are transformed into each other under the action of the migration of a proton, such as keto-enol and Yiming-Eminova isomerization. Valence tautomers is determined by the mutual transformations as a result of restructuring some of the bound electrons.

As used herein, the term “�farmacevtichesky acceptable salt” means pharmaceutically acceptable organic or inorganic salts of the compounds according to the invention. Examples of such salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannat, Pantothenate, bitartrate, ascorbate, succinate, maleate, getitemat, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesilate", aconsultant, benzolsulfonat, p-toluensulfonate and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-aftout)). The term “pharmaceutically acceptable salt” can include other molecules, such as acetate ion, succinate ion or other counterion. The counterion may be any organic or inorganic molecule that stabilizes the charge on the original connection. In addition, the pharmaceutically acceptable salt may have more than one charged atom. In the case that a lot of charged atoms is part of a pharmaceutically acceptable salt, such a salt can have multiple counterions. Consequently, the pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

If the connection according to the invention is a base, the desired pharmaceutically acceptable salt may be obtained by any suitable method known�th professionals for example, by treatment of the free base with an inorganic acid such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, etc., or organic acid such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyrenoidosa acid, such as glucuronic acid or galacturonic acid; alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid such as p-toluensulfonate acid or econsultancy acid, or etc.

If the connection according to the invention is acid, the desired pharmaceutically acceptable salt may be obtained by any suitable method, for example, by treatment of the free acid inorganic or organic base such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like, Representative examples of suitable salts are the main advantages of�are, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients constituting the composition, and/or with the body of a mammal, which administered the composition.

The term "solvate" means an Association or complex of one or more solvent molecules and compounds according to the invention. Examples of solvents that form solvates are, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" means a complex in which the solvent molecule is water.

The term "protective group" means a Deputy, who is usually used to block or protect certain functionaliy group reacting with another functional group on the compound. For example, "aminosidine group" is a substituent attached to the amino group, which is a block�ruet or protects the functional amino group in this compound. Suitable aminosidine groups are acetyl, triptorelin, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethoxycarbonyl (Fmoc). Similarly, "hydroxy-protective group" means the Deputy hydroxy-group that blocks or protects the functional hydroxy-group. Suitable protective groups are acetyl and silyl. "Carboxy-protective group" means the Deputy carboxypropyl that blocks or protects the functional carboxypropyl. Standard carboxy-protective groups are vinylsulfonate, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluensulfonyl)ethyl, 2-(p-nitrobenzylidene)ethyl, 2-(diphenylphosphino)ethyl nitroethyl, etc. a General description of the protective groups and their use can be found in the publication by T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

The term “leaving group” means a functional group which can be substituted by another functional group. Some leaving groups are well known in the art, and examples of such groups include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluensulfonyl (tosyl), triftormetilfullerenov (triflic) and triftormetilfullerenov.

Designations

Linker components:

MC=6-maleimido�Royle

Val-Cit or “vc”=valine-citrulline (representative of the dipeptide in the linker, contain no cleavable by a protease)

Citrulline=2-amino-5-freidapinto acid

RAV=p-aminobenzeneboronic (example "camouglage" linker component)

Me-Val-Cit=N-methyl-valine-citrulline (where the peptide bond of the linker has been modified to prevent its cleavage by cathepsin B)

MC(PEG)6-OH=maleimidomethyl-polyethylene glycol (can be attached to antibody cysteines).

Cytotoxic drug:

MMAE=monomethylaniline E (mol.m. (MW) 718)

MMAF=variant of auristatin E (MMAE) with a phenylalanine at the C-end of the drug (MW 731,5)

MMAF-DMAEA=MMAF with DMAEA (diethylaminoethylamine) associated amide bond with the C-terminal phenylalanine (MW 801,5)

MMAF-TEG=MMAF with tetraethylene glycol associated with the phenylalanine ester bond

MMAF-NtBu=N-t-butyl associated with C-end MMAF amide bond

DM1=N(2')-deacetyl-N(2')-(3-merkapto-1-oxopropyl)-maytansine

DM3=N(2')-deacetyl-N2-(4-merkapto-1-oxobutyl)-maytansine

DM4=N(2')-deacetyl-N2-(4-merkapto-4-methyl-1-oxobutyl)-maytansine

Other abbreviations used here have the following meanings: AE means auristatin E, Vos means N-(tert-butoxycarbonyl), cit means citrulline, dap means dalapon, DCC means 1,3-dicyclohexylcarbodiimide, DCM (was held) means dichloro methane, DEA oz�ACHAT diethylamine, DEAD means diethylazodicarboxylate, DEPC means diethylphosphoramidite, DIAD means diisopropylethylamine, DIEA means N,N-diisopropylethylamine, dil means daisosasen, DMA means dimethylacetamide, DMAP means 4-dimethylaminopyridine, DME means dimethyl ether of ethylene glycol (or 1,2-dimethoxyethane), DMF (DMF means N,N-dimethylformamide, DMSO (DMSO means dimethylsulfoxide, doe means draftin, dov means N,N-dimethylamine, DTNB means 5,5'-dithiobis(2-nitrobenzoic acid), DTPA means diethylenetriaminepentaacetic acid, DTT means of dithiothreitol, EDCl means of the hydrochloride of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EEDQ means 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS means mass spectrometry by elektrorazpredelenie, EtOAc means ethyl acetate, Fmoc means N-(9-fluorenylmethoxycarbonyl), means gly glycine, HATU means hexaflurophosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea, HOBt means 1-hydroxy-benzotriazole, HPLC (HPLC means high performance liquid chromatography, ile means isoleucine, lys is lysine means, MeCN (CH3CN) means acetonitrile, MeOH means methanol, Mtr means 4-insidepermitted (or 4-methoxytrityl), nor (nor) means (1S,2R)-(+)-norephedrine, PBS means phosphate-buffered saline (pH 7.4), PEG (PEG means polyethylene glycol, Ph means phenyl, Cronache p-nitrophenyl, MS means 6-maleimidomethyl, phe means L-phenylamine, PyBrop refers to hexaphosphate bromo-Tris-pyrrolidinone, SEC means size exclusion chromatography, Su means succinimide, TFA means trifluoroacetic acid, TLC (TLC means thin layer chromatography, UV (UV means ultraviolet radiation, and val means valine.

"Free amino acid cysteine" means a cysteine amino acid residue, which was introduced in the parent antibody, has a thiol functional group (-SH) and does not form a pair in the form intramolecular or intermolecular disulfide bridge.

The term “quantity of thiol reactivity” means a quantitative characterization of the reactivity of free cysteine amino acid residues. The amount of thiol reactivity represents the percentage of the free cysteine amino acid residues in designed on the basis of the cysteine antibody that reacts with a reagent that interacts with the thiol, with a maximum magnitude of this reactivity is taken to equal 1. For example, a free cysteine amino acid in constructed on the basis of the cysteine antibody that reacts with interacting with the thiol reagent, such as Biotin-maleimide reagent, with 100% yield with the formation of Biotin-labeled antibodies, and�States the amount of thiol reactivity, component of 1.0. Another cysteine amino acid, introduced into the same or different parent antibody that reacts with interacting with the thiol reagent, with 80% yield, is the amount of thiol reactivity, a component of 0.8. Another cysteine amino acid, introduced into the same or different parent antibody which does not react with interacting with the thiol reagent, has a value thiol reactivity equal to 0. Determination of the thiol reactivity of specific cysteine can be carried out using ELISA analysis, mass spectroscopy, liquid chromatography, autoradiography or other quantitative analytical tests.

“Parent antibody” is an antibody containing the amino acid sequence in which one or more amino acid residues are substituted by one or more cysteine residues. The parent antibody may contain the native sequence or wild-type sequence. Compared to other native antibodies, antibody wild-type or modified forms of antibodies, the parent antibody may have existing modifications of the amino acid sequence (such as additions, deletions and/or substitutions). The parent antibody may be directed against pre�interest component of the antigen target for example, a polypeptide having an important biological properties. Also discusses antibodies against polipeptidnyh antigens (such as tumor-associated glycolipid antigens; see, U.S. patent No. 5091178).

III. Compositions and methods according to the invention

The present invention relates to anti-CD79b antibodies or their functional fragments, as well as to method of their use for the treatment of hematopoietic tumors.

In one of its aspects the present invention relates to an antibody which binds, preferably specifically, to any of the above or below described polypeptides. This antibody is, but not necessarily, a monoclonal antibody, a fragment of the antibody, including Fab, Fab', F(ab')2- and Fv-fragment of dianthicola, single-domain antibody, a chimeric antibody, a humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be but are not necessarily, anywhereman with the growth-inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin, maytansinoid, derived dolastatin or calicheamicin, an antibiotic, a radioactive isotope, nucleotidase enzyme or etc. the Antibody according to the invention can be, but not necessarily, produced in Cho cells or bacterial cells and preferably induce death of the cells to which they bind. Antibodies according to the invention is used for detection, can be-detectable labeled, attached to a solid carrier, or etc.

In one of its aspects the present invention relates to humanitariannet anti-CD79b antibody, where the monovalent affinity antibodies against CD79b (e.g., affinity of the antibody used as the Fab-fragment against CD79b) is substantially the same as monovalent affinity of the murine antibody (e.g. affinity of the murine antibody, is used as the Fab-fragment against CD79b) or a chimeric antibody (e.g. affinity of himem�th antibody, used as a Fab-fragment against CD79b), comprising the sequence of the variable domain of the light and heavy chains, or comprising or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the monovalent affinity antibodies against CD79b (e.g., affinity of the antibody used as the Fab-fragment against CD79b), for example, at least in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60 times lower than the monovalent affinity of the murine antibody (e.g. affinity of the murine antibody, is used as the Fab-fragment against CD79b) or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab-fragment against CD79b), comprising the sequence of the variable domain of the light and heavy chains, or consisting of or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the monovalent affinity antibodies against CD79b (e.g., affinity of the antibody used as the Fab-fragment against CD79b), for example, less�St least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher than the monovalent affinity of the murine antibody (e.g. affinity of the murine antibody, is used as the Fab-fragment against CD79b) or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab-fragment against CD79b), comprising the sequence of the variable domain of the light and heavy chains, or consisting of or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) on figures 8A-B (SEQ ID NO: 14).

In one of its aspects the present invention relates to humanitariannet anti-CD79b antibody, where the affinity of the specified anti-CD79b antibody in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is substantially the same as the affinity of a murine antibody (e.g. affinity of the antibody used as the IgG anti-CD79b) or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab-fragment against CD79b) in its bivalent form, containing the sequence of the variable domain of the light and heavy chains, or consisting of or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates to humanitariannet anti-CD79b EN�Italo, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b), for example, at least in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60 times lower than the affinity of a murine antibody (e.g. affinity of the antibody, used as IgG anti-CD79b) or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab-fragment against CD79b) in its bivalent form, containing the sequence of the variable domain of the light and heavy chains, or consisting of or essentially consisting of the sequence shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b), for example, at least in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher than the affinity of a murine antibody (e.g. affinity of the murine antibody, is used as the IgG anti-CD79b) or a chimeric antibody (e.g. affinity of the chimeric antibody used as an IgG fragment against CD79b) in its bivalent form, containing the sequence of the variable domain of the light and heavy chains, or state�of boiling or essentially consisting of said sequence, shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14).

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.4 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.4±0,04.

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.3 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.32 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.36 nm Il� above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.4 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.44 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.48 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.5 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.3-0.5 nm. In another aspect of the present invention apply� to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.32-0.48 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0,36-0,44 nm.

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.2 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.2±0,02.

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.1 nm or higher. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g.�p, the affinity of the antibody used as the IgG anti-CD79b) is 0.12 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.14 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.16 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is of 0.18 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.2 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the gG against CD79b) is 0.22 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) 0,24 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.26 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.28 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.30 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.1-0.3 nm. In another aspect, the present invention relates�I humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.12 to 0.28 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.14 to 0.26 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0,16-0,24 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0,18-0,22 nm.

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.5 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its divalent form, e.g. aff�tension antibodies, used as IgG anti-CD79b) is 0.5±0,1.

In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.4 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.5 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.6 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.7 nm or above. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0,3-0, nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0.4-0.6 nm. In another aspect, the present invention relates to humanitariannet anti-CD79b antibody, where the affinity antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as the IgG anti-CD79b) is 0,5-0,55 nm.

In one aspect of the invention, the monovalent affinity of the murine antibodies against CD79b is substantially the same as the affinity of binding of the Fab-fragment, containing sequences of variable domains of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another aspect of the invention, the monovalent affinity of the murine antibodies against CD79b is substantially the same as the affinity of binding of the Fab-fragment, containing sequences of variable domains of antibodies produced from hybridomas deposited with the ATCC under No. HB11413 July 20, 1993, or a chimeric antibody containing the variable domains of the antibodies obtained from hybridomas deposited with the ATCC under No. HB11413 July 20, 1993

As is well known to the experts, the affinity of binding of the ligand with the receptor can be determined using any of a variety of tests and is expressed in V�e of the range of quantitative values. Accordingly, in one embodiment of the invention, the binding affinity expressed as Kd values and defines the natural affinity of binding (e.g., with minimized the avidity effects). Generally, and preferably, the binding affinity of the antibody is measured in vitro, regardless of whether it is extracellular or cell-associated environment. As described in detail in the present application, the fold difference in affinity svyazanie can be quantified as the ratio between the monovalent affinity of binding of the humanized antibody (e.g., in Fab form) and the amount of monovalent affinity of binding of the reference/compare antibody (e.g., in Fab form) (e.g., a murine antibody having the sequence of the donor hypervariable region), where the magnitude of the affinity of binding is determined in the same conditions of analysis. Thus, in one embodiment of the invention, the fold difference in binding affinity is defined as the ratio of Kd values humanized antibody in Fab form and a specified benchmark/compare Fab antibodies. For example, in one embodiment of the invention, if an antibody according to the invention (A) has an affinity that is "3 times lower than the affinity of a reference antibody (M), then if the Kd value for A is 3x,�the guise of Kd for M should be 1x, and the ratio of Kd for A to Kd of M" should be 3:1. Conversely, in one embodiment of the invention, if an antibody according to the invention (C) has an affinity that is "3-fold higher than the affinity of a reference antibody (R), then if the Kd value for C is equal to 1x, the value of Kd for R should be 3x, and the ratio of Kd to Kd With K to R" should be 1:3. To determine the affinity of binding can be applied a variety of tests known in the art, including the assays described in the present application, such as, for example, Biacore analysis, radioimmunoassays (RIA) and ELISA.

In one of its aspects the present invention relates to an antibody that binds to CD79b, where the specified antibody contains:

(a) at least one, two, three, four, five or six HVR selected from the group consisting of:

(i) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO: 131)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO: 132)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a QQSNEDPLT (SEQ ID NO: 133)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO: 134)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO: 135), and

(vi) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPVYFDY (SE ID NO: 136).

In one embodiment of the invention HVR-L1 of an antibody according to the invention contains the sequence of SEQ ID NO: 131. In one embodiment of the invention HVR-L2 of an antibody according to the invention contains the sequence of SEQ ID NO: 132. In one embodiment of the invention HVR-L3 of an antibody according to the invention contains the sequence of SEQ ID NO: 133. In one embodiment of the invention HVR-H1 of an antibody according to the invention contains the sequence of SEQ ID NO: 134. In one embodiment of the invention HVR-H2 of an antibody according to the invention contains the sequence of SEQ ID NO: 135. In one embodiment of the invention HVR-H3 of an antibody according to the invention contains the sequence of SEQ ID NO: 136. In one embodiment of the invention, the antibody according to the invention containing these sequences (in combination as described in the present application) is humanized or human.

In one of its aspects the present invention relates to an antibody that binds to CD79b, where the specified antibody contains:

(a) at least one, two, three, four, five or six HVR selected from the group consisting of:

(i) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO: 131)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO: 132)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a combintion of�th QQSNEDPLT (SEQ ID NO: 133)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO: 134)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO: 135), and

(vi) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPVYFDY (SEQ ID NO: 136); and

(b) at least one variant HVR, where the specified variant HVR sequences has a modification of at least one residue of the sequence represented in SEQ ID NO.: 131, 132, 133, 134, 135 or 136. In one embodiment of the invention HVR-L1 of an antibody according to the invention contains the sequence of SEQ ID NO: 131. In one embodiment of the invention HVR-L2 of an antibody according to the invention contains the sequence of SEQ ID NO: 132. In one embodiment of the invention HVR-L3 of an antibody according to the invention contains the sequence of SEQ ID NO: 133. In one embodiment of the invention HVR-H1 of an antibody according to the invention contains the sequence of SEQ ID NO: 134. In one embodiment of the invention HVR-H2 of an antibody according to the invention contains the sequence of SEQ ID NO: 135. In one embodiment of the invention HVR-H3 of an antibody according to the invention contains the sequence of SEQ ID NO: 136. In one embodiment of the invention, the antibody according to the invention containing these sequences (in combination as described in the present application) is humanized or human.

In �bottom of its aspects the present invention relates to an antibody, containing one, two, three, four, five or six HVR, where each HVR contains a sequence or consists of or essentially consists of a sequence selected from the group consisting of SEQ ID NO: 131, 132, 133, 134, 135 and 136, and where SEQ ID NO: 131 corresponds to an HVR-L1, SEQ ID NO: 132 corresponds to an HVR-L2, SEQ ID NO: 133 corresponds to an HVR-L3, SEQ ID NO: 134 corresponds to an HVR-H1, SEQ ID NO: 135 corresponds to an HVR-H2, and SEQ ID NO: 136 corresponds to an HVR-H3. In one embodiment of the invention, the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each, in this order, contains SEQ ID NO: 131, 132, 133, 134, 135 and 136.

Options HVR in the antibody according to the invention can have modifications of one or more residues within the HVR. In one embodiment of the invention a variant HVR-L1 contains one substitution in the following positions: A4 (K), A9 (E or S) and A10 (A or S). In one embodiment of the invention a variant HVR-L2 contains 1-5 (1, 2, 3, 4 or 5) substitutions in any one or combination of the following provisions: B2 (S or G), B3 (R or G), B4 (K, R, Y, I, H or Q), B5 (R), B6 (G, K, A, R, S or L) and B7 (R, N, T or G). In one embodiment of the invention the variant HVR-L3 contains 1-4 (1, 2, 3, or 4) substitutions in any one or combination of the following positions: C1 (N or D), C2 (N or P), C3 (D or R), C5 (S, K, A, Q, D, L or G), C6 (A, E or N), C7 (A), C8 (R) and C9 (N). In one embodiment of the invention the variant HVR-H1 contains 1-7 (1, 2, 3, 4, 5, 6 or 7) substitutions in any one or in combination of CL�blowing provisions: D1 (P), D2 (F), D3 (P, S, Y, G or N), D4 (L or V), D5 (T, R, N, K, C, G or P), D6 (R, T, K or G), D8 (F), D9 (V or L) and D10 (S, Q, N or D). In one embodiment of the invention a variant HVR-H3 comprises 1-3 (1, 2, or 3) substitutions in any one or combination of the following provisions: F4 (R or I), F6 (I or F), F7 (K, C, R, V or F), F8 (L) and F9 (S). Letter(s) in parenthesis following each position indicates(s) that is representative of the replacement amino acid; and, as will be obvious to the person skilled in the art, depending on the context of the present description, the admissibility of the substitution of other amino acids can be evaluated by routine methods known in the art and/or described in this application. In one embodiment of the invention, A9 in a variant HVR-L1 is an E. In one embodiment of the invention F6 in a variant HVR-H3 is a I. In one embodiment of the invention, F7 in a variant HVR-H3 is a R. In one embodiment of the invention F8 in a variant HVR-H3 is an L. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H3, where F6 is a I, F7 is a R and F8 is L. In a one embodiment of the invention, the antibody according to the invention contains a variant HVR-L1, where A9 is an E, and a variant HVR-H3, where F6 is a I, F7 is a R and F8 is L. In a one embodiment of the invention, A9 in a variant HVR-L1 is a S. In about�Mr. from variants of the invention, the antibody according to the invention contains a variant HVR-L1, where A9 is an S, and a variant HVR-H3, where F6 is a I, F7 is a R and F8 is L. a

In one embodiment of the invention, the antibody according to the invention contains a variant HVR-L1, where A4 is a K. In some embodiments of the invention specified variant HVR-L1 contains HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each, in order, contains the sequence of SEQ ID NO: 132, 133, 134, 135 and 136. In some embodiments of the invention, the antibody containing the specified variant HVR-L1, also contains HVR-L1, where A9 is a E or S, and/or A10 represents A or S. In some embodiments of the invention, the antibody containing the specified variant HVR-L1, also contains a variant HVR-L3, where C6 represents a E or N, and/or C7 is an A. In some embodiments of the invention, these antibodies also contain a consensus sequence of frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensual frame sequence contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is a, A, 73 Is T and/or 78 is A. In one embodiment of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In some embodiments of these antibodies consensual consisten�etelnost skeleton human skeleton light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 represents L, and/or position 47 is an F. In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73, 75, 78 and/or 80. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, position 67 is an A, position 69 is an F, position 71 is a, A, position 73 is a T, position 75 is a S, position 78 is an A, and/or position 80 is an M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In one embodiment of these antibodies consensus sequence of frame area of the human skeleton light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (in the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or position 47 is an F.

In one embodiment, izobreteny�, the antibody according to the invention contains a variant HVR-L2, where B3 represents R, B4 is K, B6 represents G, and B7 is a R. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-L2, where B3 represents R, B4 represents Y, B6 represents a K and B7 is a R. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-L2, where B3 represents R, B4 is K, and B6 is a G. In some embodiments of the invention, the antibody containing the specified variant HVR-L2, also contains HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each, in order, contains the sequence represented in SEQ ID NO: 131, 133, 134, 135 and 136. In some embodiments of the invention, the antibody containing the specified variant HVR-L2, also contains a variant HVR-L1, where A9 is an E or S and/or A10 represents A or S. In some embodiments of the invention, the antibody containing the specified variant HVR-L2, also contains a variant HVR-L3, where C6 is an E or an N-and/or C7 is an A. In some embodiments of the invention, these antibodies also contain a consensus sequence of frame area of the human heavy chain subgroup III. In one embodiment of these antibodies, the consensus sequence of frame area contains over�ENU in position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is a, A, 73 is a T and/or 78 is A. In a one embodiment of these antibodies, these antibodies also contain konsensusnomu sequence of frame area of the human light chain Ki. In some embodiments of these antibodies, the consensus sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain K1) 4 is a L and/or 47 is F. a In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73, 75, 78 and/or 80. In one embodiment of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 is an F, 71 represents A, 73 is a T, 75 represents S, 78 represents A and/or 80 is a M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In one embodiment of these antibodies, consent�SNA sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is an F.

In one embodiment of the invention, the antibody according to the invention contains a variant HVR-L3, where C5 is a K. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-L3, where C5 represents S. In some embodiments of the invention, the antibody containing the specified variant HVR-L3, also contains HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, where each, in order, contains the sequence represented in SEQ ID NO: 131, 133, 134, 135 and 136. In some embodiments of the invention, the antibody containing the specified variant HVR-L3, also contains a variant HVR-L1, where A9 is an E or S and/or A10 represents A or S. In some embodiments of the invention, the antibody containing the specified variant HVR-L3, also contains a variant HVR-L3, where C6 is an E or an N-and/or C7 is an A. In some embodiments of the invention, these antibodies also contain a consensus sequence of frame area of the human heavy chain subgroup III. In one embodiment of these antibodies, the consensus sequence of frame area contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is made�is A, 73 is a T and/or 78 is A. In a one embodiment of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In some embodiments of these antibodies, the consensus sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is F. a In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73, 75, 78 and/or 80. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 is an F, 71 represents A, 73 is a T, 75 represents S, 78 represents A and/or 80 is a M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In one embodiment of these antibodies, the consensus sequence of frame area of the human light chain Ki contain�it substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is an F.

In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H1, where D3 is P, D5 is a T, D6 represents R and D10 represents N. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H1, where D3 is P, D5 is represented by N, D6 represents R and D10 represents N. In some embodiments of the invention, the antibody containing the specified variant HVR-H1, also contains HVR-L1, HVR-L2, HVR-L3, HVR-H2 and HVR-H3, where each, in order, contains the sequence represented in SEQ ID NO: 131, 133, 134, 135 and 136. In some embodiments of the invention, the antibody containing the specified variant HVR-H1, also contains a variant HVR-L1, where A9 is an E or S and/or A10 represents A or S. In some embodiments of the invention, the antibody containing the specified variant HVR-H1, also contains a variant HVR-L3, where C6 is an E or an N-and/or C7 is an A. In some embodiments of the invention, these antibodies also contain a consensus sequence of frame area of the human heavy chain subgroup III. In one embodiment of these antibodies, consent�SNA the frame sequence field contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is a, A, 73 is a T and/or 78 is A. In a one embodiment of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In some embodiments of these antibodies, the consensus sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is F. a In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73, 75, 78 and/or 80. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 is an F, 71 represents A, 73 is a T, 75 represents S, 78 represents A and/or 80 is a M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In one embodiment of these antibodies, a consensus�Usna sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is an F.

In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H3, where F6 is a I and F8 is L. In a one embodiment of the invention, the antibody according to the invention contains a variant HVR-H3, where F6 is a I, F7 is a R and F8 is L. a In some embodiments of the invention, the antibody containing the specified variant HVR-H3, also contains HVR-L1, HVR-L2, HVR-L3, HVR-H2 and HVR-H3, where each, in order, contains the sequence represented in SEQ ID NO: 131, 132, 133, 134 and 135. In some embodiments of the invention, the antibody containing the specified variant HVR-H3, also contains a variant HVR-L1, where A9 is an E or S and/or A10 represents A or S. In some embodiments of the invention, the antibody containing the specified variant HVR-H3, also contains a variant HVR-L3, where C6 is an E or an N-and/or C7 is an A. In some embodiments of the invention, these antibodies also contain a consensus sequence of frame area of the human heavy chain subgroup III. In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain�and subgroup III contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain groups (III) 71 represents A, 73 is a T and/or 78 is A. In a one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73 and/or 78. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 is an F, 71 represents A, 73 is a T and/or 78 is A. In a one embodiment of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In some embodiments of these antibodies, the consensus sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is F. a In one embodiment of these antibodies, the consensus sequence of frame area of the human heavy chain subgroup III contains a replacement put in�and 48, 67, 69, 71, 73, 75, 78 and/or 80. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 is an F, 71 represents A, 73 is a T, 75 represents S, 78 represents A and/or 80 is a M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence of frame area of the human light chain Ki. In one embodiment of these antibodies, the consensus sequence of frame area of the human light chain Ki contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (of the consensus sequence of frame area of the human light chain Ki) 4 is a L and/or 47 is an F.

In one of its aspects the present invention relates to an antibody that contains one, two, three, four, five or all of the HVR sequences presented in the figure 9 (SEQ ID NO: 17-21) and/or figure 10 (SEQ ID NO: 22-106).

Used therapeutic agent, when administered to a host, preferably causing a weak immune response or do not cause an immune response from the specified host. In one of its variants, the present invention relates to the specified facility. So, n�example, in one of its variants the present invention relates to humanitarianlaw the antibody that produces and/or presumably produces a humoral response in humans against mouse antibodies (HAMA) at a significantly lower level compared with the antibody containing the sequence of SEQ ID NO: 10 and 14 of the individual host. In another example, the present invention relates to humanitarianlaw the antibody that produces and/or presumably develops the mammal or mammalian, non-human, the response against mouse antibodies (HAMA). In one example of the antibody according to the invention produces a response against murine antibodies to clinically acceptable level or at a lower level.

A humanized antibody according to the invention may contain one or more human and/or human consensus sequences negueruela region (for example, frame area) in the variable domain of heavy and/or light chain. In some embodiments of the invention, in human and/or human consensus sequences negueruela region contains one or more additional modifications. In one embodiment of the invention, the variable domain of the heavy chain of the antibody according to the invention contains human�forge a consensus frame sequence, which, in one embodiment of the invention, the frame represents a consensus sequence of subgroup III. In one embodiment of the invention, the antibody according to the invention contains a variant of the consensus frame of the sequence of subgroup III, modified in one position of amino acid residue. For example, in one embodiment of the invention, a variant of the consensus frame of the sequence of subgroup III may contain substitution at one or more positions selected from the provisions of 71, 73 and/or 78. In one embodiment of the invention, such substitution is R71A, N73T and/or L78A, in any combination thereof. For example, in one embodiment of the invention, a variant skeleton consensus sequences of heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73 and/or 78. In one embodiment of the invention, this replacement is V48I, F67A, I69F, R71A, N73T and/or L78A. For example, in one embodiment of the invention, a variant skeleton consensus sequences of heavy chain subgroup III contains a substitution at position 48, 67, 69, 71, 73, 75, 78 and/or 80. In one embodiment of the invention, this replacement is V48I, F67A, I69F, R71A, N73T, K75S, L78A and/or L80M. In one embodiment of the invention, the variable domain light chain antibodies according to the invention comprises a human consensus frame sequentially�th which, in one embodiment of the invention, the frame represents a consensus sequence Ki. In one embodiment of the invention, the antibody according to the invention contains a variant of the consensus frame of the sequence Ki, the modified at least one amino acid position. For example, in one embodiment of the invention, a variant of the consensus frame of the sequence Ki may contain substitution in position 4. In one embodiment of the invention, the specified substitution is M4L. For example, in one embodiment of the invention, a variant of the consensus frame of the sequence Ki may contains a substitution at position 4 and/or 47. In one embodiment of the invention the specified substitution is M4L and/or L47F.

As we all know, and as described in more detail below, the amino acid position/boundary amino acid that defines the hypervariable region of the antibody may vary depending on the environment of this amino acid and its various characteristics, known in the art (as described below). Some provisions in the variable domain can be considered as a hybrid hypervariable positions, that is, these provisions could, presumably, be in a hypervariable region in accordance with one set of criteria, and may be located outside� hypervariable region in accordance with a different set of criteria. One or more of such provisions may also be present in extended hypervariable regions (as further defined below). The present invention relates to antibodies containing modifications in these hybrid hypervariable positions. In one embodiment of the invention, such hypervariable provisions are one or more provisions 26-30, 33-35B, 47-49, 57 through 65, 93, 94, 101-102 in the variable domain of the heavy chain. In one embodiment of the invention, these hybrid hypervariable provisions are one or more provisions 24-29, 35-36, 46-49, 56 and 97 in the variable domain light chain. In one embodiment of the invention, the antibody according to the invention comprises a variant human consensus frame sequence subgroups of human antibodies, modified in one or more hybrid hypervariable positions.

In one aspect of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising a variant of a consensus of a frame sequence of a human subgroup III, modified in one or several positions 26-30, 33-35, 48-49, 58, 60-63, 93 and 101. In one embodiment of the invention, the antibody contains replacement G26P. In one embodiment of the invention, the antibody contains replacement F27Y. In one embodiment of the invention, the antibody with�keeps replacing T28P, S, Y, G or N. In one embodiment of the invention, the antibody contains replacement F29L or F29V. In one embodiment of the invention, the antibody contains replacement S30T, R, N, K, C, G, or P. In one embodiment of the invention, the antibody contains replacement A33W or A33F. In one embodiment of the invention, the antibody contains replacement M34I, V or L. In one embodiment of the invention, the antibody contains replacement S35E, Q, N or D. In one embodiment of the invention, the antibody contains replacement V48I. In one embodiment of the invention, the antibody contains replacement S49G. In one embodiment of the invention, the antibody contains replacement Y58N. In one embodiment of the invention, the antibody contains replacement A60N. In one embodiment of the invention, the antibody contains replacement D61E. In one embodiment of the invention, the antibody contains replacement S62I. In one embodiment of the invention, the antibody contains replacement V63F. In one embodiment of the invention, the antibody contains replacement A93T. In one embodiment of the invention, the antibody contains replacement D101S.

In one aspect of the invention, the antibody according to the invention contains a variable domain light chain comprising a variant of a consensus of a frame sequence of a human Kappa sub-group I, modified in one or several positions 24, 27-29, 56 and 97. In one embodiment of the invention, the antibody contains replacement R24K. In one embodiment of the invention, the antibody contains Zama�at Q27K. In one embodiment of the invention, the antibody contains replacement S28D or E. In one embodiment of the invention, the antibody contains replacement I29G, A or S. In one embodiment of the invention, the antibody contains replacement S56R, N, T or G. In one embodiment of the invention, the antibody contains replacement T97N.

In one aspect of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising a variant of a consensus of a frame sequence of a human subgroup III, modified in the provisions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or in all positions 26-30, 33-35, 48-49, 58, 60-63, 93 and 101. In one embodiment of the invention, such a modification selected from the group consisting of G26P, F27Y, T28P (S, Y, G or N), F29L (V), S30T (R, N, K, C, G or P), A33W (F), M34I (V or L), S35E (Q, N or D), V48I, S49G, Y58N, A60N, D61E, S62I, V63F, A93T and D101S. In some embodiments of the invention, the antibody according to the invention contains a variant of the consensus frame of the sequence of subgroup III, modified in the provisions 48, 67, 69, 71, 73, 75, 78 and/or 80. In one embodiment of the invention, this replacement is V48I, F67A, I69F, R71A, N73T, K75S, L78A and/or L80M.

In one aspect of the invention, the antibody according to the invention contains a variable domain light chain comprising a variant of a consensus human frame sequence Kappa subgroup I, modified in positions 1, 2, 3, 4, 5 or all Polo�relations 24, 27-29, 56 and 97. In one embodiment of the invention shown a modification selected from the group consisting of R24K, Q27K, S28D (E), I29G (A or S), S56R (N, T, or G) and T97N. In some embodiments of the invention, the antibody according to the invention contains a variant of the consensus frame of the sequence Ki, modified in position 4 and/or 47. In one embodiment of the invention the specified substitution is M4L and/or L47F.

The antibody according to the invention can contain any suitable human or human consensus frame sequence light chain, provided that the antibody will possess the desired biological properties (e.g., desired binding affinity). In one embodiment of the invention, the antibody according to the invention contains at least a portion of a frame sequence of a human light chain Kappa (or all of the specified sequence). In one embodiment of the invention, the antibody according to the invention contains at least a portion of a frame of a consensus sequence of the human Kappa sub-group I (or all of the specified sequence).

In one aspect of the invention, the antibody according to the invention contains a variable domain of heavy and/or light chain comprising a frame sequence represented in SEQ ID NO: 9 (figure 7A-B) and/or 13 (figures 8A-B).

In one of the AU�projects of the invention, the antibody according to the invention is a humanized anti-CD79b antibody, anywhereman with a cytotoxic agent. In one aspect of the invention, a humanized anti-CD79b antibody, anywhereman with a cytotoxic agent inhibits tumor progression in xenograft.

In one aspect of the invention, a humanized antibody and chimeric antibody are monovalent. In one embodiment of the invention, humanized and chimeric antibodies contain a single Fab region linked to an Fc-region. In one embodiment of the invention, the reference chimeric antibody contains a sequence of variable domains are shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14) and associated with human Fc-region. In one embodiment of the invention human Fc-region is a region of IgG (e.g., IgG1, 2, 3 or 4).

In one of its aspects the present invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 15 (SEQ ID NO: 164 to 166). In one embodiment of the invention contains a variable domain sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 15 (SEQ ID NO: 160 to 163). In one aspect of the invention, the antibody contains a sequence of CH1 and/or Fc presented on figure 15 (SEQID NO: 167 and/or 168). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164 to 166), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 15, SEQ ID NO: 160 to 163). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164 to 166), and the sequence of the CH1 and/or Fc presented on figure 5 (SEQ ID NO: 167 and/or 168). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164 to 166), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 15, SEQ ID NO: 160 to 163), and the sequence of the CH1 and/or Fc (figure 15, SEQ ID NO: 167 and/or 168).

In one of its aspects the present invention relates to an antibody containing the variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 15 (SEQ ID NO: 156-158). In one embodiment of the invention contains a variable domain sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 15 (SEQ ID NO: 152-155). In one embodiment of the invention, the specified antibody contains a sequence CL1 presented on figure 15 (SEQ ID NO: 159). In one embodiment invented�I, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 156-158), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 152-155) presented on figure 15. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO:156-158), and CL1 sequence (SEQ ID NO: 159) presented on figure 15. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 156-158), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 152-155) presented on figure 15, and CL1 sequence presented on figure 15 (SEQ ID NO: 159).

In one of its aspects the present invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 16 (SEQ ID NO: 183-185). In one embodiment of the invention contains a variable domain sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 16 (SEQ ID NO: 179-182). In one aspect of the invention, the antibody contains a sequence of CH1 and/or Fc presented on figure 16 (SEQ ID NO: 186 and/or 187). In one embodiment of the invention, the antibody according to the invention �will win variable domain of the heavy chain, contains a sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 183-185), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 16, SEQ ID NO: 179-182). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 182-185), and the sequence of the CH1 and/or Fc presented on figure 5 (SEQ ID NO: 186 and/or 187). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 183-185), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 16, SEQ ID NO: 179-182), and the sequence of the CH1 and/or Fc (figure 16, SEQ ID NO: 186 and/or 187).

In one of its aspects the present invention relates to an antibody containing the variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 16 (SEQ ID NO: 175-177). In one embodiment of the invention contains a variable domain sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 16 (SEQ ID NO: 171-174). In one embodiment of the invention, the specified antibody contains a sequence CL1 presented on figure 16 (SEQ ID NO: 178). In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 175-177), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 171-174) presented on figure 16. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 175-177), and CL1 sequence (SEQ ID NO: 178), represented in figure 16. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 175-177), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 171-174) presented on figure 16, and CL1 sequence presented on figure 16 (SEQ ID NO: 178).

In one of its aspects the present invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 17 (SEQ ID NO: 202-204). In one embodiment of the invention contains a variable domain sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 17 (SEQ ID NO: 198-201). In one aspect of the invention, the antibody contains a sequence of CH1 and/or Fc presented on figure 17 (SEQ ID NO: 205 and/or 206). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 17, SEQ ID NO: 02-204), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 17, SEQ ID NO: 198-201). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 17, SEQ ID NO: 202-204), and the sequence of the CH1 and/or Fc presented on figure 17 (SEQ ID NO: 205 and/or 206). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 17, SEQ ID NO: 202-204), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 17, SEQ ID NO: 198 to 201), and the sequence of the CH1 and/or Fc (figure 17, SEQ ID NO: 205 and/or 206).

In one of its aspects the present invention relates to an antibody containing the variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 17 (SEQ ID nos: 194-196). In one embodiment of the invention contains a variable domain sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 17 (SEQ ID NO: 190-193). In one embodiment of the invention, the specified antibody contains a sequence CL1 presented on figure 17 (SEQ ID NO: 197). In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID nos: 194-196), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR-LC (SEQ ID NO: 190-193), presented on figure 17. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID nos: 194-196), and CL1 sequence (SEQ ID NO: 197), are presented in figure 17. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID nos: 194-196), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 190-193) presented on figure 17, and CL1 sequence presented on figure 17 (SEQ ID NO: 197).

In one of its aspects the present invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 18 (SEQ ID NO: 221-223). In one embodiment of the invention contains a variable domain sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 18 (SEQ ID NO: 217-220). In one aspect of the invention, the antibody contains a sequence of CH1 and/or Fc presented on figure 18 (SEQ ID NO: 224 and/or 225). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 18, SEQ ID NO: 221-223), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 18, SEQ ID NO: 217-220). One in�options of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 18, SEQ ID NO: 221-223), and the sequence of the CH1 and/or Fc presented on figure 18 (SEQ ID NO: 224 and/or 225). In one embodiment of the invention, the antibody according to the invention contains a variable domain of the heavy chain comprising the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 18, SEQ ID NO: 221-223), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 18, SEQ ID NO: 217-220), and the sequence of the CH1 and/or Fc (figure 18, SEQ ID NO: 224 and/or 225).

In one of its aspects the present invention relates to an antibody containing the variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 18 (SEQ ID NO: 213 to 215). In one embodiment of the invention contains a variable domain sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 18 (SEQ ID NO: 209-212). In one embodiment of the invention, the specified antibody contains a sequence CL1 presented on figure 18 (SEQ ID NO: 216). In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213 to 215), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 209-212) presented on figure 18. In one embodiment of the invention, the antibody according to ISO�the acquisition contains a variable domain light chain, comprising a sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213 to 215), and CL1 sequence (SEQ ID NO: 216), presented on figure 18. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence of HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213 to 215), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 209-212) presented on figure 18, and CL1 sequence presented on figure 18 (SEQ ID NO: 216).

In one aspect of the invention, the antibodies according to the invention are designed on the basis of cysteine antibodies in which one or more amino acids of a parent antibody are replaced with a free cysteine amino acid, as described in the application WO 2006/034488 and in the application for U.S. patent 2007/0092940 (which is completely introduced into the present description by reference). Thus can be constructed in any form is anti-CD79b antibodies, i.e., the mutated antibody. For example, the Fab-fragment of the parent antibody may be designed so that it was a designed based on the cysteine Fab, denoted here "uoFab". Similarly, can be constructed of the parent monoclonal antibody, which is Mab". It should be noted that mutation at one site leads to the incorporation of a single cysteine residue in uoFab and �ulacia at the two sites leads to the inclusion of two cysteine residues in Mab, due to the dimeric nature of the antibody IgG. Constructed of cysteine-based anti-CD79b antibodies according to the invention are monoclonal antibodies, humanized or chimeric monoclonal antibodies, and antigen-binding fragments of antibodies, hybrid polypeptides and analogs that predominantly bind to cell-associated CD79b polypeptides. Designed on the basis of cysteine antibody may alternatively include an antibody containing cysteine described in the antibody or Fab, and such a design can be obtained after construction sequence and/or selection of antibodies, without the need for modification of the parent antibody, where such construction is carried out through the creation of antibodies by the method of phage view and selection of such antibodies or by means of constructing frame sequences and constant regions of light and/or heavy chain de novo. Designed on the basis of cysteine antibody contains one or more free cysteine amino acids having the amount of thiol reactivity in the range of 0.6-1.0, 0.7 to 1.0 or 0.8 to 1.0. Free cysteine amino acid is a cysteine residue, which was introduced into the parent antibody and is not part of a disulfide bridge. Designed based on the cysts�ina antibody can be used to attach cytotoxic and/or imaging compounds in the website introduced cysteine through, for example, maleimide or halogenoacetyl. Nucleophilic reactivity of the thiol functional groups of the Cys residue with maleimide group approximately 1000 times higher than the reactivity of any other functional groups of amino acids in the protein, such as amino group of lysine residues or N-terminal amino group. Thiol-specific functional group in iodization and maleimide reagents may react with amino groups, but at higher pH (>9,0), and this reaction takes a longer time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

In one aspect of the invention, constructed on the basis of cysteine anti-CD79b antibody according to the invention contains a cysteine introduced at any one of the following provisions, where provisions in the light chain are numbered in Cabatu (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), and the provisions in the heavy chain numbered according to the European numbering system (including the Fc-region) (see Kabat et al. (1991), supra), where the constant region of light chain, represented and emphasized in the figures 24A, 25A, 26A, 27A, 28, 48A and 49A, starts from the position 109 (numbering according Kabuto), and the constant region of the heavy chain, represented and emphasized in the figures 24B, 25B, 26B, 27B, 28B, 48B and 49B, begins at position 118 (in accordance with the European system the NUMA�ation (EU)). Such situation may also be the position in the sequential numbering of amino acids of a full-size light chain or the heavy chain shown in figures 24-28, 48 and 49. In one embodiment of the invention, the anti-CD79b antibody contains a cysteine introduced in LC-V205C (the number on Cabatu: Val 205; sequence number 209 figure 27A 49A and means Cys entered in this position). Cysteine introduced in the light chain is shown in bold and double underlined below in figures 27A and 49A. In one embodiment of the invention, the anti-CD79b antibody contains a cysteine introduced into the HC-A118C (number EU: Ala 118; number Cabatu 114; ordinal 118 figure 24B, 25B, 26B, 28B, or means 48B Cys introduced at this position). Cysteine introduced in the heavy chain are shown in bold and double underlined below in the figures 24B, 25B, 26B, 28B, or 48B. In one embodiment of the invention, the anti-CD79b antibody contains a cysteine introduced in Fc-S400C (number EU: Ser 400; room for Kabuto 396; sequence number 400 in the figures 24B, 25B, 26B, 28B, or means 48B Cys introduced at this position). In other embodiments of the invention, the cysteine introduced in the heavy chain (including the Fc-region), is present in any one of the following provisions (in accordance with numbering Kabata, and in brackets, in accordance with the EU numbering): 5, 23, 84, 112, 114 (room for EU 118), 116 (room 120 EU), 278 (room 282 EU), 371 (room for EU 375) or 396 (room for EU 400). Thus, modification�the issues of the amino acids in these positions for a parent humanized anti-CD79b antibody according to the invention are: V5C, A23C, A84C, S112C, A114C (the number on A118C EU), T116C (the number on T120C EU), V278C (the number on EU V282C), S371C (the number on EU S375C) or S396C (the number on EU S400C). Thus, modifications of amino acids in these positions for a parent chimeric anti-CD79b antibody according to the invention are: Q5C, K23C, S84C, S112C, A114C (the number on A118C EU), T116C (the number on T120C EU), V278C (the number on EU V282C), S371C (the number on EU S375C) or S396C (the number on EU S400C). Thus, modifications of amino acids in these positions for a parent antibodies against CD79b dog-like apes (anti-cynoCD79b) according to the invention are: Q5C, T23C, S84C, S112C, A114C (the number on A118C EU), T116C (the number on T120C EU), V278C (the number on EU V282C), S371C (the number on EU S375C) or S396C (the number on EU S400C). In other embodiments of the invention the cysteine introduced in the light chain is the cysteine at any of the following provisions: (in accordance with numbering Kabata): 15, 110, 114, 121, 127, 168, 205. Thus, modifications of amino acids in these positions for a parent humanized anti-CD79b antibody according to the invention are: V15C, V110C, S114C, S121C, S127C, S168C, or V205C. Thus, modifications of amino acids in these positions for a parent chimeric anti-CD79b antibody according to the invention are: L15C, V110C, S114C, S121C, S127C, S168C or V205C. Thus, modifications of amino acids in these positions for a parent anti-cynoCD79b antibody according to the invention are: L15C, V110C, S114C, S121C, S127C, S168C or V205C.

Water of its aspects the present invention includes constructed on the basis of cysteine anti-CD79b antibody, containing one or more free cysteine amino acids, where the specified constructed on the basis of cysteine anti-CD79b antibody binds to a CD79b polypeptide, and where the specified antibody is produced by a method comprising substituting one or more amino acid residues of a parent anti-CD79b antibody by cysteine, where the specified parent antibody comprises at least one HVR sequence selected from:

(a) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO: 131) or KASQSVDYEGDSFLN (SEQ ID NO: 137);

(b) HVR-L2 containing a sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO: 132);

(c) HVR-L3, containing the sequence C1-C9, where C1-C9 is a QQSNEDPLT (SEQ ID NO: 133);

(d) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO: 134);

(e) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO: 135), and

(f) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPVYFDY (SEQ ID NO: 136) or TRRVPIRLDY (SEQ ID NO: 138).

In some of its aspects the present invention relates to constructed on the basis of cysteine anti-CD79b antibody containing the amino acid sequence that is at least about 80%, and alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or 100% identical to the amino acid sequence constructed on the basis of cysteine antibody having a full-sized sequence described in the present application, or the amino acid sequence constructed on the basis of cysteine antibody not containing the signal peptide, and is described in the present application.

In yet another aspect, the present invention relates to the selection is developed on the basis of cysteine anti-CD79b antibody containing the amino acid sequence that is encoded by the nucleotide sequence, hybridizes with the complement of the DNA molecule encoding (a) is constructed on the basis of cysteine antibody having a full-sized amino acid sequence described in the present application, (b) amino acid sequence constructed on the basis of cysteine antibody not containing the signal peptide, and is described in the present application, (c) an extracellular domain of a transmembrane protein is constructed on the basis of cysteine antibodies, whether or not containing a signal peptide, as described in the present application, (d) amino acid sequence encoded by any of sequences described herein nucleic acid, or (e) any other specifically defined fragment of a full-sized aminokislot�th sequence is constructed on the basis of cysteine antibodies, described in this application.

In its particular aspects the present invention relates to the selection is developed on the basis of cysteine anti-CD79b antibody that does not contain N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence described in the present application. In the present application also describes methods of obtaining such antibodies, where these methods include culturing host cells containing a vector comprising encoding the corresponding nucleic acid molecule, under conditions suitable for expression of the specified constructed on the basis of cysteine antibodies, and the selection is designed on the basis of cysteine antibodies from cell culture.

In another aspect, the present invention relates to the selection is developed on the basis of cysteine anti-CD79b antibody which is an antibody with deletionism transmembrane domain or with an inactivated transmembrane domain. In the present application also describes methods of obtaining such antibodies, where these methods include culturing host cells containing a vector comprising encoding the corresponding nucleic acid molecule, under conditions suitable for p�Russia specified constructed on the basis of cysteine antibodies, and the selection is designed on the basis of cysteine antibodies from cell culture.

In other of its aspects the present invention relates to isolated chimeric and designed on the basis of cysteine anti-CD79b antibodies containing any of the products described here are designed on the basis of cysteine antibodies associated with a heterologous polypeptide (non CD79b). Examples of such chimeric antibodies contain any of the products described here are designed on the basis of cysteine antibodies associated with a heterologous polypeptide, such as, for example, the sequence epitope tag or an Fc-region of immunoglobulin.

Constructed of cysteine-based anti-CD79b antibody may be a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be but are not necessarily, anywhereman with the growth-inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin, maytansinoid, derived dolastatin or calicheamicin, an antibiotic, a radioactive isotope, nucleotidase enzyme or etc. the Antibody according to the invention can be, but not necessarily, about�ucirvine in CHO cells or bacterial cells, and preferably these antibodies inhibit the growth or proliferation of cells with which they are associated, or induce the death of these cells. Antibodies according to the invention is used for diagnostic purposes, can be-detectable labeled, attached to a solid carrier, or etc.

In other of its aspects the present invention relates to vectors containing DNA encoding any of these anti-CD79b antibodies and anti-CD79b antibodies constructed with cysteine. The present invention also relates to the cell host containing any of the vectors. So, for example, cells of the host cells may be CHO cells, E. coli or yeast cells. The present invention also relates to a method for producing any of the polypeptides described herein, and the method comprises culturing host cells under conditions suitable for expression of the desired polypeptide, and secretion of the desired polypeptide from the cell culture.

Designed on the basis of cysteine antibody can be used for cancer treatment, and such antibodies are antibodies specific to cell surface receptors and transmembrane receptors, and tumor-associated antigens (TAA). Such antibodies can be used as naked antibodies (unconjugated with drug or � molecule-tagged) or conjugates of the antibody-drug" (ADC). Designed on the basis of cysteine antibodies according to the invention can be site-specifically and functionally attached to the reagent that reacts with a thiol. Reacting with the thiol reagent can be a multifunctional linker reagent, the reagent-label for the capture reagent is a fluorophore and an intermediate connection "drug-linker". Designed on the basis of cysteine antibody may be labeled with the label apparently detected, immobilized on a solid phase carrier and/or conjugative molecule drugs. The reactivity of the thiol group can be reported to any antibody that can be done by replacing the amino acids of the reactive cysteine amino acids in the regions of light chains, selected from the following regions of the amino acid sequence: L10-L20, L105-L115, L109-L119, L116-L126, L122-L132, L163-L173, L200-L210; and in areas of heavy chains selected from the following areas of the amino acid sequence: H1-H10, H18-H28, H79-H89, H107-H117, H109-H119, H111-H121, and in the Fc-region at sites selected from H270-H280, H366-H376, H391-401, where the numbering of amino acids starts from the position 1 to the numbering system Kabat (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) and further proceeds as described in WO2006034488; US 2007/0092940. The reactivity of the thiol group can be�ü reported to the domains of antibody, such as a constant domain of the light chain (CL) and constant domains of the heavy chain, CH1, CH2 and CH3. Cysteine substitutions, giving the magnitude of the reactivity of the thiol group of 0.6 and higher may be made in the constant domains of the heavy chain α, δ, ε, γ and μ of intact antibodies: IgA, IgD, IgE, IgG and IgM, respectively, including the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Such antibodies and their use is described in WO2006/034488, US 2007/0092940.

Designed on the basis of cysteine antibodies according to the invention preferably retain the antigen-binding capacity of the parent wild-type counterparts. Thus, constructed on the basis of cysteine antibodies have the ability to bind, preferably specifically with antigens. Such antigens include, for example, opukholeobrazovanie antigens (TAA), proteins of cell surface receptors and other cell surface molecules, transmembrane proteins, signaling proteins, factors regulating cell survival, factors that regulate proliferation of cells, molecules associated with the development or differentiation of tissue (for example, molecules that are known or suspected to participate in such development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in the formation of blood vessels, and molecules, Assoc�iroanya with angiogenesis (e.g., molecules that are known or suspected to participate in angiogenesis). Tumor-associated antigen may be a factor cluster of differentiation (i.e. protein CDS, including, but not limited to, CD79b). Constructed of cysteine-based anti-CD79b antibody according to the invention retain antigen-binding ability of the parent analogues anti-CD79b antibody. Thus, constructed on the basis of cysteine anti-CD79b antibody according to the invention have the ability to bind, preferably specifically, with CD79b antigens, including isoforms beta and/or alpha-human anti-CD79b antibodies, if these antigens are expressed on the surface of cells, including, but not limited to, b cells.

In one aspect of the invention, the antibodies according to the invention can be anywhereman with any molecule with a tag that can be covalently linked to the antibody through a reactive molecule, activated group or a reactive thiol group of cysteine (Singh et al. (2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY; Lundblad, R. L. (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, FL). The attached label may have the following functions, namely: (i) producing the detected signal; (ii) interaction with a second� label and thereby the modification of the detected signal, the transmitted first or second label, e.g., by FRET (transfer fluorescence resonance energy); (iii) stabilize interactions or increase affinity of binding, with antigen or ligand; (iv) impact on mobility, e.g. electrophoretic mobility or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) the formation of immobilized molecules, and thereby modulating the affinity to the ligand, the binding of the antibody to the antigen or the formation of ionic complexes.

Labeled antibodies, constructed on the basis of cysteine, can be used in diagnostic assays, e.g., for detecting expression of interest antigen-specific cells, tissues, or serum. For use in diagnostic purposes specified antibody is usually labeled apparently detected molecule. There are various labels that can be essentially divided into the following categories:

Radioisotopes (radionuclides), such as3N,11C,14C,18F,32P,35S,64Cu,68Ga86Y94Tc,111In123I,124I,125I,131I,133I,133Xe177Lu,211At or213Bi. Labeled with radioisotopes antibodies can be used in the visualization experiments Retz�of Perov target. The antibody may be labeled with reagents, ligands that bind or form a chelate complex or any other complex with a radioisotope, where said reagent is able to react with thiol cysteine entered in the specified antibody, in accordance with the methods described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al. Ed. Wiley-Interscience, New York, NY, Pubs. (1991). Chelating ligands, which can form complex with metal ions, are DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). Radionuclides may be attached by forming a complex with the conjugate “antibody-drug” according to the invention (Wu et al. (2005) Nature Biotechnology 23(9):1137-1146).

Linker reagents such as DOTA-maleimide (4-maleimidomethyl-DOTA) can be obtained by reaction between aminobenzyl-DOTA with 4-maleimidomethyl acid (Fluka) activated with isopropylcarbamate (Aldrich), in accordance with the procedure described Axworthy et al. (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807). DOTA-maleimide reagents react with free cysteine amino acid residues of antibodies constructed with cysteine, and contribute to the formation of complex metal-ligand at the indicated antibody (Lewis et al. (1998) Bioconj. Chem. 9:72-86). Reagents for labeling chelating linker, such as DOTA-NHS (mono-N-hydroxy-succinimide� l,4,7,10-tetraazacyclododecane-l,4,7,10-vs acid), are commercially available (Macrocyclics, Dallas, TX). Visualization of receptor-target radionuclide-labeled antibodies allows the identification of the activation path through the detection and quantification of progressive accumulation of antibodies in tumor tissue (Albert et al. (1998) Bioorg. Med. Chem. Lett. 8:1207-1210). Conjugated radioactive metals can be found inside the cells after degradation lysosomes.

Chelate complexes with metal suitable for labeling antibodies in experiments by imaging are described in U.S. patents 5342606; 5428155; 5316757; 5480990; 5462725; 5428139; 5385893; 5739294; 5750660; 5834456; and in the publications of Hnatowich et al. (1983) J. Immunol. Methods, 65:147-157; Meares et al. (1984) Anal. Biochem. 142:68-78; Mirzadeh et al. (1990) Bioconjugate Chem. 1:59-65; Meares et al. (1990) J. Cancer l990, Suppl. 10:21-26; Izard et al. (1992) Bioconjugate Chem. 3:346-350; Nikula et al. (1995) Nucl. Med. Biol. [0378] 22:387-90; Camera et al. (1993) Nucl. Med. Biol. 20:955-62; Kukis et al. (1998) J. Nucl. Med. 39:2105-2110; Verel et al. (2003) J. Nucl. Med. 44:1663-1670; Camera et al. (1994) J. Nucl. Med. 21:640-646; Ruegg et al. (1990) Cancer Res. 50:4221-4226; Verel et al. (2003) J. Nucl. Med. 44:1663-1670; Lee et al. (2001) Cancer Res. 61:4474-4482; Mitchell, et al. (2003) J. Nucl. Med. 44:1105-1112; Kobayashi et al. (1999) Bioconjugate Chem. 10:103-111; Miederer et al. (2004) J. Nucl. Med. 45:129-137; DeNardo et al. (1998) Clinical Cancer Research 4:2483-90; Blend et al. (2003) Cancer Biotherapy &Radiopharmaceuticals 18:355-363; Nikula et al. (1999) J. Nucl. Med. 40:166-76; Kobayashi et al. (1998) J. Nucl. Med. 39:829-36; Mardirossian et al. (1993) Nucl. Med. Biol. 20:65-74; Roselli et al. (1999) Cancer Biotherapy &Radiopharmaceuticals, 14:209-20.

Fluorescent labels, such as the chelates formed of rare earth metals (chelate�, formed by europium), fluoresceine several types, including FITZ, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine several types including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; Texas red and their analogues. Fluorescent labels can be anywhereman with antibodies by methods described, for example, in the manual Current Protocols in Immunology, supra. Fluorescent dyes and fluorescent reagents-tags are commercially available reagents supplied by firms Invitrogen/Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

Different label “enzyme-substrate” are available or described in the literature (U.S. patent 4275149). The enzyme generally catalyzes a chemical transformation of a chromogenic substrate, which can be measured by various methods. For example, the enzyme may catalyze a color change of the substrate, which can be measured spectrophotometrically. Alternative enzyme may alter the fluorescence or chemiluminescence of the substrate. Methods for quantifying changes in the fluorescence intensity as described above. Chemiluminescent substrate undergoes electronic excitation under the action of a chemical reaction, then he can emit light, which can then be measured (e.g. chemiluminometer), or to inform ene�Gia fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., Firefly luciferase and bacterial luciferase; U.S. patent 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g. glucose oxidase, galactosidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase and xanthine-oxidase), lactoperoxidase, microbiocides, etc. Methods of conjugation of enzymes to antibodies are described in the publications O'sullivan et al. (1981) "Methods for the Preparation of Enzyme - Antibody Conjugates for use in Enzyme Immunoassay", in Methods in Enzym. (ed J. Langone &H. Van Vunakis), Academic Press, New York, 73:147-166.

Examples of combinations of the “enzyme-substrate” are, for example:

(i) horseradish peroxidase (HRP) with its substrate hydrogen peroxide, where the specified hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or hydrochloride 3,3',5,5'- tetramethylbenzidine (TMB));

(ii) alkaline phosphatase (ALP) with the chromogenic substrate para-nitrophenylphosphate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Specialists known and various other combinations of enzyme-substra�”. General description of them can be found in U.S. patents 4275149 and 4318980.

The label can be directly anywhereman with an amino acid side chain, with an activated amino acid side chain, with the antibody, is developed on the basis cysteine, etc. for example, the antibody may be anywhereman with Biotin and any of the labels above three broad categories can be anywhereman with Avidya or streptavidin, or Vice versa. Biotin selectively binds with the streptavidin, and thus, this label may be indirectly anywhereman with the antibody. Alternative, for implementation of indirect conjugation of the label with the polypeptide variant, the polypeptide variant kongugiruut with a small hapten (e.g., digoxin) and one of the notches of the above-mentioned various types kongugiruut with antiJapanese polypeptide variant (e.g., antibody against digoxin). Thus, can be achieved indirect conjugation of the label with the polypeptide variant (Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, San Diego).

The antibody according to the invention can be used in any known analytical method such as ELISA, in the analysis of competitive binding, direct and indirect sandwich-analyses and in the analyses performed by immunoprecipitation (Zola, (1987) Monoclonal Antibodies: A Manual of Technique, pp.147-158, CRC Press, Inc.).

Detectable label can be used to determine the localization, to visualize and to quantify binding events or recognition. Labeled antibodies according to the invention can recognize receptors on the cell surface. Another example of application-detectable-labeled antibodies is the immune method of immobilization on spheres, including the conjugation of a sphere with a fluorescently labeled antibody and detection of the fluorescence signal after binding of the ligand. Similar methods for the detection of binding, for measuring and detecting interaction of the antibody with the antigen used, the effect of surface plasmon resonance (PPR).

Detected markers, such as fluorescent dyes and chemiluminescent dyes (Briggs et al. (1997) "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids", J. Chem. Soc, Perkin Trans. 1:1051-1058), give the detected signal, and are typically used for labeling of antibodies, preferably having the following properties, namely: (i) the labeled antibody should produce a signal with a very high intensity with low background signal, so that a small number of antibodies could be detected with a highly sensitive method in cell-free or cell-based analysis; and (ii) the labeled antibody should be photostable so that fluorescentsignal could be detected be observed and recorded without the use of a significant level of optical bleaching. In methods that use the binding of labeled antibodies with the cell membranes or cell surfaces, especially live cells, the tag, preferably (iii) should have good solubility in water, which allows to achieve effective conjugate concentration and high sensitivity detection, and (iv) must be non-toxic to live cells, in order to avoid disruption of normal metabolic processes in cells or premature cell death.

Direct quantification of the fluorescence intensity of the cells and counting of fluorescent tagging, for example, event binding conjugates the peptide-dye” with the cell surface, may be implemented on a system FMAT® 8100 HTS System (Applied Biosystems, Foster City, Calif.), which allows for automatic mixing and reading, and non-radioactive assays on live cells or spheres (Miraglia, "Homogeneous cell - and bead-based assays for high throughput screening using fluorometric microvolume assay technology", (1999) J. of Biomolecular Screening 4:193-204). The use of labeled antibodies also provides testing for binding to a cell surface receptor, assays with immune immobilization, fluorescent solid-phase assays (FLISA), analysis by rassal�of caspase (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-apoptosis also been other ideas where in vivo", (1998) Proc. Natl. Acad. Sci. USA 95:618-23; US 6372907), analysis of apoptosis (Vermes, "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V" (1995) J. Immunol. Methods 184:39-51) and analyzed for cytotoxicity. Method fluorimetric analysis at the level of micro volumes can be applied to identify positive or negative regulation under the action of a molecule delivered to the cell surface (Swartzman, "A homogeneous and multiplexed down immunoassay for high-throughput screening using fluorometric microvolume assay technology", (1999) Anal. Biochem. 271:143-51).

Labeled antibodies according to the invention can be used as imaging of biological markers and probes in various methods and technologies Biomedicine and molecular imaging, such as (i) MRI (visualization using magnetic resonance method); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al. (2004) Bioconjugate Chem. 15:41-49; (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound examination. Immunoscintigraphy is a visualization technique in which antibodies labeled with radioactive substances are administered to an animal or person, and the image determine the place of localization of the antibody in the animal or human body (U.S. patent 6528624). Visualizers� biomarkers can be objectively measured and evaluated, as an indicator of normal biological processes, pathogenic processes or pharmacological responses to therapeutic treatment. Biological markers may be markers of several types: markers of type 0, which represent a natural long-known markers of this disease and linearly correlated with known clinical indices, for example, assessment of inflammation of the synovia in rheumatoid arthritis, conducted by NMR-tomography; markers of type I, which allow to detect the effect of therapeutic treatment in accordance with the existing mechanism, even if this mechanism can not be associated with clinical outcome; the markers of type II, which act as a “surrogate” endpoint where changes in the biomarker or modify the signal coming from the biomarker, allow to predict a favorable clinical effect for "confirmation" of the target response, such as bone erosion in rheumatoid arthritis as measured by computed tomography (CT). Thus, using imaging biomarkers can be obtained pharmacodynamic (PD) therapeutic data relating (i) to expression of the target protein, (ii) binding a therapeutic agent to a protein target, i.e. selectivity, and (iii) the clearance and half-life; and farmacocin�critical data. The advantages of in vivo imaging biomarkers compared with the laboratory of biological markers are: the possibility of their use for the implementation of non-invasive treatment, the possibility of quantitative evaluation and application for examination of the whole organism, the possibility of multiple doses of and analysis, that is, at different points in time, and the possibility of applying the results obtained in preclinical studies (for small animals), to conduct clinical trials (for humans). In some cases, biological visualization allows not to carry out a series of experiments or to minimize the number of experiments on animals in preclinical studies.

Methods of labeling peptides are well known in the art. Cm. Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997) Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Glazer et al. (1975) Chemical Modification of Proteins. Laboratory Techniques in Biochemistry and Molecular Biology (T. S. Work and E. Work, Eds.) American Elsevier Publishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”, Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter, Berlin and New York; and Wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); De Leon-Rodriguez et al. (2004) Chem.Eur. J. 10:1149-1155; Lewis et al. (2001) Bioconjugate Chem. 12:320-324; Li et al. (2002) Bioonjugate Chem. 13:110-115; Mier et al. (2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labeled with two molecules, the fluorescent reporter and the quencher is in close proximity to each other, are involved in the resonant energy transfer fluorescence (FRET). Reporter groups usually are fluorescent dyes that are excited by light at certain wavelengths and transfer the energy to the acceptor or quencher, i.e., the group with the corresponding Stokes shift, providing radiation with maximum brightness. Fluorescent dyes are molecules with more pronounced aromaticity, such as fluorescein and rhodamine, and their derivatives. Fluorescent reporter may be partially or largely suppressed by the molecule-quencher in the intact peptide. After cleavage of the peptide peptidase or protease may be detected increase in the fluorescence intensity (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248:18-34).

Labeled antibodies according to the invention can also be used as a means for affinity purification. In this method, the labeled antibody is immobilized on a solid phase, such as Sephadex resin or filter paper, by methods well known in the art. Immobilized antibody is subjected to contact with about�RASCOM, containing the purified antigen, and then the carrier is washed with a suitable solvent to remove almost all of the material in the sample except the purified antigen, and attach to the immobilized polypeptide variant. Finally, the carrier is washed with another suitable solvent, such as glycine buffer, pH 5.0, which releases the polypeptide antigen of the variant.

Reagents for labeling usually have reactive functional groups which may react (i) directly with the cysteine thiol of the antibody, is developed on the basis of cysteine, formation of labeled antibodies, (ii) with a linker reagent with the formation of intermediate compounds “linker-label” or (iii) a linker with the antibody with the formation of labeled antibodies. Reactive functional groups of reagents for labeling are maleimid, halogenoacetyl, gadacemashiiiii ester (e.g., NHS, N-hydroxysuccinimide), isothiocyanate, sulphonylchloride, 2,6-dichlorotriazinyl, panafcortelone ether and phosphoramidite, although can also be used and other functional groups.

Representative reactive functional group is N-hydroxysuccinimidyl (NHS), in which carboxypropyl replaced apparently detected by a label, e.g., Biotin or a fluorescent dye�eat. The NHS-ester of the specified labels may be preformed, isolated, purified and/or characterized, or it may be formed in situ and subjected to a reaction with the nucleophilic group of an antibody. Usually carboxyl form labels activated by reaction between a specific combination of carbodiimide reagents, for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, or uronium reagent, for example, TSTU (tetrafluoroborate O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea, HBTU (hexaflurophosphate O-benzotriazole-1-yl)-N,N,N'n'-tetramethylurea) or HATU (hexaflurophosphate O-(7-asobancaria-l-yl)-N,N,N',N'-tetramethylurea); and an activator such as 1-hydroxy-benzotriazole (HOBt), and N-hydroxysuccinimide, with obtaining NHS-ester label. In some cases, the label and the antibody can be linked by in situ activation markers and interaction with the antibody with obtaining conjugate label-antibody” in one stage. Other activating and binding reagents are TBTU (hexaflurophosphate 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethylurea), TFFH (2-forexceptional N,N',N",N'"-tetramethylene), PyBOP (hexaflurophosphate the benzotriazole-1-yl-oxy-Tris-pyrrolidinone), EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (l-(mesitylene-2-sulfonyl)-3-nitro-lH-l,2,4-triazole and arylsulfonate, for example, .

Connection "albumin-binding peptide-Fab" according to the invention

In one aspect of the invention, the antibody according to the invention is attached to the albumin-binding protein. The plasma protein binding can be an effective means of improving the pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Peptides binding to serum albumin, (ABP), can alter the pharmacodynamics of hybrid proteins containing the active domains, for example, modify the absorptive capacity of the fabric, its permeability and diffusion. These pharmacodynamic parameters can be modulated by specific selection of the appropriate peptide sequence that binds to serum albumin (application for U.S. patent 2004/0001827). The number of albumin-binding peptides were identified by a screening method of phage view (Dennis et al. (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J. Biol Chem. 277:35035-35043; WO 01/45746). Compounds according to the invention include ABP sequences described by (i) Dennis et al. (2002) J. Biol Chem. 277:35035-35043, tables III and IV, page 35038; (ii) in the application for U.S. patent 20040001827, paragraph [0076], SEQ ID nos: 9-22; and (iii) WO 01/45746, pp. 12-13: which is introduced into the present description by reference. Albumin-SV�binding (ABP)-Fab were constructed by joining the albumin-binding peptide to the C-Terminus of the heavy chain Fab in a stoichiometric ratio of 1:1 (1 ABP/1 Fab). It was shown that the binding of these ABP-Fab to albumin increases the half-life of antibodies in rabbits and mice more than 25 times. Therefore, the above-described reactive Cys residues may be introduced in these ABP-Fab and used for site-specific conjugation with cytotoxic drugs followed by animal studies in vivo.

Representative sequences of the albumin-binding peptide include, but are not limited to, the amino acid sequence represented in SEQ ID NO: 246-250:

CDKTHTGGGSQRLMEDICLPRWGCLWEDDF SEQ ID NO: 246

QRLMEDICLPRWGCLWEDDF SEQ ID NO: 247

QRLIEDICLPRWGCLWEDDF SEQ ID NO: 248

RLIEDICLPRWGCLWEDD SEQ ID NO: 249

DICLPRWGCLW SEQ ID NO: 250

Conjugates of the antibody-drug"

In another aspect, the present invention relates to immunoconjugates or to conjugate "antibody-drug" (ADC) containing the antibody, anywhereman with a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e. radioconjugates). In another aspect, the present invention relates to methods of using such immunoconjugate�s. In one aspect of the invention immunoconjugate contains any of the aforementioned anti-CD79b antibodies covalently linked to a cytotoxic agent or to detect the agent.

In one aspect of the invention, an anti-CD79b antibody according to the invention binds to the same epitope on CD79b is associated with another CD79b antibody against. In another embodiment of the invention, an anti-CD79b antibody according to the invention binds to the same epitope on CD79b, which is bound Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC under number HB11413 July 20, 1993, monoclonal antibodies containing the variable domains of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or chimeric antibody containing the variable domain of the antibody obtained from a hybrid HB11413 deposited with the ATCC on 20 July 1993, and constant domains from IgG1, or the variable domains of monoclonal antibody comprising sequences of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another embodiment of the invention, an anti-CD79b antibody according to the invention binds to the same epitope on CD79b is associated with other anti-CD79b antibody (i.e., CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT107-2 (AbD Serotec Catalog #MCA2209), antibody against human CD79b (BD Biosciences Catalog #557592; San Jose, CA)).

In another aspect of the invention, an anti-CD79b ant�body according to the invention binds to an epitope on CD79b, differing from the epitope is associated with other anti-CD79b antibody. In another embodiment of the invention, an anti-CD79b antibody according to the invention binds to an epitope on CD79b, wherein the epitope is associated with Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC under number HB11413 July 20, 1993, monoclonal antibodies containing the variable domains of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or chimeric antibody containing the variable domain of the antibody obtained from a hybrid HB11413 deposited with the ATCC on 20 July 1993, and constant domains from IgG1, or the variable domains of monoclonal antibody comprising sequences of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another embodiment of the invention, an anti-CD79b antibody according to the invention binds to an epitope on CD79b, wherein the epitope is associated with other anti-CD79b antibody (i.e., CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT107-2 (AbD Serotec Catalog #MCA2209), antibody against human CD79b (BD Biosciences Catalog #557592; San Jose, CA)).

In another aspect of the invention, an anti-CD79b antibody according to the invention is different from (i.e. not them) Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC under number HB11413 July 20, 1993, monoclonal antibodies containing variable �Omani SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or a chimeric antibody containing the variable domain of the antibody obtained from a hybrid HB11413 deposited with the ATCC July 20, 1993 and constant domains from IgG1, or the variable domains of monoclonal antibody comprising sequences of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another aspect of the invention, an anti-CD79b antibody according to the invention is different from (i.e. not them) Fab-fragment of another anti-CD79b antibody (i.e., CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT107-2 (AbD Serotec Catalog #MCA2209), antibodies against human CD79b (BD Biosciences Catalog #557592; San Jose, CA)).

In one aspect of the invention, the antibody according to the invention specifically binds to CD79b animal of the first species, but not specifically binds to CD79b animal of another species. In one embodiment of the invention, the animal of the first type is the person and/or Primate (e.g., dog-like monkey), and the animals of the second type is the family of mice animal (e.g., mouse) and/or an animal of the family dog. In one embodiment of the invention, the animal of the first species is human. In one embodiment of the invention, the animal of the first species is a Primate, for example, dog-like monkey. In one embodiment of the invention, the animal of the second species is an animal of the family of mice, such as mice. In one embodiment, the image�moving animal of the second species is an animal of the family dog.

In one of its aspects the present invention relates to compositions containing one or more antibodies according to the invention and a carrier. In one embodiment of the invention the specified carrier is a pharmaceutically acceptable carrier.

In one of its aspects the present invention relates to nucleic acids encoding anti-CD79b antibody according to the invention.

In one of its aspects the present invention relates to vectors containing the nucleic acid according to the invention.

In one of its aspects the present invention relates to the cell host containing a nucleic acid or vector according to the invention. The vector may be a vector of any type, for example, a recombinant vector such as an expression vector. This may be used by the host cell of any kind. In one embodiment of the invention the cells of the host are prokaryotic cells, such as E. coli. In one embodiment of the invention the cells of the host are eukaryotic cells, e.g. mammalian cells, such as cells of the Chinese hamster ovary (Cho) cells.

In one of its aspects the present invention relates to methods for producing antibodies according to the invention. For example, the present invention relates to a method for producing an anti-CD79b antibody (to�oroe, as defined in the present application, includes a full-size sequence and its fragments), where the specified method comprises the expression in a suitable the host cell a recombinant vector according to the invention, encoding the indicated antibody (or its fragment), and the allocation of the specified antibodies.

In one of its aspects the present invention relates to industrial product that contains a container and a composition contained in the container, where said composition contains one or more anti-CD79b antibodies according to the invention. In one embodiment of the invention said composition contains a nucleic acid according to the invention. In one embodiment of the invention a composition comprising an antibody, also contains the media that, in some embodiments of the invention that is pharmaceutically acceptable. In one embodiment of the invention, the article of manufacture according to the invention also contains instructions for introduction of the composition (e.g., antibody) to an individual.

In one of its aspects the present invention relates to a kit containing a first container comprising a composition comprising one or more anti-CD79b antibodies according to the invention; and a second container containing a buffer. In one embodiment of the invention, the specified buffer is pharmaceutically acceptable�MYM. In one embodiment of the invention a composition comprising an antibody antagonist, also includes media that in some embodiments of the invention is pharmaceutically acceptable. In one embodiment of the invention, the kit also contains instructions for introduction of the composition (e.g., antibody) to an individual.

In one of its aspects the present invention relates to the use of anti-CD79b antibodies according to the invention for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to the use of nucleic acids according to the invention for the preparation of a medicament for therapeutic and/or prophylactic Le� " s disease, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to the use of the expression vector according to the invention for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (OL�) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to the use of host cell according to the invention for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to the use of industrial products according to the invention for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic N�L, untreatable the NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to the use of the kit according to the invention for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention cancer, tumor and/or cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to a method of inhibiting growth of cells expressing CD79b, where said method comprises contacting these cells with an antibody according to the invention, resulting in inhibition of growth of these cells�K. In one embodiment of the invention, the antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of therapeutic treatment of a mammal having a cancerous tumor that contains cells expressing CD79b, where the method includes introduction to the specified mammal a therapeutically effective amount of the antibody according to the invention, and thus effective treatment of the specified mammal. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of treating or preventing a cell proliferative disorder associated with increased levels of expression of CD79b, where the specified method comprises administering to the individual in need of such treatment, an effective amount of an antibody according to the invention, and thus effective treatment or prevention of a specified cell-proliferative disorder. In one embodiment of the invention the specified cell-proliferative disorder is cancer. One in�options of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of inhibiting growth of cells whose growth is at least partly dependent on the growth-potentiating action CD79b, where said method comprises contacting these cells with an effective amount of an antibody according to the invention, and thus the inhibition of growth of these cells. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method of therapeutic treatment of a tumor in a mammal, the growth of which is at least partially dependent on the growth-potentiating action CD79b, where said method comprises contacting these cells with an effective amount of an antibody according to the invention, and thus effective treatment of the indicated tumor. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

In one of its aspects the present invention relates to a method for treating cancer�, comprising administering to the patient a pharmaceutical composition containing described here immunoconjugate acceptable diluent, carrier or excipient. In one embodiment of the invention the specified cancer is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. In one embodiment of the invention, the patient is administered with a cytotoxic agent in combination with compound-conjugate "antibody-drug".

In one of its aspects the present invention relates to a method of inhibiting the proliferation of b cells, comprising treating cells immunoconjugates containing the antibody according to the invention, under conditions conducive to binding immunoconjugate with CD79b. In one embodiment of the invention, the disease associated with proliferation of b-cells selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable devil�incomei NHL chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. In one embodiment of the invention In a cell is a xenograft. In one embodiment of the invention, the specified treatment is carried out in vitro. In one embodiment of the invention, the specified treatment is carried out in vivo.

In one of its aspects the present invention relates to a method of determining the presence of CD79b in a sample suspected to contain CD79b, where the specified method comprises treating a specified specimen, the antibody according to the invention, and determining the level of binding of the indicated antibody to CD79b in a specified sample, where the specified binding of the antibody to CD79b in a specified sample is indicative of the presence of the indicated protein in a given sample. In one embodiment of the invention the specified sample is a biological sample. In another embodiment of the invention the specified biological sample contains cells. In one embodiment of the invention the biological sample is taken from a mammal suffering from or suspected to suffer b-cell disorder and/or cell-proliferative disorder, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), viole�ing NHL recurrent aggressive NHL, recurrent asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to a method of diagnosing a cellular proliferative disorder associated with increased number of cells, such as b cells expressing CD79b, where said method comprises contacting the test cells in a biological sample with any of the above antibodies; determining the level of antibody bound to test cells in the sample by detecting binding of an antibody to CD79b; and comparing the level of antibody bound to cells in a control sample, where the level of bound antibodies normalize by the number of CD79b-expressing cells in the test and control samples and where a higher level of bound antibody in the sample compared to a control sample, indicates the presence of cell-proliferative disorder associated with cells expressing CD79b.

In one of its aspects the present invention relates to a method for the detection of soluble CD79b in shelter� or serum, where said method comprises contacting the test sample of blood or serum from the mammal, preferably suffering from b-cell-proliferative disorder with an anti-CD79b antibody according to the invention, and the detection of increased level of soluble CD79b in the tested sample compared to a control sample of blood or serum taken from a healthy mammal. In one embodiment of the invention the method of detection can be applied for the diagnosis of b-cell proliferative disorder associated with increased levels of soluble CD79b in the blood or serum of a mammal.

In one of its aspects the present invention relates to a method of binding antibodies according to the invention with a cell that expresses CD79b, where said method comprises contacting the specified cell with the antibody according to the invention. In one embodiment of the invention, the specified antibody anywhereman with a cytotoxic agent. In one embodiment of the invention, the specified antibody anywhereman with the growth-inhibitory agent.

The methods according to the invention can be applied to the treatment of any suitable pathological state, for example, a condition in which the cells and/or tissues Express CD79b. In one embodiment of the invention, in the method according to Fig�the plants, the cell-target is a hematopoietic cell. For example, a hematopoietic cell can be a cell selected from the group consisting of lymphocytes, leukocytes, platelets, erythrocytes and natural killer cells. In one embodiment of the invention, in the method according to the invention, the cell-target is a b-cell or T-cell. In one embodiment of the invention, in the method according to the invention, the cell-target is a cancer cell. So, for example, cancer cells may be selected from the group consisting of lymphoma cells, leukemia or myeloma.

The methods according to the invention may also include additional processing steps. For example, in one embodiment of the invention the method also includes a step in which the target cell and/or the target tissue (e.g., cancer cell) irradiated or treated with a chemotherapeutic agent.

As described in the present application, CD79b is a signaling component of the b-cell receptor. Accordingly, in one embodiment of the methods according to the invention, the specified cell-target (e.g., a cancer cell) is the cell in which is expressed CD79b, compared to a cell that does not Express CD79b. In another embodiment of the invention specified by the cell-target is a cancer cell in which watches�I an increased level of expression of CD79b, compared to normal non-cancer cell tissue of the same type. In one embodiment of the invention, the method according to the invention is aimed at the destruction of the target cells.

In other of its aspects the present invention relates to vectors containing DNA encoding any of the antibodies described herein. The present invention also relates to the cell host containing any such vector. So, for example, cells of the host cells may be CHO cells, E. coli or yeast cells. The present invention also relates to a method for producing any of the antibodies described herein, where the method includes culturing host cells under conditions suitable for expression of the desired antibody and isolation of the desired antibody from the cell culture.

In yet another aspect, the present invention relates to the compositions described herein containing an anti-CD79b antibody in combination with a carrier. The specified media is, but not necessarily, a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to the use described herein antibodies against CD79b polypeptide for the preparation of a medicament to treat a condition that is susceptible to antibodies against CD79b polypeptide.

Another aspect of the present invention is �omposite, containing a mixture of compounds is an antibody-drug of formula I, where the average load of the drug to the antibody is approximately 2-5 or 3-4.

In another aspect, the present invention relates to pharmaceutical compositions comprising a compound of the ADC of formula I, mixture of compounds of the ADC of formula I or their pharmaceutically acceptable salts or solvates, and a pharmaceutically acceptable diluent, carrier or excipient.

In another aspect, the present invention relates to pharmaceutical combinations comprising compound ADC of formula I and a second compound having anti-cancer or other therapeutic properties.

In another embodiment, the present invention relates to a method of preventing or inhibiting the proliferation of tumor or cancer cells, where the specified method comprises treating the cell with a conjugate "antibody-drug of formula I or its pharmaceutically acceptable salt or solvate in an amount effective for preventing or inhibiting the proliferation of tumor or cancer cells.

In another aspect, the present invention relates to a method of treating cancer, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising the ADC of formula I.

In another �howl aspect, the present invention relates to industrial products, that is, the sets containing conjugate "antibody-drug", the container and the liner in the container or label with instructions for treatment.

In one of its aspects the present invention relates to a method of producing compound-conjugate "antibody-drug of formula 1, where said method comprises the steps: (a) reaction between cysteine groups introduced in designed on the basis of cysteine antibody with a linker reagent with the formation of intermediate compounds antibody-linker Ab-L; and (b) reaction of interaction of Ab-L with an activated molecule drug D with the formation of the conjugate "antibody-drug"; or stages: (c) reaction between the nucleophilic group of the molecule of the drug with a linker reagent with the formation of the intermediate connection "drug-linker" D-L; and (d) reaction between D-L-cysteine group, introduced in designed on the basis of cysteine antibody to form a conjugate "antibody-drug".

In one of its aspects the present invention relates to the analysis for the detection of cancer cells, comprising: (a) treatment of cells with conjugate "constructed on the basis of cysteine anti-CD79b antibody-drug", and (b) the extent svyazyvanie� connection-conjugate "is constructed on the basis of cysteine anti-CD79b antibody-drug" with the specified cell.

A.Anti-CD79b antibodies

In one of its variants the present invention relates to anti-CD79b antibodies, which can be applied here as therapeutic agents. Representative antibodies are polyclonal, monoclonal, humanized, bespecifically and heteroconjugate antibodies.

1. Polyclonal antibodies

Polyclonal antibodies are preferably produced in animals after multiple subcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevant antigen and an adjuvant. They can be used for conjugating the relevant antigen (and in particular, if you use synthetic peptides) with a protein which is immunogenic for species that undergo immunization. So, for example, the antigen can be conjugated with keyhole lymph snails (KLH), serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, (conjugated through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic acid anhydride, SOCl2or R1N=C=NR, where R and R1represent different alkyl groups.

Animal immunestimulating, immunogenic conjugates or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant of freind and by subcutaneous injection of a solution in many areas. After a month immunize animals again in many areas by subcutaneous injection of 1/5-1/10 of the original amount of peptide or conjugate in complete adjuvant of franda. After 7-14 days the animals take blood and analyze the serum for antibody titer. Then immunize animals again before reaching the plateau of the title. Conjugates can also be obtained in recombinant cell culture in the form of hybrid proteins. In addition, to enhance the immune response can also be used aggregating agents such as alum.

2. Monoclonal antibodies

Monoclonal antibodies can be obtained using the hybrid technology, first described by Kohler et al. Nature, 256:495 (1975), or they can be obtained by recombinant DNA methods (U.S. patent No. 4816567).

To obtain a hybrid mouse or other suitable animal host, such as a hamster, immunize, as described above, for helping them lymphocytes that produce or are capable of producing antibodies, specifically binding to the protein used for immunization. Alternatives�of the lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and subjected to fusion with myeloma cells using a suitable agent for the merger, such as polyethylene glycol, resulting in formation of a hybrid cell (Goding,Monoclonal Antibodies: Principles and Practice,pp.59-103 (Academic Press, 1986)).

The thus obtained hybrid cells are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also referred to as the "merger partner"). For example, if the parental myeloma cells do not contain the enzyme hypoxanthine-guanine-phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for the production of hybridomas typically include hypoxanthine, to produce remissions in childhood and thymidine (Wednesday hat), that is, substances that prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells, called partners in the merger, are cells that are able to be effectively merge, support stable production of high levels of antibodies indicated the selected antibody-producing cells, and are sensitive to a selective medium on which the tryouts are on naslite parent cell. Preferred myeloma cell lines are mouse�nye myeloma lines, such as cell lines, cells derived from murine tumors MORSE-21 and MPC-11, available at the Institute Salk Institute Cell Distribution Center, San Diego, California USA, cells and SP-2 and derivatives thereof, for example, the cells of the X63-Ag8-653, available from the American type culture collection, Manassas, Virginia, USA. For producing human monoclonal antibodies are also used in human myeloma cell lines and heteromyidae cell line “mouse-man” (KozborJ. Immunol., 133:3001 (1984) & Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp.About 51 To 63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium for the growth of hybrid cells examined for the production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybrid cells, determined, preferably, by immunoprecipitation or by conducting in vitro analysis of the binding, such as radioimmunoassay (RIA) or solid-phase immunofermentnyi analysis (ELISA).

The affinity of binding of the monoclonal antibody can be, for example, determined using analysis of Scatchard described by Munson et al.,Anal. Biochem., 107:220 (1980).

After identification of hybrid cells which produce antibodies with the desired specificity, affinity and/or activity, the clones can be subclavian by conducting procedures limiting dilution and �antivirusni by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media which are designed to serve this purpose are, for example, medium (D-MEM or RPMI-1640. In addition, hybrid cells may be grown in vivo as ascites tumors in animals, e.g., by i.p. injection of these cells in mice.

Monoclonal antibodies, secreted by subklonov, suitably separated from the culture medium, ascites fluid or serum by carrying out standard procedures purification of antibodies, such as, for example, affinity chromatography (e.g., protein A or G-protein-sepharose), or ion-exchange chromatography, chromatography on hydroxiapatite, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies may be readily isolated and sequenced in accordance with standard procedures (e.g., by using oligonucleotide probes that are able to specifically communicate with the genes encoding the heavy and light chains of murine antibodies). A preferred source of such DNA are hybrid cells. After isolation of this DNA can be integrated into expression vectors, which are then transferout in the host cell, such as E. coli cells, simian COS cells, cells of the Chinese hamster ovary (Cho) cells or myeloma cells that in other cases� not produce protein antibodies resulting in in these recombinant cells-the owners are synthesized monoclonal antibodies. Discussion recombinant expression of an antibody-coding DNA in bacteria can be found in articles Skerra et al.,Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun,Immunol. Revs., 130:151-188 (1992).

In another embodiment of the invention, monoclonal antibodies or fragments of antibodies can be isolated from phage libraries of antibodies generated by the methods described by McCafferty et al.,Nature, 348:552-554 (1990). In the work of Clackson et al.Nature, 352:624-628 (1991) and Marks et al.,J. Mol. Biol.222:581-597 (1991) described the allocation of murine and human antibodies, respectively, using phage libraries. In later publications describe the production of high-affinity (nm order) human antibodies by permutation of genes chain antibodies (Marks et al.Bio/Technology, 10:779-783 (1992)), as well as through co-infection and recombination in vivo used as a strategy for constructing very large phage libraries (Waterhouse et al.Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these methods are an acceptable alternative to traditional hybrid technology for obtaining monoclonal antibodies used for isolation of monoclonal antibodies.

The DNA that encodes the antibody, can be modified for the production of polypeptides chimeric or hybrid antibodies, n�example, by replacing the sequence encoding the constant domains (CHand CL) the heavy chain and light chain of a human antibody, homologous sequences of mouse antibodies (U.S. patent No. 4816567 and Morrison, et al.,Proc. Natl Acad. Sci. USA, 81:6851 (1984), or by joining to the immunoglobulin coding sequence all coding sequences (or part thereof) for nimmanahaeminda polypeptide (heterologous polypeptide). Such sequences nimmanahaeminda polypeptides are used to replace the constant domains of an antibody, or they are used to replace the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site having specificity for one antigen and another antigen-binding site having specificity for a different antigen.

3.Human and humanized antibodies

Anti-CD79b antibody according to the invention can also comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (e.g., murine antibodies) are chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies), colorinterior minimum sequence derived from nonhuman immunoglobulin. Humanized antibodies are human immunoglobulins (recipient antibody) in which residues originating from the hypervariable region (CDR) of the antibodies of the recipient are replaced by residues originating from the hypervariable region (CDR) of a non-human antibody (donor antibody) such as mouse antibody, rat antibody, rabbit antibody having the desired specificity, affinity and binding capacity. In some cases the remains of the frame region (Fv) of the human immunoglobulin are replaced by corresponding inhuman remnants. Humanized antibodies can also comprise residues that are not found in the recipient antibody or in the imported CDR sequences or frame area. In General, a humanized antibody may include substantially all or at least one, and typically two, variable domain, in which all or almost all regions correspond to the CDR regions of non-human immunoglobulin, and all or nearly all FR are FR from a human immunoglobulin consensus sequence. A humanized antibody also includes, but not necessarily, at least a portion of constant region of immunoglobulin (Fc), typically a human immunoglobulin� [Jones et al. (1986)Nature, 321:522-525; Riechmann et al. (1998)Nature332:323-329 Presta andCurr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally speaking, a humanized antibody has one or more amino acid residues introduced into it from a source, not a person. These inhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be performed mainly by the method of winter (Winter) and the staff of [Jones et al.,Nature321:522-525 (1986); Riechmann et al.,Nature, 332:323 to 327 (1988); Verhoyen et al.,Science239:1534-1536 (1988)], by replacing sequences of the rodent CDR or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. patent No. 4816567), in which mainly the smaller part, compared with the variable domain of an intact human antibody is replaced by the corresponding sequence from a non-human antibody. Actually, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues substituted by residues originating from analogous sites of antibodies rodents.

When POPs�Denmark humanized antibodies to reduce antigenicity and HAMA response (the formation of human anti-mouse antibody) when the antibody for therapeutic treatment of a person, it is very important to choose the variable domains of both light and heavy chains of human antibodies. The decrease in the level NAMA-response or preventing such a response is an important aspect of clinical development of suitable therapeutic agents. See, for example, Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described in this application, the present invention relates to antibodies that have been humanized to reduce or prevent NAMA-response. Versions of these antibodies can also be obtained with routine methods known in the art, some of which are described in detail below. In accordance with the so-called method of “fitting”, the sequence of the variable domain of the antibody rodent sceneroot along the entire library of known sequences of variable domains of human antibodies. Then identify the sequence of the V-domain of a human antibody which is most similar to the sequence of the rodent, and take as the human frame region (FR) to create a humanized antibody (Sims et al.,J. Immunol. 151:2296 (1993); Chothia et al.,J. Mol. Biol. 196:901 (1987)). In the other method uses specific movement�ing region, derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. This same frame area can be used for several different humanized antibodies (Carter et al.,Proc. Natl. Acad. Sci., USA, 89:4285 (1992); Presta et al.,J. Immunol, 151:2623 (1993)).

For example, the amino acid sequence of the antibodies described in this application can serve as the initial (parental) sequences for the diversification of the sequence(s) of a frame region and/or hypervariable region. Selected frame sequence, which is attached with the original hypervariable sequence, here called acceptor human skeleton sequence. Acceptor human skeleton sequences can be obtained or can occur from a human immunoglobulin (regions VL and/or VH), and preferably the human acceptor frame sequences can be obtained or can occur from human consensus frame of the sequence, such as a frame sequence, as has been demonstrated, have minimal immunogenicity, or do not have immunogenicity in humans.

If the acceptor is derived from human immunoglobulin�on, the human frame sequence can be, but not necessarily, selected on the basis of its homology with the donor frame sequence by aligning donor frame sequence with different human frame sequences in the collection of human skeleton sequences, and selection of the most homologous frame sequence as acceptor.

In one embodiment of the invention the human consensus frame region come from a consensus of frame sequences, the VH subgroup III and/or VL Kappa subgroup I.

Thus, acceptor human skeleton VH region can contain one, two, three or all of the following frame sequence:

FR1 containing EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 143),

FR2 containing WVRQAPGKGLEWV (SEQ ID NO: 144),

FR3 containing RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYC (SEQ ID NO: 147), where X1represents A or R, X2is a T or N, and X3represents A or L,

FR4 containing WGQGTLVTVSS (SEQ ID NO: 146).

Examples of consensual frame of the VH regions are:

the consensus frame region of a human VH subgroup I minus CDR on Kabuto (SEQ ID NO: 108);

the consensus frame region of a human VH subgroup I minus extended hypervariable region (SEQID NO: 109-111);

the consensus frame region of a human VH subgroup II minus CDR on Kabuto (SEQ ID NO: 112);

the consensus frame region of a human VH subgroup II minus extended hypervariable region (SEQ ID NO: 113-115);

the consensus frame region of a human VH subgroup III minus CDR on Kabuto (SEQ ID NO: 116);

the consensus frame region of a human VH subgroup III minus extended hypervariable region (SEQ ID nos: 117-119);

acceptor of a frame region of a human VH minus CDR on Kabuto (SEQ ID NO: 120);

acceptor of a frame region of a human VH minus extended hypervariable region (SEQ ID NO: 121-122);

acceptor frame area 2 human VH minus CDR on Kabuto (SEQ ID NO: 123); or

acceptor frame area 2 human VH minus extended hypervariable region (SEQ ID NO: 124-126).

In one embodiment of the invention, the acceptor of a frame region of a human VH comprises one, two, three or all of the following frame sequence:

FR1 containing EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 143),

FR2 containing WVRQAPGKGLEWV (SEQ ID NO: 144),

FR3 containing RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO: 145),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO: 148),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 149),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCS (SEQ ID NO: 150) or

RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO: 151),

FR4 containing WGQGTLVTVSS (SEQ ID NO: 146).

Acceptor frame area of human VL can� to contain one, two, three or all of the following frame sequence:

FR1 containing DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 139),

FR2 containing WYQQKPGKAPKLLIY (SEQ ID NO: 140),

FR3 containing GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 141),

FR4 containing FGQGTKVEIKR (SEQ ID NO: 142).

Examples of consensual frame of the VL sequences are:

the consensus of a frame sequence of a human VL Kappa subgroup I (SEQ ID NO: 127);

the consensus of a frame sequence of a human VL Kappa subgroup II (SEQ ID NO: 128);

the consensus of a frame sequence of a human VL Kappa subgroup III (SEQ ID NO: 129); or

the consensus of a frame sequence of a human VL Kappa subgroup IV (SEQ ID NO: 130).

Although the acceptor sequence may be identical to the human frame sequence, regardless of whether it is from a human immunoglobulin or a human consensus frame of the sequence, but the present invention is described acceptor sequence, which, compared with human immunoglobulin sequence or human consensus frame sequence, may contain existing amino acid substitutions. These existing substitutions compared to the human immunoglobulin sequence or a consensus frame� sequence, preferably are minimal, and usually only differ by four, three, two amino acid residues or a single amino acid residue.

The remains of the hypervariable regions of non-human antibody administered in a frame acceptor human VL and/or VH. So, for example, can be included residues corresponding to residues on CDR Cabatu, the remnants of the hypervariable loops on Chothia, Abm residues, and/or contact residues. This may be included, but not necessarily, the remains of the extended hypervariable region: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3).

Although the introduction of residues hypervariable region is discussed in the description of the present invention, however, it should be noted that such introduction can be carried out by various methods, for example, nucleic acid that encodes the desired amino acid sequence can be obtained by introducing mutations into the nucleic acid encoding the sequence of murine variable domain, with subsequent replacement of frame balances on the remains of the acceptor human skeleton region, or by introducing mutations into the nucleic acid sequence encoding a human variable domain, followed by replacement of residues hypervariable domain remains on nicelove�climate antibodies, or by the synthesis of the nucleic acid encoding the desired sequence, etc.

In the examples described here are the options with the attached hypervariable region were obtained by the method of Kunkel mutagenesis of nucleic acid encoding the human acceptor sequence, using a separate oligonucleotide for each hypervariable region (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). Appropriate substitutions may be introduced in a frame and/or a hypervariable region of routine methods for the correction and recovery of the desired interactions "hypervariable region-antigen".

Phage (famine) representation (also referred to here in some cases, rahovym presentation) can be applied as a convenient and rapid method of producing and screening of many different potential variants of the antibodies in the library, obtained by randomization of the sequences. However, specialists and other known methods of obtaining and screening of modified antibodies.

The technology of phage (fahmideh) represent an effective means for producing and selecting novel proteins, binding to a ligand such as an antigen. The use of phage technology (fahmideh) representation allows to obtain large libraries of protein variants that can be quickly�ro sorted on the sequence, are associated with the molecule-target with high affinity. Nucleic acids that encode polypeptide variants, usually attached to a nucleic acid sequence that encodes a viral envelope protein, such as a protein encoded by the gene III, or protein encoded by gene VIII. Were developed monovalent system fahmideh representations, in which a nucleic acid sequence encoding a protein or polypeptide attached to a nucleic acid sequence that encodes part of a protein encoded by the gene III (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In a monovalent system fahmideh the submission of the hybrid gene is expressed at a low level, and wherein the protein III gene of the wild type expressed so that infectivity of the particles is preserved. Methods of producing peptide libraries and screening these libraries are described in many patents (e.g., U.S. patent No. 5723286, in U.S. patent No. 5432018, in U.S. patent No. 5580717, in U.S. patent No. 5427908 and in U.S. patent No. 5498530).

Libraries of antibodies or antigen-binding polypeptides were obtained by various methods, including modification of one gene by embedding a randomized DNA sequences or cloning of a family of related genes. Methods of introducing the antibodies or the antigen-binding frame�tov using the technology of phage representation described in U.S. patents №№ 5750373, 5733743, 5837242, 5969108, 6172197, 5580717 and 5658727. Then the library sceneroot for the presence of antibodies or antigen-binding proteins with desired properties.

Methods to replace the selected amino acids at the level of the matrix nucleic acid are well known in the art and some are described in the present application. For example, the remains of the hypervariable region can be replaced by the method of the Councel. See, for example, Kunkel et al., Methods Enzymol. 154:367-382 (1987).

The sequence of oligonucleotides includes one or more constructed sets of codons for modifiable residues of the hypervariable region. The set of codons is a set of different nucleotide triplet that is used to encode the desired amino acid variants. Sets of codons can be represented by symbols that indicate specific nucleotides or equimolar mixtures of nucleotides, are presented below in accordance with the IUB code.

Codes IUB

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T) H

N (A or C or G or T).

For example, in a series of codons DVK, D can be nucleotides A or G or T; V can be A or G or C, and K can be G or T. This Noboa� codons may be 18 different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Sets of oligonucleotides or primers can be synthesized by standard methods. A set of oligonucleotides can be synthesized, for example, by solid-phase synthesis and may contain sequences that represent all possible combinations of nucleotide triplets that are included in that set of codons, which encode the desired group of amino acids. Synthesis of oligonucleotides with "degeneracy" of selected nucleotides in certain positions is well known in the art. Such sets of nucleotides having certain sets of codons, can be synthesized on commercially available nucleic acid synthesizers (available, for example, Applied Biosystems, Foster City, CA), or they can be purchased from suppliers (for example, Life Technologies, Rockville, MD). Therefore, a set of synthesized oligonucleotides with a specific set of codons, typically includes a variety of oligonucleotides with different sequences, wherein the set of codons in the full-size sequence. The oligonucleotides used in accordance with the present invention have the sequence that gibridizatsiya with the matrix nucleic acid of the variable domain, and can also include restriction sites for cloning.

In one of these �of erodov nucleic acid sequence, encoding variants of amino acids can be created by oligonucleotide-mediated mutagenesis. This method is well known in the art and described by Zoller et al. Nucl Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variants of amino acids, is produced by hybridizing set of oligonucleotides, containing the sets of codons, with matrix DNA, where the specified matrix is a single-stranded form of the plasmid containing the sequence of the matrix nucleic acid variable region. After hybridization, for the synthesis of a full-sized second complementary strand of a matrix using DNA polymerase, which allows to embed the oligonucleotide primer, and the chain will contain the sets of codons, provided a set of oligonucleotides.

Usually use oligonucleotides with a length of at least 25 nucleotides. Optimal oligonucleotide has 12 to 15 nucleotides that are completely complementary to the matrix on either side of the nucleotide(s) encoding(s) mutation(s). This will ensure proper hybridization of the oligonucleotide with single-stranded molecule of the matrix DNA. The oligonucleotides can be readily synthesized by methods known in the art, and described in the publication of Crea et al., Proc. NAT'l. Acad. Sci. USA, 75:5765 (1978).

DNA-matrix on ispolzovaniem, derived from vectors bacteriophage M13 (suitable commercially available vectors M13mp18 and M13mp19), or by using vectors that contain a single-stranded origin of replication of phage described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is mutated, may be integrated into one of these vectors with obtaining single-stranded matrix. Obtaining single-stranded matrix is described in sections 4.21-4.41 of the manual Sambrook et al., see above.

For the modification of the native DNA sequence of the oligonucleotide hybridizing with single-stranded matrix in appropriate hybridization conditions. Then, for the synthesis of the complementary strand of the matrix is carried out using the oligonucleotide as a primer, add a DNA polymerizing enzyme, usually DNA polymerase T7 or fragment maple DNA polymerase I. the result is heteroduplex molecule in which one strand of DNA encodes the mutated form of gene 1, and the other chain (the original matrix) encodes the native unmodified gene sequence 1. Then such heteroduplex molecule is transferred into a suitable host cell, usually in prokaryotes, such as E. coli JM101. After cell cultivation, sown on tablets with agarose and sceneroot using oligonucleotide primer, radioactively labeled with 32-phosphate to C�for the purposes of identification of bacterial colonies, containing mutated DNA.

The just described method may be modified in order to create homoduplexes molecules in which both strands of the plasmid contain the mutation(s). This modification is carried out as follows: single-stranded oligonucleotide hybridizing with single-stranded matrix as described above. A mixture of three deoxyribonucleotides, namely desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTT), together with a modified timezonebias, denoted by dCTP-(aS) (which can be obtained from Amersham). This mixture was added to the complex matrix-oligonucleotide". After addition of DNA polymerase to this mixture is formed strand of DNA identical to the matrix, except for the mutated bases. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which will ensure the protection of DNA from cleavage restrictive endonuclease. After the formation of single-stranded gap in the chain-matrix of the double-stranded heteroduplex under the action of the corresponding restricteduse enzyme, a chain-matrix can be hydrolysed by ExoIII nuclease or another appropriate nuclease in position behind the mutagenic region(s) of the site(s). Then the reaction is stopped, resulting in a molecule that is only single-stranded halfway. For�eat get full size of double-stranded DNA homoduplex using DNA polymerase in the presence of all four deoxyribonucleotide-triphosphates, ATR and DNA-ligase. Then this homoduplex molecule can be transferred into a suitable host cell.

As shown above, the sequence of the set of oligonucleotides has a length sufficient for hybridization with the matrix nucleic acid, and may also, but need not, contain restriction sites. DNA-matrix can be obtained by using vectors that originate from vectors bacteriophage M13 or vectors containing single-stranded origin of replication of phage, as described in the publication Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is mutated, must be embedded in one of these vectors with obtaining single-stranded matrix. Obtaining single-stranded matrix is described in sections 4.21-4.41 of the manual Sambrook et al., see above.

In accordance with another method of binding to the antigen can be restored in the process of humanization of antibodies through the selection of running coupled hypervariable regions (see application No. 11/061841, filed February 18, 2005). This method includes the introduction of nonhuman hypervariable regions in frame acceptor sequence, and then introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor skeleton of the sequence. Alternative the introduction of one or NESCO�kih amino acid substitutions may be accompanied by a modification of the acceptor in the frame sequence.

In accordance with another method library can be obtained by constructing sets of upstream and downstream oligonucleotides, where each of these sets has a plurality of oligonucleotides with different sequences, formed by a series of codons that are present in the sequence of the oligonucleotides. The sets of upstream and downstream oligonucleotides, together with the sequence of the matrix nucleic acid of the variable domain can be used in polymerase chain reaction with the formation of libraries of PCR products. PCR products can be called "clusters of nucleic acids" because they can be attached to other related or unrelated to nucleic acid sequences, for example, to proteins of the viral envelope and the dimerization domains, using well-developed methods of molecular biology.

The sequence of the PCR primers includes one or more constructed sets of codons in positions accessible to solvent, and in various positions in a wide range hypervariable region. As described above, the set of codons is a set of different sequences of nucleotide triplets are used to encode the desired variants of amino acids.

Suitable anti�La, meet the necessary criteria, and selected through the appropriate stages of screening/selection can be extracted and cloned standard recombinant methods.

It is also important that humanized antibodies retain high affinity binding to antigen and other desired biological properties. To achieve this objective, in accordance with the preferred method, humanized antibodies are obtained by analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional model of the immunoglobulin are publicly available and known in the art. There are also computer programs for illustration and representation of the probable three-dimensional combinatorial structures of selected sequences of the candidate immunoglobulin. The study of this representation allows us to consider the likely role of these residues in the functioning sequence of the candidate immunoglobulin, that is, to analyze the influence of these residues on the ability of the immunoglobulin candidate to contact the antigen. In this method, FR residues can be selected from sequences of the recipient and import sequences and combined �AK, to obtain an antibody with the desired properties, such as increased affinity for the antigen(s) target(s). Basically, immediate and greatest impact on binding to the antigen have the remains of the hypervariable region.

Also examines various forms of a humanized anti-CD79b antibody. For example, the humanized antibody may be an antibody fragment, such as Fab, which kongugiruut, but not necessarily, with one or more cytotoxic agents with education immunoconjugate. Alternative humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

Alternatively, humanization can be obtained from human antibodies. For example, at the present time can be obtained from transgenic animals (e.g., mice) that after immunization will be capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin produced. So, for example, indicated that the homozygous deletion of the gene in the region of the junction of the heavy chain of the antibody (JH) in chimeric mice and in mice with a mutated germ line results in complete inhibition of endogenous production of antibodies. Transfer of arrays of genes of human immunoglobulin germ line of such mice with m�targeted germ line leads to the production of human antibodies after injection of antigen. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggemann et al.,Year in Immuno.7:33 (1993); U.S. patents NN 5545806, 5569825, 5591669 (all from GenPharm); 5545807; and WO 97/17852.

Alternative technology phage submission (McCafferty et al.,Nature348:552-553 [1990]) can be used for producing human antibodies and fragments of antibodies in vitro from sets of genes of variable domain immunoglobulin (V) from unimmunized donors. In accordance with this method, the genes of domain V of antibodies cloned with preservation of the reading frame in either the major or minor gene of the protein shell of filamentous phage, such as M13 or fd, and present on the surface ragovoy particles as functional fragments of the antibodies. Because filamentous particle contains a single-stranded copy of the DNA genome of the phage, the selection is conducted on the basis of the functional properties of antibodies, also allows you to select the gene encoding the antibody with these properties. Thus, the phage mimics some properties of b-cells. Phage representation can be implemented in a variety of formats, described in the publications Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology3:564-571 (1993). For phage representation can be used several sources of V-gene segments. In the publication Clackson et al.,Nature, 352:624-628 (1991) described the allocation of various arrays antileprotic of oxazolone from a small randomised combinatorial library of V genes obtained from the spleen of immunized mice. Can be also designed a set of V genes from unimmunized human donors can be obtained antibodies against a diverse array of antigens (including autoantigens), mainly the methods described in the publications Marks et al.,J. Mol. Biol.222:581-597 (1991), or Griffith et al.,EMBO J.12:725-734 (1993). See, also U.S. patent Nos. 5565332 and 5573905.

As discussed above, human antibodies may also be produced using in vitro activated b cells (see, also U.S. patent NO. 5567610 and 5229275).

4.Fragments of antibodies

In some cases it is desirable to use non-antibodies, and fragments thereof. The smaller size of the fragments allows for rapid clearance, which can improve access to solid tumors.

To obtain fragments of antibodies have been developed various methods. Traditionally, these fragments are formed as a result of proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods24:107-117 (1992) and Brennan et al.,Science, 229:81 (1985)). However, these fragments are produced directly by recombinant cell host. Fab, Fv and scFv fragments of antibodies can be expressed in E. coli and secretariats from E. coli, which facilitates the production of these fragments in large quantities. fragments of antibodies can�t be isolated from phage libraries of antibodies, discussed above. Alternative Fab'-SH fragments can be directly isolated from E. coli and chemically linked to form F(ab')2-fragments (Carter et al.,Bio/Technology10:163-167 (1992)). According to another approach, F(ab')2-fragments can be isolated directly from a culture of recombinant host cell. Fab and F(ab')2-a fragment with a longer half-life in vivo, which contain the residue that binds to the epitope of the receptor "salvation" described in U.S. patent No. 5869046. Specialists and other known methods of obtaining fragments of antibodies. In other embodiments of the invention, the preferred antibody is a single chain Fv fragment (scFv). Cm. WO 93/16185, U.S. patent No. 5571894 and U.S. patent No. 5587458. Fv and sFv fragments are present only in species with intact combined sites that do not contain constant regions, and therefore they are suitable to reduce the level of nonspecific binding in their application in vivo. Can be designed hybrid sFv-proteins with obtaining hybrid effector protein at the amino - or carboxy-end of the sFv. SeeAntibody Engineering, ed. Borrebaeck, see above. The antibody fragment may also be “single-chain antibody”, for example, the antibody described in U.S. patent No. 5641870. Such single-stranded fragments of antibodies can be monospecifičeskoj or bespecifically.

5. B�specific antibodies

Bespecifically antibodies are antibodies that have the binding specificity of at least two different epitopes. Representative bespecifically antibodies can bind to two different epitopes of the protein CD79b described in the present application. Other such antibodies may be a combination of CD79b-binding site with a binding site for other proteins. Alternatively, a branch of antibodies against CD79b can be combined with the branch that is associated with the triggering molecule on a leukocyte such as a molecule T-cell receptor (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so that the cellular protective mechanisms have been focused CD79b-expressing cells and localized in these cells. Bespecifically antibodies can also be used to determine localize cytotoxic agents to cells expressing CD79b. These antibodies have CD79b-connecting branch and the branch, which is associated with a cytotoxic agent (such as, for example, saporin, an antibody against interferon-α, vinylchlorid, a chain of ricin, methotrexate or hapten labeled with a radioactive isotope). Bespecifically antibodies can be obtained in the form of full-size antibodies or fragments of antibodies (e.g., bespecifically F(ab')2-antibodies).

In WO 96/16673 described bespecifically anti-ErbB2/anti-FcγRIII antibody and U.S. patent No. 5837234 described bespecifically anti-ErbB2/anti-FcγRI antibody. Bespecifically anti-ErbB2/Fcα antibody is described in WO98/02463. In U.S. patent No. 5821337 described bespecifically anti-ErbB2/anti-CD3 antibody.

Methods of obtaining bespecifically antibodies known in the art. Traditional production of full-size bespecifically antibodies is based on the coexpression of two pairs of heavy chain-light chain immunoglobulin, where the two chains have different specificity (Millstein et al.,Nature, 305:537-539 (1983)). These hybridomas (quadroma), due to the randomized set of heavy and light chains of immunoglobulin, produce a potential mixture of 10 different antibody molecules, of which only one molecule has the “right” bespecifically structure. Cleaning such a “right” of the molecule, which is usually carried out by stepwise conducting affinity chromatography, provides a challenge and gives a low yield of product. A similar procedure is described in WO 93/08829 and publication in Traunecker et al., EMBO J., 10:3655-3659 (1991).

In accordance with another approach, the variable domains of the antibodies with the desired specificnosti binding (combined sites “antibody-antigen”) is attached to the sequences of the constant domain of immunoglobulin. This �prisoedinenie is preferably carried out with a constant domain of the heavy chain Ig, containing at least a portion of a hinge region, CN2 and pN3. In this case it is preferable that this hybrid had the first constant region of the heavy chain (CN1) containing the site necessary for binding to the light chain is present in at least one of the hybrids. DNA encoding the hybrid heavy chain immunoglobulin and, if necessary, the light chain of the antibody, inserted into separate expression vectors, and cotransfected in a suitable host cell. This provides a high degree of flexibility when adjusting the ratios of the three polypeptide fragments in embodiments of the invention, in which the unequal content of the three polypeptide chains used in this design give optimal outputs need especifismo antibodies. However, you can embed the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal proportions gives a high output, or if such ratios do not have significant effect on the output of the combination circuit.

In a preferred embodiment of this approach, bespecifically antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one branch, and hybrid pairs of heavy� chain-light chain immunoglobulin (providing a second binding specificity), in the other branch. It was found that this asymmetric structure facilitates the separation of desired especifismo connections from unwanted combinations of immunoglobulin chains, because the presence of light chain immunoglobulin in only one half bespecifically molecules provides an easy way of his selection. This method is described in WO 94/04690. A more detailed description of obtaining bespecifically antibodies see, for example, Suresh et al.,Methods in Enzymology, 121:210 (1986).

In accordance with the second approach, described in U.S. patent No. 5731168, the boundary between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers isolated from recombinant cell culture. This border preferably contains at least a portion of the domain WithN3. In this method a small side chains of one or several amino acids in the edge region of the first antibody molecule replaced larger side chains (e.g. tyrosine or tryptophan). In the edge region of the second molecule antibodies create compensatory "cavities" that are identical or similar to the size of the larger(s) side(s) chain(s), by replacing the large side chains of amino acids smaller side chains (e.g., alanine or threonine). This allows to increase the yield of heterodimers on to other unwanted end-products, such as homodimer.

Bespecifically antibodies include cross-linked antibodies or their “heteroconjugate”. For example, one of the antibodies in the specified heteroconjugate may be associated with Avidya and the other with Biotin. Such antibodies have, for example, proposed for the delivery of immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Antibodies-heteroconjugate can be obtained by any standard methods of cross-stitching. Suitable cross-linking agents are well known in the art and described in U.S. patent No. 4676980 along with various methods of cross-stitching.

Methods of producing bespecifically antibodies of fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained by chemical binding. In the work of Brennan et al.,Science, 229:81 (1985) describes a procedure wherein intact antibodies are subjected to proteolytic cleavage with the formation of F(ab')2-fragments. These fragments regenerate in the presence of the agent, forming aitiology complex, such as sodium arsenite, to stabilize the adjacent dithiolo and prevent the formation of intermolecular disulfide bonds. Then, the resulting Fab'-fragments are converted into derivatives of dinitrobenzoate (TNB).After that, one of the derivatives of Fab'-TNB again converted into Fab'-thiol by restoring mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB, resulting in bespecifically antibody. Such produced bespecifically antibodies can be used as agents for the selective immobilization of enzymes.

Recent advances in this field allow direct selection of E. coli fragments, Fab'-SH, which can be chemically linked to form bespecifically antibodies. In the work of Shalaby et al.,J. Exp. Med. 175:217-225 (1992) describes the production of a fully humanized molecule F(ab')2especifismo antibodies. Each Fab'fragment was separately secreted from E. coli and subjected to direct chemical binding in vitro with the formation of especifismo antibodies. Thus, the obtained bespecifically antibody has the ability to bind to cells, sverkhekspressiya the ErbB2 receptor and normal human T-cells and also triggers the lytic activity of human cytotoxic lymphocytes against the tumor target human breast cancer.

Were also described various methods of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, bespar� - cific antibodies have been produced using “latinovich lightning. Kostelny et al.,J. Immunol., 148(5):1547-1553 (1992). Peptides latinboy lightning, originating from proteins Fos and Jun, were attached to the Fab'-parts of two other antibodies by ligating genes. Homodimeric antibodies were restored in the hinge region with the formation of monomers and then re-oxidized with the formation of heterodimers of antibody. This method can also be used for the production of homodimeric antibodies. Technology “dentical” described by Hollinger et al.,Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993), provides an alternative mechanism for obtaining fragments especifismo antibodies. These fragments contain VHattached to VLby means of a linker that is too short for pairing between the two domains on the same chain. In accordance with this VLand VHdomains of one fragment are forced to pair with the complementary VLand VH-domains of another fragment, thereby forming two antigen-binding site. Was also described another strategy to obtain fragments especificacao antibodies using single-chain Fv(sFv) dimers. Cm. Gruber et al.,J. Immunol., 152:5368 (1994).

Also considers the antibodies with more than two valencies. So, for example, can be obtained and thespecification antibodies. Tutt et al.,J. Immunol.147:60 (1991).

6. Heteroconjugate antibodies/p>

Heteroconjugate antibodies also included in the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies, for example, assumed to target immune system cells to unwanted cells [U.S. patent No. 4676980], and therefore they can be used for the treatment of HIV infection [WO 91/00360, WO 92/200373 and EP 03089]. It is known that antibodies can be produced in vitro by the methods of synthesis of proteins, including methods using a cross-cross-linking agents. For example, immunotoxins can be constructed by carrying out the reaction of disulfide exchange or the formation of thioether linkages. Examples of suitable reagents for this purpose are aminothiols and methyl-4-mercaptopyrimidine, and such reagents are described, for example, in U.S. patent No. 4676980.

7. Polyvalent antibodies

Multivalent antibody may be faster internalizacao (and/or it may be faster catabolism) than bivalent antibody with antigen expression in the cell bound to the antibody. Antibodies according to the invention can be multivalent antibodies (which do not belong to the IgM class) with three or more antigen-binding sites (e.g. tetravalent antibodies), which can be easily produced by recomb�Nantou expression of the nucleic acid encoding the polypeptide chain of the antibody. Multivalent antibody may contain the dimerization domain and three or more antigen-binding sites. The preferred dimerization domain comprises (or consists of) an Fc-region or a hinge region. In this case, the antibody may contain an Fc region and three or more antigen-binding sites from amino end to the Fc-region. Described here is the preferred multivalent antibody comprises (or consists of) three to about eight antigen-binding sites, and preferably four antigen-binding site. Multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the specified(s) polypeptide(s) chain(s) contains(at) two or more variable domains. For example, the polypeptide(s) chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide(s) chain(s) may contain: chain VH-CH1-flexible linker-VH-CH1-Fc region"; or chain VH-CH1-VH-CH1-Fc region". The polyvalent antibody according to the invention also, �predpochtitelno, contains at least two (and preferably four) of the polypeptide variable domain of the light chain. Described here multivalent antibody may, for example, contain from about two to eight polypeptides variable domain of the light chain. These polypeptides variable domain of the light chain contains a variable domain light chain as well, but not necessarily, the domain is CL.

8. The design of antibodies with effector functions

It may be desirable to modify the antibody according to the invention in order to impart effector functions, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc-region of an antibody. Alternative or additionally, cysteine(C) residue(s) may be introduced(s) in the Fc-region that will lead to the formation of messagewith disulfide bonds in this region. Thus, the obtained homodimeric the antibody may possess enhanced ability to internalize and/or increased capacity for complement-mediated cytolysis of cells and increased antibody-dependent cellular cytotoxicity (ADCC). Cm. Caron et al.,J. Exp Med.176:1191-1195 (1992) and Shopes, B.J. Immunol.148:2918-2922 1992). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained using heterobifunctional cross-cross-linking agents described in the publication of Wolff et al.,Cancer Research53:2560-2565 (1993). Alternative can be constructed antibody having two Fc-region, resulting in can be enhanced complement lysis and enhanced ADCC. Cm. Stevenson et al.,Anti-Cancer Drug Design3:219-230 (1989). To increase the half-life of antibodies in serum, the specified antibody (and in particular, in its fragment) may be inserted epitope that binds to a receptor "salvation", for example as described in U.S. patent No. 5739277. As used herein, the term "epitope that binds to a receptor "salvation" means an epitope of the Fc-region of IgG molecules (e.g., IgG1, IgG2, IgG3or IgG4), which is responsible for increasing the half-life of IgG molecules in serum in vivo.

9.Immunoconjugate

The present invention also relates to immunoconjugates (which are synonyms of the terms "conjugate antibody-drug" or "ADC"), comprising the antibody, anywhereman with a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or LM�now one of origin or its fragments), or a radioactive isotope (i.e. radioactive conjugate).

In some embodiments of the invention immunoconjugate contains the antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents used to obtain these immunoconjugates described above. Enzymatically active toxins and fragments thereof which can be used for these purposes are the A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin a-chain, exotoxin (from Pseudomonas aerugiNOa), A-chain of ricin a-chain abrina And-chain medecine, alpha sarcin, proteins Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of Momordica charantia, Curtin, krotin, inhibitor Sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, anomity and trichothecene. For the production of radioactively conjugated antibody can be used by various radionuclides. Examples of such radionuclides are212Bi131I,131In90Y and186Re. Conjugates of the antibody and cytotoxic drugs are made using a variety of bifunctional protein-binding agents, such as N-Succinimidyl-3-(2-pyridylthio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-azido�of unity (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, immunotoxin ricin can be obtained as described in the publication Vitetta et al.Science, 238:1098 (1987).14C-labeled 1-isothiocyanatobenzene-3- acid (MX-DTPA) is representative chelating agent for conjugating radionuclide to the antibody. Cm. WO 94/11026.

The present invention also addresses the conjugates of the antibody and one or more low molecular weight toxins, such as calicheamicin, auristatin peptides, such as monomethylaniline (MAE)(a synthetic analog of dolastatin) maytansinoid, such as DM1, trichoton and S, and the derivatives of these toxins having a toxic activity.

Representative immunoconjugate - conjugate "antibody-drug"

Immunoconjugate (or "conjugate antibody-drug" ("ADC")) according to the invention can be immunoconjugate formula I, shown below, where the antibody conjugative (i.e., covalently linked) with one or more molecules of the drug (D) through, but not necessarily, the linker (L). The ADC may include conjugates "thio-Mab-Lek�rstone ("TDC").

Ab-(L-D)pI

Accordingly, the antibody may be anywhereman with a drug, either directly or through a linker. In formula I R is a weighted average of the number of molecules of the drug to the antibody, where the specified number may be, for example, from about 1 to 20 molecules of the drug to the antibody, and in some embodiments of the invention from 1 to about 8 drug molecules to the antibody. The present invention relates to compositions containing a mixture of compounds is an antibody-drug of formula I, where the average load of the drug to the antibody is approximately 2-5 or 3-4.and.

Representative linkers

The linker may contain one or more linker components. A representative of the linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit), alanine-phenylalanine ("ala-phe"), p-aminobenzeneboronic ("PAB"), and components resulting from conjugation with linker reagents: N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", also denoted here "MCC") and N-Succinimidyl-(4-�odacity)aminobenzoate ("SIAB"). Experts know the various linker components, some of which are described below.

The linker may be a "contain no cleavable linker" facilitating release of the drug in the cell. So, for example, can be used linkers that are sensitive to the effects of acid (e.g., hydrazon); linkers that are sensitive to the action of proteases (e.g. peptidases); photochemically unstable linkers; dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. patent No. 5208020).

In some embodiments of the invention, the linker represented by the following formula II:

-Aa--Ww--Yy-,

where a represents the extension component;andinteger 0 or 1; W is an amino acid; w independently represents an integer from 0 to 12; Y means the GS spacer component; y is 0, 1 or 2, and Ab, D, and p are defined above for formula I. Representative variants of such linkers described in the application U.S. 2005-0238649 A1, which in its entirety is introduced into the present description by reference.

In some embodiments of the invention, the linker component may contain "lengthening component that binds the antibody with a different linker component or molecule drugs. A representative of the extension components is presented below (where waves�flock line indicates sites of covalent binding with the antibody):

In some embodiments of the invention, the linker component may contain amino acid component. In one such amino acid variants of the invention component provides the cleavage of the linker by the protease, which facilitates the drug release from immunoconjugate after it is processed by intracellular proteases, such as lysosomal enzymes. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784. Representative amino acid components include, but are not limited to, dipeptide, Tripeptide, tetrapeptide and Pentapeptide. Representative dipeptides are valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-val-cit). Representative tripeptides are glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid component may contain natural amino acid residues, as well as minor amino acids and non-natural amino acid analogs, such as citrulline. Amino acid components can be designed and optimized in their selectivity against fermentativnogo cleavage by specific enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or plazminovoj protease.

In some embodiments of the invention, the link�RNA component may contain "GS spacer" component, which antibody binds to a molecule of the drug, either directly or through the extension of the component and/or amino acid component. The GS spacer component may be "carolinensis" or "nesamierinamais" component. "Nesamierinamais" GS spacer component is a component where a portion of the GS spacer component or all of this component remain associated with a molecule of the drug after enzymatic (e.g., proteolytic) cleavage of the ADC. Examples nesmolkayuschee GS spacer components are, but not limited to, glycine GS spacer component and glycine-glycine GS spacer component. Also consider other combinations of peptide spacers-sensitive sequence-specific enzymatic cleavage. So, for example, enzymatic digestion ADC containing a glycine-glycine GS spacer component, a protease associated with tumor cells, will lead to the release of a molecule "glycine-glycine-drug" from the rest of the ADC. In one of these options a molecule of glycine-glycine-drug undergoes a one-step hydrolysis in the tumor cell, which leads to cleavage of the glycine-glycine GS spacer component from pier�Kula medicines.

"Carolinensis" GS spacer component provides the release of a molecule medicines without carrying out single-stage hydrolysis. In some embodiments of the invention, the GS spacer linker component contains p-aminobenzyl group. In one such options p-aminobenzoyl alcohol attached to the amino acid component through the amide bond, carbamate, methylcarbamate, or a carbonate formed by the reaction of interaction of benzyl alcohol with a cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment of the invention the GS spacer component is p-aminobenzeneboronic (PAB). In some embodiments of the invention, the phenylene portion of a p-aminobenzyl group is substituted with Qm where Q represents C1-C8alkyl, O-(C1-C8alkyl), halogen, nitro or cyano; m is an integer from 0 to 4. Other examples carolinensis GS spacer components are, but not limited to, aromatic compounds that, by their electronic properties similar to p-aminobenzoylamino alcohol (see, for example, request US 2005/0256030 A1), such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para-aminobenzoate. Can spacers be used that undergo cyclization after hydro�iza amide bond, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo ring[2.2.1] and bicyclo[2.2.2]-system (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and amide 2-aminophenylamino acid (Amsberry, et al. (1990) J. Org. Chem. 55:5867). Examples carolinensis spacers used in the ADC, are amine-containing medications, substituted in α-position of glycine (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

In one embodiment of the invention specified the GS spacer element below is a branched bis(gidroximetil)styrene (BHMS), which can be used to incorporate and release multiple drugs and which has the structure:

,

where Q represents C1-C8alkyl, O-(C1-C8alkyl), halogen, nitro or cyano; m is an integer from 0 to 4; n is 0 or 1; and p is the number from 1 to about 20.

In another embodiment of the invention the linker L may be a dendritic linker type used for covalent binding more than one molecule of the drug with the antibody through branching multifunctional linker molecules (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar attributed�e of the drug to the antibody, that is the load that corresponds to the efficiency of the ADC. Thus, if constructed on the basis of cysteine antibody contains only one reactive thiol group of cysteine, through a dendritic linker can be attached to a large number of molecules of the drug.

A representative of the linker components and their combinations are presented below for the ADC of formula II:

Linker components, including lengthening, GS spacer and amino acid components, can be synthesized by methods known in the art, for example, by methods described in the application US 2005-0238649 A1.

b. A representative molecule drug

(1) Maytansine and maytansinoids

In some embodiments of the invention immunoconjugate contains antibody, anywhereman with one or more molecules maytansinoid. Maytansinoid are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from East African shrews Maytenus serrata (U.S. patent No. 3896111). Then it was discovered that certain microbes also produce maytansinoids, such as maytansine and esters of p-3-maytansine (U.S. patent No. 4151042). Synthetic maytansine� and its derivatives and analogues described, for example, in U.S. patents№№ 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533.

Molecules maytansinoid medicines are attractive molecules of medicines for use in the conjugates of the antibody-drug" because they (i) can be relatively easily obtained by fermentation or chemical modification or derivatization products of fermentation, (ii) are suitable for derivatization with functional groups suitable for conjugation through the joining disulfide and ridiculing of linkers to antibodies, (iii) are stable in plasma, and (iv) are effective against various tumor cell lines.

Maytansine compounds that can be used as maytansinoids medicines, are well known in the art and can be isolated from natural sources by known methods, or they can be obtained by methods of genetic engineering and fermentation (US 6790952; US 2005/0170475; Yu et al. (2002) PNAS 99:7968-7973). Maytansine and its analogs can also be obtained by known methods of synthesis.

Representative maytansinoid drugs drugs are those having a modified aromatic ring, such kaaks-19-dechloro (U.S. patent 4256746)(obtained by reduction ansamitocins P2 case lithium); C-20-hydroxy (or C-20-desmethyl) +/-C-19-Deshler (U.S. patents Nos. 4361650 and 4307016) (obtained by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20 dermatox, C-20-acyloxy (-OCOR), +/-dechloro (U.S. patent No. 4294757)(obtained by acylation using acylchlorides), and molecules having modifications at other positions.

Representative molecules maitaining medicines are molecules having modifications such as: C-9-SH (U.S. patent 4424219)(obtained by reaction between maytansine with H2S or P2S5); C-14-alkoxymethyl(dermatox/CH2OR)(U.S. patent 4331598); C-14-gidroximetil or acyloxymethyl (CH2OH or CH2OAc)(U.S. patent 4450254)(obtained from Nocardia); C-15-hydroxy/acyloxy (U.S. patent 4364866)(obtained by transformation maytansine under the action of Streptomyces); C-15-methoxy (U.S. patent Nos. 4313946 and 4315929)(isolated from Trewia nudlflora); C-18-N-desmethyl (U.S. patents Nos. 4362663 and 4322348)(obtained by demethylation maytansine under the action of Streptomyces); and 4,5-deoxy (U.S. patent 4371533)(obtained by the reduction of maytansine the titanium trichloride/LAH).

It is known that many of the provisions in maytansinoid compounds, depending on the type of link that can be used as the position of the accession. For example, for images�of ester bonds, particularly suitable are the C-3 position having a hydroxyl group, C-14 position modified with gidroximetil, C-15 position modified hydroxyl group, and C-20 position having a hydroxyl group (US 5208020; US RE39151; US 6913748; US 7368565; US 2006/0167245; US 2007/0037972).

Molecules maytansinoid medicines are molecules having the structure:

,

where the wavy line indicates the covalent binding of the sulfur atom of the molecule maytansinoid drug and the linker of the ADC. R can independently represent H or C1-C6alkyl. Alkalinous chain linking amide group to the sulfur atom, can be metanil, etanol or propyl, i.e., where m is 1, 2 or 3 (US 633410; US 5208020; US 7276497; Chari et al. (1992) Cancer Res. 52:127-131; Liu et al. (1996) Proc. Natl. Acad. Sci USA 93:8618-8623).

In the present description addresses all of the stereoisomers of molecules maytansinoid drug compounds according to the invention, i.e. any combination of R - and S-configurations at the chiral carbons of D. In one embodiment of the invention, the molecule maytansinoid medicines has the following stereochemical structure:

.

Representative embodiments of the molecules maytansinoid medicines are: DM1, DM3 and DM4, having the structures:

,

where the wavy line indicates the covalent binding of the sulfur atom of the molecule of the drug with a linker (L) conjugate "antibody-drug" (WO 2005/037992; US 2005/0276812 A1).

Other representative conjugates "maytansinoid-antibody-drug" have the following structure (where Ab represents the antibody, and p is from 1 to about 8):

Representative conjugates ""antibody-drug", where DM1 is attached through a BMPEO linker to a thiol group of the antibody have the following structure and symbols:

,

where Ab is an antibody; n is 0, 1 or 2, and p is 1, 2, 3, or 4.

Immunoconjugate containing maytansinoid, methods for their preparation and their therapeutic use are described, for example, Erickson, et al. (2006) Cancer Res. 66(8):4426-4433; in U.S. patents Nos. 5208020, 5416064, in the application US 2005/0276812 A1 and in European patent EP V, which fully entered into the present application by reference.

Conjugates of an antibody-maytansinoid” is produced by chemical binding of the antibody to the molecule maytansinoid, where the specified binding does not lead to a significant reduction of the biological activity of the antibodies or molecules mA�casinoid. See, for example, U.S. patent No. 5208020 (which in its entirety is introduced into the present description by reference). Maytansinoid can be synthesized by known methods, or they can be isolated from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 and in other patents and non-patent publications described above, and such maytansinoids are maytansine and analogues maytansine with modifications in the aromatic ring or at other positions of the molecule maytansine, such as various esters maytansine.

To create conjugates of an antibody-maytansinoid" can be used a variety of linker groups, known in the art, including, for example, the groups described in U.S. patent No. 5208020 or in European patent EP V, and in the publication of Chari et al., Cancer Research 52:127-131 (1992) and in the application for U.S. patent US 2005/016993 A1, which is fully introduced into the present description by reference. Conjugates of an antibody-maytansinoid" containing linker component SMCC may be prepared as described in application for U.S. patent US 2005/0276812 A1, in the section "Conjugates of the antibody-drug means and methods". Linker groups are disulfide groups, thioether groups, groups that are sensitive to the effects of acid photochemically unstable group, groups, sensitivity�date to the action of peptidases, or group that is sensitive to the action of esterase described in the aforementioned patents. Additional linker groups are described and illustrated in this application.

Conjugates of an antibody-maytansinoid" can be obtained using a variety of bifunctional protein-binding agents, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). In some embodiments of the invention binding agents to create disulfide bonds are N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) or N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP).

The linker can be attached to the molecule maytansinoid in different positions, depending on the type of communication. For example, the ester linkage may be formed by reaction with a hydroxyl group standartisierte binding. This reaction can be carried out in position C-3 having a hydroxyl group in position C-14, modified gidroximetil, a-15, a modified hydroxyl group, and in position C-20, with a hydroxyl group. In one embodiment of the invention the coupling is formed at the position C-3 maytansine or equivalent.

(2) Auristatin and dolastatin

In some embodiments of the invention immunoconjugate contains antibody, anywhereman with dolastatin or peptide analogue of dolastatin or their derivatives, for example, auristatin (U.S. patents Nos. 5635483, 5780588). It was found that dolastatin and auristatin negatively affect the dynamics of the formation of microtubules, GTP hydrolysis, and division of nuclei and cells (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anti-cancer (U.S. patent 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Molecules dolastatins or auristatin medicines can be attached to the antibody N-(amino)-end-or P-(carboxymethyl)-end of the molecule peptide drugs (WO 02/088172).

Representative embodiments of auristatin are molecules medicines containing attached to the N end of monomethylaniline, DE and DF (application US 2005/0238649 described by Senter et al., Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented March 28, 2004, such application is fully introduced into the present description by reference).

A peptide molecule of the drug may be selected from compounds of formulas (DEand DFpresented below:

,

where the wavy line in DEand DFindicates the site of covalent attachment to the antibody or to a component of the antibody-linker;

and at each position, independently:

R2selected from H and C1-C8alkyl;

R3selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkyl-aryl, C1-C8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkyl-aryl, C1-C8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

or R4and R5taken together form a carbocyclic ring and have the formula -(CRaRb)n-, where Raand Rbindependently selected from H, C1-C8the alkyl and C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R6selected �W H and C 1-C8alkyl;

R7selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkyl-aryl, C1-C8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

each R8independently selected from H, OH, C1-C8alkyl, C3-C8carbocycle and O-(C1-C8alkyl);

R9selected from H and C1-C8alkyl;

R10selected from aryl or C3-C8heterocycle;

Z represents O, S, NH or NR12where R12represents C1-C8alkyl;

R11selected from H, C1-C20of alkyl, aryl, C3-C8heterocycle, -(R13O)m-R14or -(R13O)m-CH(R15)2;

m is an integer of 1-1000;

R13represents C2-C8alkyl;

R14represents H or C1-C8alkyl;

R15in each case independently represents H, COOH, -(CH2)n-N(R16)2, -(CH2)n-SO3H or -(CH2)n-SO3-C1-C8alkyl;

R16in each case independently represents H, C1-C8alkyl or -(CH2)n-COOH;

R18selected from-C(R8)2-C(R8)2-aryl, -C(R8)2-C(R8)sub> 2-(C3-C8heterocycle), and-C(R8)2-C(R8)2-(C3-C8carbocycle); and

n is an integer from 0 to 6.

In one embodiment of the invention R3, R4and R7independently represent an isopropyl or sec-butyl, and R5represents-H or methyl. In a preferred embodiment of the invention, each of R3and R4represents isopropyl, R5represents-H, and R7represents sec-butyl.

In another embodiment of the invention, each of R2and R6is methyl, and R9represents-H.

In yet another embodiment of the invention R8in each case represents-OCH3.

In a representative embodiment of the invention, each of R3and R4represents isopropyl, each of R2and R6is methyl, R5represents-H, R7represents sec-butyl, R8in each case represents-OCH3and R9represents-H.

In one embodiment of the invention, Z is-O - or-NH-.

In one embodiment of the invention R10represents aryl.

In a representative embodiment of the invention R10represents phenyl.

In a representative embodiment of the invention, if Z represents-O-, R represents-H, methyl or tert-butyl.

In one embodiment of the invention, when Z is-NH, R11represents-CH(R15)2where R15represents -(CH2)n-N(R16)2and R16represents-C1-C8alkyl or -(CH2)n-COOH.

In another embodiment of the invention, when Z is-NH, R11represents-CH(R15)2where R15represents -(CH2)n-SO3H.

Representational option of auristatin formula DEis MMAE, where the wavy line indicates the covalent binding of the conjugate "antibody-drug" with a linker (L):

Representational option of auristatin formula DFis MMAF, where the wavy line indicates the covalent binding of the conjugate "antibody-drug" with a linker (L) (see US application 2005/0238649 and publication Doronina et al. (2006) Bioconjugate Chem. 17:114-124):

In other representative embodiments are monomethylamine compounds having phenylalaninamide-modification at C-end of the Pentapeptide auristatin medicines (WO 2007/008848), and monomethylamine compounds having phenylalanine modification of the side chain at C-end of the Pentapeptide and�restating medicines (WO 2007/008603).

Other molecules of the drug are the following MMAF derivatives, where the wavy line indicates the covalent binding of the conjugate "antibody-drug" with a linker (L):

In one aspect of the invention, the hydrophilic groups are, but are not limited to, esters of triethyleneglycol (TEG), presented above, which can be attached to a molecule of the drug in R11. Not limited to any particular theory, we can only note that the hydrophilic groups enhance the internalization of molecules of the drug and prevent its agglomeration.

Representative versions of the ADC of formula I containing auristatin/dolastatin or their derivatives described in the application US 2005-0238649 and publication in Doronina et al. (2006) Bioconjugate Chem. 17:114-124, which fully entered into the present description by reference. Representative versions of the ADC of formula I containing MMAE or MMAF and various linker components have the following structure (where “Ab” is an antibody; p is from 1 to about 8, “Val-Cit” or “vc” is a dipeptide valine-citrulline, and “S” represents a sulfur atom). It should be noted that in some descriptions of the structure of the ADC is connected�about with the sulfur atom, the antibody is designated "Ab-S", which indicates only the bond with the sulfur atom, but does not indicate what specific sulfur atom attached to several molecules of the linker-drug". In the following structures of the bracket on the left can also be set to the left of the sulfur atom Ab and S, and such entry will be equivalent to the formula for the ADC according to the invention presented in this description.

Representative versions of the ADC of formula I containing MMAF and various linker components include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. It is interesting to note that immunoconjugate containing MMAF attached to an antibody through a linker, which undergoes proteolytic cleavage, have activity comparable to the activity immunoconjugates containing MMAF attached to an antibody through proteoliticeski degradable linker. See, Doronina et al. (2006) Bioconjugate Chem. 17:114-124. In such cases, the drug release, obviously, is due to the decomposition of the antibody in the cell. Cm. below.

Typically, the peptide molecules of the drug can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be formed, for example, by the method of synthesis in the liquid phase (see E. Schröder K. nd Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known to specialists in the field of peptide synthesis. Molecules auristatin/dolastatin medicines can be obtained by methods described in the application U.S. US 2005-0238649 A1; U.S. patents №№ 5635483; No. 5780588; in publications Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

In particular, molecules auristatin/dolastatin medicines formula DFsuch as MMAF and derivatives thereof, can be obtained by methods described in the application U.S. 2005-0238649 A1 and the publication Doronina et al. (2006) Bioconjugate Chem. 17:114-124. Molecules auristatin/dolastatin medicines formula DEsuch as MMAE and their derivatives, can be obtained by methods described in the publication Doronina et al. (2003) Nat. Biotech. 21:778-784. Molecule "drug-linker" MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be suitably synthesized by routine methods, e.g., as described in the publication Doronina et al. (2003) Nat. Biotech. 21:778-784, and in the publication of patent application No. US 2005/0238649 A1, and then they can be anywhereman with interest the antibody.

(3) Calicheamicin

In other embodiments of the invention immunoconjugate contains antibody, anywhereman with one or more Molek�Lamy calicheamicin. The antibiotics of the family of calicheamicin capable of producing double-stranded DNA breaks in subpicomolar concentrations. Description of obtaining conjugates collection calicheamicin can be found in U.S. patents№№ 5712354, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all patents owned by the company American Cyanamid Company). Structural analogues calicheamicin, which can be used for this purpose include, but are not limited to, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θ1I(see Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents owned by the company American Cyanamid). Another anticancer drug, which can be anywhereman antibody is a means QFA, which is an antifolate. Calicheamicin and QFA have intracellular active sites and have difficulty passing through the plasma membrane. Therefore, the absorption of these agents cells by internalization, mediated by antibody leads to significant enhancement of their cytotoxic effects.

C. Other cytotoxic funds

Other antitumor agents that can be anywhereman with the antibody, are BCNU, streptozocin, vincristine and 5-fluorouracil, the family of agents known collectively set�KS LL-E, described in U.S. patents №№ 5053394, 5770710 and espiramicina (U.S. patent No. 5877296).

Enzymatically active toxins and fragments thereof which can be used for this purpose are the A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin a-chain, exotoxin (from Pseudomonas aerugiNOa), A-chain of ricin a-chain abrina And-chain medecine, alpha sarcin, proteins Aleurites fordii, protein diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of Momordica charantia, Curtin, krotin, inhibitor Sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, anomity and trichothecene. See, for example, the application WO 93/21232, published 28 October 1993

The present invention also addresses immunoconjugate formed by the antibody and a compound having nucleotidase activity (e.g. a ribonuclease or DNA endonuclease such as a deoxyribonuclease; Dnazol).

In some embodiments of the invention immunoconjugate may contain highly radioactive atom. For the production of radioactively conjugated antibody can be used by various radioactive isotopes. Examples of such radionuclides are211At,131I,125I,90Y186Re,188Re,153Sm212Bi32P,212Pb and radioactive isotopes of Lu. If the specified immunoconjugate is used for detection, it mo�et to contain a radioactive atom for scintigraphic studies, for example,99mTc or123I, or a spin label for imaging by nuclear magnetic resonance (NMR) spectroscopy (also known as Mr-visualization, MRR), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be included in immunoconjugate known methods. For example, the peptide can be synthesized by biological methods, or it can be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 instead of hydrogen. Labels such as99mTc or123I,186Re,188Re and111In can be attached via a cysteine residue of the peptide. Yttrium-90 can be attached via a lysine residue. For the introduction of iodine-123 can be used IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57). Other methods are described in detail in the publication "Monoclonal Antibodies in ImmuNOcintigraphy" (Chatal, CRC Press 1989).

In some embodiments of the invention immunoconjugate can contain an antibody, anywhereman with a prodrug-activating enzyme which converts a prodrug (e.g., peptidase chemotherapeutic agent, see WO 81/01145) to an active drug such as an anticancer drug. Such th�conjugate can be used in antibody-dependent mediated by the enzyme Pro-drug therapy (“ADEPT”). Enzymes that can be anywhereman with the antibody, include, but are not limited to, alkaline phosphatase, which can be used for converting phosphate-containing prodrug into the free drug; arylsulfatase, which can be used for converting sulfate-containing prodrug into the free drug; sitoindosides, which can be used for converting non-toxic 5-fertilizin in anti-cancer drug, namely 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which can be used for converting peptide-containing prodrug into free drugs; D-alanismorissette, which can be used for the conversion of a prodrug containing a D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase, which can be used for converting glycosylated prodrug into the free drug; β-lactamase, which can be used for the conversion of medicines, derivatizing β-lactams, into free drugs; and penicillin-amidase, such as penicillin V amidase or penicillin G-s�Aza, which can be used for the conversion of medicines, derivatizing of nitrogen atoms of amine phenoxyacetyl or fenilatsetilenom groups, respectively, into free drugs. Enzymes can be covalently attached to antibodies by methods of recombinant DNA, are well known in the art. See, for example, Neuberger et al., Nature, 312:604-608 (1984).

d. Download drugs

Download medicinal product is denoted by p, i.e. the average number of molecules of drug per antibody in a molecule of the formula I. the Download of the drugs may be from 1 to 20 molecules of the drug (D) to the antibody. The conjugate "antibody-drug" (ADC) of formula I are sets of antibodies conjugated with various molecules of the drug, from 1 to 20. The average number of molecules of drug per antibody in preparations of ADC obtained as a result of conjugation reactions may be characterized by standard means such as mass spectroscopy, ELISA analysis and HPLC. May also be defined quantitative distribution of the ADC, expressed as "p". In some instances, separation, purification and characterization of homogeneous ADC, where p is a certain value obtained for the ADC with different drug loading �of means, can be carried out using reverse-phase HPLC or electrophoresis. For example, the pharmaceutical composition is an antibody-drug of formula I can be a heterogeneous mixture of conjugates with antibodies attached to 1, 2, 3, 4 or more molecules of the drug.

For some conjugates of the antibody-drug" p may be limited to the number of binding sites on the antibody. For example, if the attachment comes through cysteine thiol, as in the representative embodiments described above, the antibody may have only one or more thiol groups of cysteine, or it can have only one or several sufficiently reactive thiol groups through which may be joined by the linker. In some embodiments of the invention, a higher loading of the drug, for example, p>5, can lead to aggregation, insolubility, toxicity or loss of the ability of some conjugates of the antibody-drug" to penetrate cells. In some embodiments of the invention, loading of the drug on the ADC according to the invention is in the range of from 1 to about 8; from about 2 to about 6 or from 3 to 5. Indeed, it has been shown that for some ADC optimum ratio of molecules of medicinal CP�of DSTV on the antibody may be less than 8, and may be approximately from 2 to 5. Cm. application USA 2005-0238649 A1.

In some embodiments of the invention molecule drugs in amounts less than theoretical maximum kongugiruut with the antibody in the reaction process of conjugation. An antibody may contain, for example, lysine residues that do not react with the intermediate connection "drug-linker or linker reagent, which is discussed below. Typically, the antibody does not contain high levels of free and reactive thiol groups of cysteine, which may be associated with a molecule of the drug, and indeed, most of the thiol groups of cysteine residues in antibodies are present in the form disulfide bridges. In some embodiments of the invention, the antibody may be restored under the action of a reducing agent, such as dithiotreitol (DTT) or tricarbonylchromium (TCEP), in conditions of partial or full recovery with the formation of reactive thiol groups of cysteine. In some embodiments of the invention, the antibody is subjected to the reaction was performed under denaturing conditions with the formation of reactive nucleophilic groups such as lysine or cysteine.

Download ADC (ratio drug/antibody) can be adjusted by various methods, for example, by (i) limiting the molar�about excess intermediate connection "drug-linker or linker reagent relative to antibody, (ii) limits the reaction time or reaction temperature of conjugation, and (iii) partial or limiting recovery for modification of thiol groups of cysteine.

It should be noted that in the case where the intermediate connection "drug-linker or linker reagent, and then with a reagent such as molecule drugs that responds more than one nucleophilic group, the resulting product is a mixture of compounds of the ADC with the distribution of one or more drug molecules attached to the antibody. The average number of molecules of the drug to the antibody may be calculated for the mixture using the "sandwich"ELISA-analysis using an antibody which is specific for the antibody for the drug. Individual ADC molecules in the mixture can be identified using mass spectroscopy, and separated by HPLC, e.g., hydrophobic chromatography (see e.g., McDonagh et al. (2006) Prot. Engr. Design &Selection, 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling the location of drug attachment in antibody-drug conjugates”, Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, Marc 2004). In some embodiments of the invention, a homogeneous ADC with loading of one drug can be extracted from the mixture to conjugation by electrophoresis or chromatography.

e. Some ways to get immunoconjugates

The ADC of formula I may be obtained in several ways with the reactions of organic chemical synthesis under appropriate conditions and using reagents known in the art, where these methods include: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent with the formation of Ab-L, via a covalent bond, and then the reaction of interaction with the molecule of the drug D; and (2) reaction of a nucleophilic group of a molecule of the drug with a bivalent linker reagent with the formation of D-L through a covalent bond, and then the reaction of interaction with the nucleophilic group of an antibody. Representative methods of obtaining the ADC of formula 1 using the reaction described in the application US 2005-0238649 A1, which is fully introduced into the present description by reference.

Nucleophilic groups present on the antibody include, but are not limited to: (i) N-terminal amino group, (ii) the side chain amino group, e.g. lysine, (iii) the thiol group of the side chain, for example, cysteine�, and (iv) a hydroxyl or amino sugars, where the specified antibody is glycosylated. Amino groups, thiol groups, and hydroxyl groups are nucleophilic and can undergo reaction with the formation of covalent bonds with electrophilic groups on linker molecules or linker reagents including: (i) active esters such as NHS-esters, HOBt-esters, halogenfree and acid halides; (ii) alkyl - and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group. Some antibodies have recovered messageview disulfides, i.e. cysteine bridges. For conjugation with linker reagents antibodies may be made reactive by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylchromium (TSER), with the result that such antibodies will be fully or partially restored. Each cysteine bridge, theoretically, can form two reactive thiol nucleophil. The antibody can be introduced additional nucleophilic groups by modifying lysine residues, for example, by reaction of interaction of lysine with 2-aminothiophenol (reagent trout) that will lead to the transformation of the amine into a thiol. Reactive thio�new groups can be incorporated into the antibody by introducing one two, three, four, or more cysteine residues (e.g., obtaining variants of antibodies containing one or more non-natural cysteine amino acid residues).

Conjugates of the antibody-drug” according to the invention can also be obtained by reaction between the electrophilic group of an antibody, such as a carbonyl group of an aldehyde or ketone with a nucleophilic group of the linker reagent or drug. Suitable nucleophilic groups on the linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, a hydrazine carboxylate, and originated. In one embodiment of the invention, the antibody is modified to include an electrophilic group that can react with nucleophilic substituents on the linker reagent or drug. In another embodiment of the invention sugar glycosylated antibodies can be oxidized, for example, periodate oxidizing agents with the formation of aldehyde or ketone groups which may react with the amine group of linker reagents or drug molecules. Received minovia group Chippawa the base can form a stable bond, or they can be recovered, for example, borohydride reagents to form stable �minovich ties. In one embodiment of the invention, reaction of the carbohydrate portion of a glycosylated antibody with galactosialidosis or metaperiodate sodium can lead to the formation of carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, the antibodies of the invention containing N-terminal serine or treningowy residues that can react with metaperiodate sodium with the formation of aldehyde instead of the first amino acid (Geoghegan &Stroh (1992), Bioconjugate Chem. 3:138-146; U.S. patent No. 5362852). This aldehyde can interact with the molecule of a drug or with a linker nucleophile.

Nucleophilic groups on the molecule pharmaceuticals include, but are not limited to, amino, thiol, hydroxyl, hydrazide, Joksimovi, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines groups able to react with the formation of covalent bonds with electrophilic groups on linker molecules and linker reagents including: (i) active esters such as NHS-esters, HOBt-esters, halogenfree and gelegenheid acids; (ii) alkyl - and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group.

�Oroch speaking, the considered compounds according to the invention are, but are not limited to: ADC obtained using the following cross-linking reagents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate) which are commercially available (for example, come Pierce Biotechnology, Inc., Rockford, IL., U. S. A; see pages 467-498, 2003-2004 Applications Handbook and Catalog).

Immunoconjugate containing the antibody and cytotoxic agent may be obtained using a variety of bifunctional protein-binding agents, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, immunotoxin ricin can be obtained as described in the publication Vitetta et al. Science, 238:1098 (1987).14With-�ECENA 1-isothiocyanatobenzene-3- acid (MX-DTPA) is representative chelating agent for conjugating radionuclide to the antibody. Cm. WO 94/11026.

Alternative hybrid protein containing the antibody and cytotoxic agent may be obtained, for example, recombinant techniques or peptide synthesis. The recombinant DNA molecule may contain a region encoding the antibody and the cytotoxic portion of the conjugate either adjacent each other or separated by a region encoding a linker peptide which does not adversely impact on the desired properties of the conjugate.

In yet another embodiment of the invention, the specified antibody can be konjugierte with the “receptor” (such as streptavidin) for his pre-targeting the tumor, where the specified conjugate “antibody-receptor is administered to the patient, followed by removal of unbound conjugate from the circulation using the agent for clearance, and then enter a “ligand” (e.g. avidin) that is conjugated with a cytotoxic agent (e.g., radionucleotides).

Representative immunoconjugate - conjugates "thio-antibody-drug"

a. Getting designed on the basis of cysteine anti-CD79b antibodies

DNA encoding variant amino acid sequence is constructed on the basis of cysteine anti-CD79b antibodies and parent anti-CD79b antibody according to the invention, receive by various methods, which include, but are not og�anywaysa them isolation from a natural source (in the case of the natural amino acid sequence variants), obtaining a drug by using site-directed (or oligonucleotide-mediated) mutagenesis (Carter (1985) et al. Nucl Acids Res. 13:4431-4443; Ho et al. (1989) Gene (Amst.) 77:51-59; Kunkel et al. (1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al. (1998) J. Biol. Chem. 273:20252-20260), PCR mutagenesis (Higuchi, (1990) in PCR Protocols, pp. 177-183, Academic Press; Ito et al. (1991) Gene 102:67-70; Bernhard et al. (1994) Bioconjugate Chem. 5:126-132; and Vallette et al. (1989) Nuc. Acids Res. 17:723-733), and clustered mutagenesis (Wells et al. (1985) Gene 34:315-323) previously obtained DNA encoding the polypeptide. Protocols, kits and reagents for the implementation of mutagenesis are commercially available, for example, such as a kit for site-directed mutagenesis QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations introduced using oligonucleotide-directed mutagenesis using double stranded plasmid DNA as template by PCR mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al. (1983) Methods Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500). Variants of recombinant antibodies can also be constructed by modification of DNA restriction fragments or by using extension PCR with overlapping conducted using synthetic oligonucleotides. Mutagenic primers encode the replacement(s) of cysteine codons. Standard methods m�of Magenta can be applied for the production of DNA, encoding such mutant antibodies constructed with cysteine (Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993).

The technology of phage submission (McCafferty et al., (1990)Nature348:552-553) can be used for producing human anti-CD79b antibodies and fragments of antibodies in vitro from sets of genes of variable domain immunoglobulin (V) from unimmunized donors. In accordance with this method, the genes of domain V of antibodies cloned with preservation of the reading frame in either the major or minor gene of the protein shell of filamentous phage, such as M13 or fd, and present on the surface ragovoy particles as functional fragments of the antibodies. Because filamentous particle contains a single-stranded copy of the DNA genome of the phage, the selection is conducted on the basis of the functional properties of antibodies, also allows you to select the gene encoding the antibody with these properties. Thus, the phage mimics some properties of b-cells (Johnson et al. (1993) Current Opinion in Structural Biology 3:564-571; Clackson et al. (1991)Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; Griffith et al. (1993) EMBO J. 12:725-734; US 5565332; US 5573905; US 5567610; US 5229275).

Anti-CD79b antibody may be chemically synthesized using known Oligopeptide method of synthesis, or they can be obtained and purified R�combinatin method. The corresponding amino acid sequence or part thereof can be obtained by direct solid-phase peptide synthesis (Stewart et al.,Solid-Phase Peptide Synthesis, (1969) W. H. Freeman Co., San Francisco, CA; Merrifield, (1963) J. Am. Chem. Soc., 85:2149-2154). Protein synthesis in vitro can be carried out manually or by automated method. Automated solid-phase synthesis method may be implemented, for example, using t-BOC - or Fmoc - protected amino acids on a peptide synthesizer Applied Biosystems (Foster City, CA) according to the manufacturers instructions. The various parts of the anti-CD79b antibody or CD79b polypeptide can be obtained by chemical synthesis and by the combined method of chemical or enzymatic synthesis with producing the desired anti-CD79b antibody or CD79b polypeptide.

To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments are formed as a result of proteolytic cleavage of intact antibodies (Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al. (1985) Science, 229:81), or they are produced directly by recombinant cell host. Fab, Fv and scFv-fragments of anti-CD79b antibodies can be expressed in E. coli and secretariats from E. coli, which facilitates the production of these fragments in large quantities. Fragments of antibodies can be isolated from phage bib�of iotek antibodies discussed above. Alternative Fab'-SH fragments can be directly isolated from E. coli and chemically linked to form F(ab')2-fragments (Carter et al., Bio/Technology 10:163-167 (1992)), or they can be isolated directly from a culture of recombinant host cells. Anti-CD79b antibody may be single-chain Fv fragment (scFv) (WO 93/16185; US 5571894; US 5587458). Fragment anti-CD79b antibody may also be a "linear antibody" (US 5641870). Such fragments, linear antibodies can be monospecifičeskoj or bespecifically.

As described below, mainly producing anti-CD79b antibody by culturing cells transformed or transfected with a vector containing nucleic acid encoding an anti-CD79b antibody. DNA encoding anti-CD79b antibody may be obtained from a cDNA library isolated from the tissue, which, obviously, contains mRNA anti-CD79b antibodies and expresses her detektiruya level. Accordingly, the DNA of a human anti-CD79b antibody or CD79b polypeptide can usually be obtained from a cDNA library isolated from human tissue. The gene encoding anti-CD79b antibody may also be obtained from a genomic library or by application of known methods of synthesis (e.g., automated nucleic acid synthesis).

The methods of construction, selection and accessed� preparations according to the invention allow to obtain designed on the basis of cysteine anti-CD79b antibodies which react with an electrophilic functional group. These methods also allow to obtain compounds of conjugates of antibodies, such as compound-conjugate "antibody-drug" (ADC), with molecules of drugs in certain designed selective sites. Reactive cysteine residues on the surface of antibodies allow specific conjugation of molecules of the drug by thiol-reactive group, such as maleimide or halogenoacetyl. Nucleophilic reactivity of the thiol functional groups of the Cys residue with maleimide group approximately 1000 times higher than the reactivity of any other functional groups of amino acids in the protein, such as amino group of lysine residues or N-terminal amino group. Thiol-specific functional group in iodization and maleimide reagents may react with amino groups, but at higher pH (>9,0), and this reaction takes a longer time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The amount of free thiol in the protein can be evaluated using standard analysis of an Elman. Immunoglobulin M is an example of a disulfide-bonded pentamer, and immunoglobulin G is an example of a protein with internal disulfide bridges associated with the subunits. In proteins,such as the specified protein for the production of reactive free thiol requires reduction of the disulfide bonds under the action of a reagent such as dithiothreitol (DTT) or selenium (Singh et al. (2002) Anal. Biochem. 304:147-156). Such a procedure may lead to the loss of tertiary structure of the antibody and the specificity of binding to the antigen.

Analysis PHESELECTOR (phage ELISA for selection of reactive thiols) allows the detection of reactive cysteine groups in antibodies in the format of phage ELISA, which facilitates the design of antibody-based cysteine (Junutula, J. R. et al. (2008) J. Immunol Methods 332:41-52; WO 2006/034488; US 2007/0092940). On the surface of the hole is applied is constructed on the basis of cysteine antibody, and then incubated with fagbemi particles and add HRP-labeled second antibody, followed by determination of optical density. The mutant proteins presented on the phage can be skanirovana fast, reliable and highly effective method. This same method can be obtained from libraries constructed on the basis of cysteine antibodies that can be subjected to selective binding to identify the inclusion of appropriate reactive sites of free Cys from randomized phage libraries of proteins, antibodies or other proteins. This method involves reacting cysteine mutant proteins presented on �Agay, with an affinity reagent or reporter group, which also interacts with the thiol.

Analysis PHESELECTOR allows the screening of reactive thiol groups in the antibody. As an example of this is the identification of an option AS this method. To identify a larger number of options thio-Fab with reactive thiol groups can be carried out effectively search for a full-sized Fab molecules. To identify and quantify the accessibility of solvent to the amino acid residues in the polypeptide that was used a parameter such as the relative accessibility of the surface. The availability of the surface can be expressed as surface area (Å2), which can be contacted with the solvent molecule, for example, with water. The space occupied by the water, expressed approximately as a sphere of radius of 1.4 Å. In the package of crystallographic programs SSR that are either free or require a license payment Company on the development of the program CCP4, Daresbury Laboratory, Warrington, WA44AD, United Kingdom, Fax: (+44) 1925 603825, or by Internet: www.ccp4.ac.uk/dist/html/INDEX.html), uses algorithms to calculate the surface accessibility of each amino acid of a protein with known x-ray crystallographic coordinates ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Acta. Cryst. D50:760-763). Two of represent�tive software modules, with the help of which do not calculate surface distance, are program AREAIMOL and SURFACE developed on the basis of algorithms B. Lee & F. M. Richards (1971) J. Mol. Biol. 55:379-400. The program AREAIMOL allows to determine the surface accessibility of the protein to solvent as the center point of the sphere probe (representing the solvent molecule), when this point of "trying out" van der velcovsky the surface of the protein. The program AREAIMOL allows us to calculate the surface area available for solvent, by generating points on the surface of a large sphere around each atom (at a distance from the center of the atom, equal to the sum of the radii of the atom and probe) and eliminating those points that are equivalent to the areas associated with neighboring atoms. The program AREAIMOL to find an available solvent for the size of the atoms in the PDB coordinate and systematize the available area of the remainder of the circuit and the whole molecule. Square (or difference of squares), is available for individual atoms can be registered in the output pseudo-PDB file. The program AREAIMOL uses only one radius for each element and recognizes only a limited number of distinct elements.

Programs AREAIMOL and SURFACE allow to achieve absolute availability, i.e. the number of angstroms (Å) in the square. The relative accessibility of the surface is calculated by CP�neniu with the accessibility of the peptide with a characteristic amino acids in this polypeptide. Standard peptide is the Tripeptide Gly-X-Gly, where X is an interesting amino acid, and this standard peptide should have an “extended” conformation, i.e. conformation, similar to beta-chains. This extended conformation maximizes the accessibility of the residue X. the Computed available area is divided into available area for standard peptide in the Tripeptide Gly-X-Gly and get a quotient that represents the relative ease of access. The percentage of availability is a relative availability multiplied by 100. Another representative algorithm to calculate the distance to the surface based on SOLV-module program xsae (Broger, C., F. Hoffman-LaRoche, Basel), which allows to calculate the relative accessibility of amino acid residues for the water sector, based on x-ray coordinates of the polypeptide. The relative surface accessibility of each amino acid in the antibody can be calculated using the data on its crystal structure (Eigenbrot et al. (1993) J. Mol. Biol. 229:969-995).

DNA that encodes constructed on the basis of cysteine antibodies may be readily isolated and sequenced in accordance with standard procedures (e.g., by using oligonucleotide probes that are able to specifically communicate with the genes encoding the heavy and light chains m�shinih antibodies). Hybrid cells serve as a source of such DNA. After DNA extraction can be placed into expression vectors, which are then transferout in the host cell, such as E. coli cells, simian COS cells, cells of the Chinese hamster ovary (Cho) cells or other cells of mammalian hosts, such as myeloma cells (U.S. patent 5807715; patent application U.S. 2005/0048572 and 2004/0229310), which usually do not produce protein antibodies, resulting in these recombinant cells-the owners are synthesized monoclonal antibodies.

After design and selection, designed on the basis of cysteine antibodies, for example, thio-Fab with highly reactive unpaired Cys residues, namely "free cysteine amino acid residues, can be obtained by (i) expression in bacteria, e.g. E. coli system (Skerra et al. (1993) Curr. Opinion in Immunol. 5:256-262; Plückthun (1992) Immunol. Revs. 130:151-188) or in the cell culture system of mammals (WO 01/00245), for example, in cells of the Chinese hamster ovary (Cho) cells; and (ii) purification by standard methods of protein purification (Lowman et al. (1991) J. Biol. Chem. 266(17):10982-10988).

The thiol group of Cys designed to react with electrophilic linker reagents and intermediate compounds "drug-linker" with the formation of the conjugates are "constructed on the basis of the cysteine antibodies�about-drug" and the other labeled antibodies, constructed with cysteine. Residues Cys, which are present in the constructed on the basis of cysteine antibodies and in the parent antibody, and which mate and form Mirzayeva and noticeplease disulfide bonds, do not have any reactive thiol groups (if they have not been treated with the reducing agent) and does not react with electrophilic linker reagents or intermediate compounds “drug-linker”. The newly introduced Cys residue may remain unpaired, and can react to form a conjugate with an electrophilic linker reagent or intermediate connection "drug-linker", such as "drug-maleimide". Representative intermediate compounds of "a drug-linker" is MC-MMAE, MC-MMAF, MC-vc-PAB-MMAE and MC-vc-PAB-MMAF. Structural provisions of the engineered Cys residues in the heavy and light chains are numbered according to the serial numbering system. This serial numbering system correlates with the numbering system Kabat (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), starting from N-Terminus, compared to a numbering scheme in Cabatu (bottom row), where inserts are labeled a,b,c. Using a numbering system Kabata, the actual Lin�of ina amino acid sequence may contain fewer or more amino acids due to a shortening of, or insertion into a FR or CDR of the variable domain. Sites designed on the basis of the cysteine variants of the heavy chain identified by applying sequential numbering and numbering scheme Kabata.

In one embodiment of the invention constructed on the basis of cysteine anti-CD79b antibody is produced by a method including:

(a) replacing one or more amino acid residues of a parent anti-CD79b antibody by cysteine; and

(b) determining the reactivity of thiol groups of cysteine anti-CD79b antibody by reaction of the interaction is constructed on the basis of cysteine antibody with a reagent that reacts with a thiol.

Designed on the basis of cysteine antibody may react with a reagent that reacts with a thiol, better than the parent antibody.

Free cysteine amino acid residues can be localized to the heavy or light chains, or in constant or variable domains. Fragments of antibodies, such as Fab, can also be constructed by replacing one or more amino acids of a fragment of the antibody one or more cysteine amino acids, with formation constructed on the basis of cysteine fragments of antibodies.

In another embodiment, the present invention relates to a method for producing (creating) constructed on the basis of cysteine anti-CD79b antibodies, where the specified method in�includes:

(a) introducing one or more cysteine amino acids into a parent anti-CD79b antibody with getting designed on the basis of cysteine anti-CD79b antibody; and

(b) determining the ability of thiol groups constructed on the basis of cysteine antibodies to react with a reagent reactive with a thiol;

where the specified constructed on the basis of cysteine antibody may react with a reagent that reacts with a thiol, better than the parent antibody.

Stage (a) of the method of producing designed on the basis of cysteine antibodies may include:

(i) mutagenesis nucleic acid sequence that encodes constructed on the basis of cysteine antibody;

(ii) the expression is constructed on the basis of cysteine antibody; and

(iii) isolation and purification of the specified constructed on the basis of cysteine antibodies.

Stage (b) the production method constructed on the basis of cysteine antibody may include the expression is constructed on the basis of cysteine antibody to the viral particle selected from a phage or fahmideh particles.

Stage (b) the production method constructed on the basis of cysteine antibodies may also include:

(i) reaction of interaction is constructed on the basis of cysteine antibody with an affinity reagent that reacts with a thiol, to obtain affinity-labeled with�konstruirovanija on the basis of cysteine antibodies; and

(ii) measuring the level of binding of the affinity labelled, constructed on the basis of cysteine antibodies with the environment to capture.

In another embodiment, the present invention relates to a method of screening designed on the basis of cysteine antibodies with highly reactive unpaired cysteine amino acids on the reactivity of their thiol groups, where the method includes:

(a) introducing one or more cysteine amino acids into a parent antibody with getting designed on the basis of cysteine antibodies;

(b) reaction of interaction is constructed on the basis of cysteine antibody with an affinity reagent that reacts with a thiol, to obtain affine labeled, constructed on the basis of cysteine antibodies;

(C) measuring the level of binding of the affinity labelled, constructed on the basis of cysteine antibodies with the environment to capture; and

(d) determining the ability of thiol groups constructed on the basis of cysteine antibodies to react with a reagent that reacts with a thiol.

Stage (a) of the screening method is designed based on the cysteine antibodies may include:

(i) mutagenesis nucleic acid sequence that encodes constructed on the basis of cysteine antibody;

(ii) the expression is constructed on the basis of the CSA�eine antibodies; and

(iii) isolation and purification of the specified constructed on the basis of cysteine antibodies.

Stage (b) of the screening method is designed based on the cysteine antibodies may include the expression is constructed on the basis of cysteine antibodies on viral particle selected from a phage or fahmideh particles.

Stage (b) of the screening method is designed based on the cysteine antibodies may also include:

(i) reaction of interaction is constructed on the basis of cysteine antibody with an affinity reagent that reacts with a thiol, to obtain affine labeled, constructed on the basis of cysteine antibody; and

(ii) measuring the level of binding of the affinity labelled, constructed on the basis of cysteine antibodies with the environment to capture.

b. Constructing variants of the anti-CD79b IgG-based cysteine

Cysteine was introduced at position 118 of the heavy chain (in accordance with the European numbering system) (equivalent to position 118 of the heavy chain, sequential numbering) in a full-sized chimeric parent monoclonal anti-CD79b antibodies or to position 205 of the light chain (in accordance with numbering Kabata) (equivalent to position 209 of the light chain, sequential numbering) in a full-sized chimeric parent monoclonal anti-CD79b antibody by methods of introducing cysteine, described in this� the application.

Below have been received, constructed on the basis of cysteine antibody containing the cysteine at position 118 of the heavy chain (in accordance with the European numbering system): (a) thio-MA79b.v17-HC(A118C) with the sequence of the heavy chain (SEQ ID NO: 228) and light chain sequence (SEQ ID NO: 229), figure 24; (b) thio-MA79b.v18-HC(A118C) with the sequence of the heavy chain (SEQ ID NO: 230) and light chain sequence (SEQ ID NO: 231), figure 25; (c) thio-MA79b.v28-HC(A118C) with the sequence of the heavy chain (SEQ ID NO: 232) and light chain sequence (SEQ ID NO: 233), figure 26; (d) thio-MA79b-HC(A118C) with the sequence of the heavy chain (SEQ ID NO: 236) and light chain sequence (SEQ ID NO: 237), figure 28; and (e) thio-anti-cynoCD79b-HC(A118C) with the sequence of the heavy chain (SEQ ID NO: 244) and with the light chain sequence (SEQ ID NO: 245), figure 48.

Below have been received, constructed on the basis of cysteine antibody containing the cysteine at position 205 of the light chain (in accordance with numbering Kabata): (a) thio-MA79b-LC(V205C) with the sequence of the heavy chain (SEQ ID NO: 234) and light chain sequence (SEQ ID NO: 235), figure 27; and (b) thio-anti-cynoCD79b(ch10D10)-LC(V205C) with the sequence of the heavy chain (SEQ ID NO: 299) and with the light chain sequence (SEQ ID NO: 300), figure 49.

These are designed on the basis of cysteine monoclonal antibodies were expressed in Cho cells (ovarian kitayskog� hamster) by temporarily fermentation in the environment, containing 1 mm cysteine.

In one embodiment of the invention humanized designed on the basis of cysteine anti-CD79b antibody MA79b contain one or more of the following sequences of the heavy chain with a free cysteine amino acid (SEQ ID NO: 251-259, table 2).

Table 2
Comparison of sequences of the heavy chain of humanized designed based on the cysteine variants of the anti-CD79b antibody MA79b, numbered in accordance with a sequential numbering system, numbering Kabata and the European numbering system
SequenceConsecutive numberingThe numbering on CabatoThe European numbering systemSEQ ID NO:
EVQLCESGGGV5CV5C251
LRLSCCASGYTA23CA23C252
MNSLRCEDTAVA88CA8C 253
TLVTVCSASTKS116CS112C254
VTVSSCSTKGPA118CA114CA118C255
VSSASCKGPSVT120CT116CT120C256
WYVDGCEVHNAV282CV278CV282C257
KGFYPCDIAVES375CS371CS375C258
PPVLDCDGSFFS400CS396CS400C259

In one embodiment of the invention chimeric constructed on the basis of cysteine anti-CD79b antibody MA79b contain one or more of the following sequences of the heavy chain with a free cysteine amino acid (SEQ ID NO: 260-268, table 3).

Table 3
Comparison of sequences of the heavy chain is designed on the basis of the cysteine variants of the anti-CD79b antibody chMA79b, numbered in accordance with a sequential numbering system, numbering Kabata and the European numbering systemSequenceConsecutive numberingThe numbering on CabatoThe European numbering systemSEQ ID NO:EVQLCQSGAEQ5CQ5C260VKISCCATGYTK23CK23C261LSSLTCEDSAVS88CS84C262TSVTVCSASTKS116CS112C263VTVSSCSTKGPA118CA114C A118C264VSSASCKGPSVT120CT116CT120C265WYVDGCEVHNAV282CV278CV282C266KGFYPCDIAVES375CS371CS375C267PPVLDCDGSFFS400CS396CS400C268

In one embodiment of the invention chimeric constructed on the basis of cysteine anti-CD79b antibodies, namely anti-cynoCD79b(ch10D10), contain one or more of the following sequences of the heavy chain with a free cysteine amino acid (SEQ ID NO: 269-277, table 4).

Table 4
Comparison of sequences of the heavy chain is designed on the basis of the cysteine variants of the anti-CD79b antibody, anti-cynoCD79b(ch10D10), numbered in accordance with a sequential numbering system, numbering Kabata and European si�topic numbering
SequenceConsecutive numberingThe numbering on CabatoThe European numbering systemSEQ ID NO:
EVQLCESGPGQ5CQ5C269
LSLTCCVTGYST23CT23C270
LNSVTCEDTATS88CS84C271
TTLTVCSASTKS111CS112C272
LTVSSCSTKGPA113CA114CA118C273
VSSASCKGPSVT115CT116CT120C274
WYVDGCEVHNAV282C V278CV282C275
KGFYPCDIAVES370CS371CS375C276
PPVLDCDGSFFS395CS396CS400C277

In one embodiment of the invention humanized designed on the basis of cysteine anti-CD79b antibody MA79b contain one or more of the following light chain sequences with a free cysteine amino acid (SEQ ID NO: 278-284, table 5).

Table 5
Comparison of sequences of the humanized light chain is designed on the basis of the cysteine variants of the anti-CD79b antibody MA79b, numbered in accordance with a sequential numbering system and the numbering Cabato
SequenceConsecutive numberingThe numbering on CabatoSEQ ID NO:
SLSASCGDRVTV15CV15C278
EIKRTCAAPSVV114CV110C279
TVAAPCVFIFPS118CS114C280
FIFPPCDEQLKS125CS121C281
DEQLKCGTASVS131CS127C282
VTEQDCKDSTYS172CS168C283
GLSSPCTKSFNV209CV205C284

In one embodiment of the invention chimeric constructed on the basis of cysteine anti-CD79b antibody MA79b contain one or more of the following light chain sequences with a free cysteine amino acid (SEQ ID NO: 285-291, table 6).

Table 6
Comparison of sequences of light chain chimeric designed based on the cysteine variants of the anti-CD79b antibody MA79b, numbered in accordance with�tvii with sequential numbering system and the numbering Cabato
SequenceConsecutive numberingThe numbering on CabatoSEQ ID NO:
SLAVSCGQRATL15CL15C285
ELKRTCAAPSVV114CV110C286
TVAAPCVFIFPS118CS114C287
FIFPPCDEQLKS125CS121C288
DEQLKCGTASVS131CS127C289
VTEQDCKDSTYS172CS168C290
GLSSPCTKSFNV209CV205C291

In one embodiment of the invention constructed on the basis of cysteine anti-CD79b antibodies, namely anti-cynoCD79b(ch10D10), contain one or more of the following on�of sledovatelnot light chain with a free cysteine amino acid (SEQ ID NO: 292-298, table 7).

Table 7
Comparison of sequences of the light chain is designed on the basis of the cysteine variants of the anti-CD79b antibody, anti-cynoCD79b(ch10D10), numbered in accordance with a sequential numbering system and the numbering Cabato
SequenceConsecutive numberingThe numbering on CabatoSEQ ID NO:
SLAVSCGQRATL15CL15C292
EIKRTCAAPSVV114CV110C293
TVAAPCVFIFPS118CS114C294
FIFPPCDEQLKS125CS121C295
DEQLKCGTASVS131CS127C296
VTEQDCKDSTYS172CS168C 297
GLSSPCTKSFNV209CV205C298

c. Labeled and designed on the basis of cysteine anti-CD79b antibodies

Constructed of cysteine-based anti-CD79b antibodies may be site-specifically and efficiently attached to the reagent that reacts with a thiol. Reagent that reacts with the thiol may be a multifunctional linker reagent to the capture reagent that is used as an affinity label (e.g., a reagent Biotin-linker"), a detectable label (e.g. a fluorophore reagent), reagent for immobilization on a solid phase (e.g., SEPHAROSE™, polystyrene or glass) or an intermediate connection "drug-linker". One example of a reagent that reacts with a thiol is N-ethylmaleimide (NEM). In a representative embodiment of the invention, the reaction of interaction of thio-Fab with the reagent Biotin-linker is formed biotinylating thio-Fab, through which can be detected and determined the presence and reactivity of the introduced cysteine residue. As a result of the reaction between thio-Fab with a multifunctional linker reagent is formed thio-Fab with a functional linker that can react with a molecule of drug�about the funds or other label. As a result of the reaction between thio-Fab with an intermediate connection "drug-linker" conjugate formed "thio-Fab-drug".

Described here are representative methods can be applied, mainly for identification and production of antibodies, mainly of other proteins through the stages described here for constructing and screening.

This method can be applied for the conjugation of other thiol-reactive reagents, in which the reactive group is, for example, maleimide, jodatime, pyridylsulfonyl or other reacting with a thiol conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). Reagent that reacts with a thiol, can be a molecule of the drug, a fluorophore such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for an imaging or radioactive metal used in therapy, piptadenia or hepatically label or detectable label, or an agent that modifies the clearance, such as various isomers of polyethylene glycol, a peptide that binds to a third component, or another carbohydrate illiberally agent.

d. The application is designed based on the cysteine anti-CD79b antibodies

Constructed of cysteine-based anti-CD79b antibodies and their conjugates can be used as therapeutic and/or diagnostic agents. The present invention also relates to methods for the prevention, treatment, therapy or reducing one or more symptoms associated with b-cell-mediated disorder. In particular, the present invention also relates to methods for the prevention, treatment, therapy or reducing one or more symptoms associated with cell-proliferative disorder such as cancer, e.g., lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. The present invention also relates to methods of diagnosis CD79b-mediated disorder or the predisposition to develop such disorders, and to methods of identifying antibodies and antigen-binding fragments of antibodies, which are mainly associated with b-cell-�associirovannyy CD79b polypeptides.

In another embodiment, the present invention relates to the application designed on the basis of cysteine anti-CD79b antibody for the preparation of a medicament to treat a condition that is susceptible to b-cell-mediated disorder.

e. Conjugates "constructed on the basis of cysteine antibody-drug" (conjugates "thio-antibody-drug" (TDC))

In another aspect, the present invention relates to compound-conjugate "antibody-drug" containing constructed on the basis of cysteine anti-CD79b antibody (Ab), and molecule auristatin medicines (D), where the specified constructed on the basis of cysteine antibody linked to D of the linker molecule (L) through one or more free cysteine amino acids, where the specified connection has the formula I:

Ab-(L-D)pI,

where p is 1, 2, 3 or 4; and where constructed on the basis of cysteine antibody produced by the method comprising substituting one or more amino acid residues of a parent anti-CD79b antibody by one or more free cysteine amino acids.

In another aspect of the present invention�worn to the song containing a mixture of compounds is an antibody-drug of formula I, where the average load of the drug to the antibody is from about 2 to about 5 or from 3 to 4.

In the figures 24-28 48-49 and presents options conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" (ADC), where the molecule auristatin medicines attached to the introduced cysteine group in light chain (LC-ADC) or heavy chain (HC-ADC).

Possible advantages conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug are increased security (higher therapeutic index"); improved pharmacokinetic parameters; saving megaplay disulfide bonds of the antibody, which may stabilize the conjugate and maintain its conformation, promoting the active binding; the ability to identify sites of conjugation of the drug; and the possibility of obtaining conjugates "constructed on the basis of cysteine antibody-drug" as a result of the reaction of conjugation constructed on the basis of cysteine antibody reagent "drug-linker", which leads to the formation of a more homogeneous product.

Linkers

"Linker", "linker component and�and "communication" means a chemical group, containing covalent bond or a chain of atoms that covalently bind the antibody to the molecule of the drug. In various embodiments of the invention, the linker is designated L. “Linker” (L) is a bifunctional or multifunctional molecule that can be used to bind one or more molecules of the drug (D) and molecules of antibody (Ab) with the formation of conjugates of the antibody-drug” (ADC) of formula I. Conjugates of the antibody-drug” (ADC) is usually obtained by use of a linker having a reactive functional group for binding with the drug and the antibody. The thiol of a cysteine introduced in the antibody (Ab) can form a bond with an electrophilic functional group of the linker reagent, the molecules of a drug or intermediate connection "drug-linker".

In one aspect of the invention, the linker has a reactive site containing an electrophilic group that reacts with the nucleophilic cysteine is present on the antibody. The thiol of cysteine specified antibody reacts with an electrophilic group on a linker and forms a covalent bond with the linker. Suitable electrophilic groups include, but are not limited to, maleimide and halogenate�ing group.

The linkers are bivalent radical, such as alkerdeel, Allen, heteroaryl, molecules, such as -(CR2)nO(CR2)n-, the repeating unit of aryloxy (e.g., polietilene, PEG, polymethylenes) and alkylamino (e.g., polyethylenimine, Jeffamine™); and an ester of a dibasic acid and amides including succinate, succinamide, diglycolate, malonate and caproamide.

Designed on the basis of cysteine antibody is subjected to reaction with linker reagents or intermediate compounds "drug-linker", electrophilic functional groups, such as maleimide or α-halogencarbonic, in accordance with the methods of conjugation are described on page 766 the publication of Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773, and according to the Protocol described in example 6.

The linker may consist of one or more linker components. A representative of the linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzeneboronic ("PAB"), N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"), N-Succinimidyl-(4-iodates)aminobenzoate ("SIAB"), ethyleneoxy-CH2CH2O - as or n�many repetitive elements ("EO" or "PEO"). Specialists and other known linker components, some of which are described in this application.

In one embodiment of the invention, the linker L in the ADC has the formula:

-Aa--Ww--Yy-,

where:

But a lengthening component covalently linked to a cysteine thiol of the antibody (Ab);

andis 0 or 1;

each W independently represents an amino acid,

w independently represents an integer from 0 to 12,

Y means the GS spacer component covalently linked to a molecule of the drug; and

y is 0, 1 or 2.

Lengthening component

Lengthening component (-A-), when present, is capable of binding an antibody with an amino acid (-W-). In this case, the antibody (Ab) has a functional group that can form a bond with a functional group of the extension component. Suitable functional groups that may be present on the antibody and which may be either natural or chemically synthesized, are, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. In one aspect of the invention functional groups of the antibody are sulfhydryl or amino. Sulfhydryl groups can be generated by restoring the intramolecular �sulfides the antibody binds. Alternative sulfhydryl groups can be formed through interaction of the lysine amino group of the antibody molecules in the presence of 2-aminothieno (reagent trout) or other sulfhydryl-forming reagent. In one embodiment of the invention, the antibody (Ab) has a free thiol group of cysteine, which can form a bond with an electrophilic functional group of the extension component. A representative of the extension components in the conjugates of formula I are represented by formulas II and III, where Ab-, -W-, -Y-, -D, w and y are defined above, and R17is a divalent radical selected from (CH2)rWith3-C8carbocycle, -O-(CH2)rarylene, (CH2)rarylene, Allen-(CH2)r, (CH2)r-(C3-C8-carbocycle), -(C3-C8-carbocyclic)-(CH2)r-, C3-C8-heterocyclyl, (CH2)r-(C3-C8-heterocyclyl), (C3-C8-heterocyclyl)-(CH2)r-, -(CH2)rC(O)NRb(CH2)r, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-, (CH2)C(O)NRb(CH2CH2O)r, (CH2)rC(O)NRb(CH2CH2O)r-CH2, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-CH2- and -(CH2CH2O)rC(O)NRb(CH2)rwhere Rbrepresents H, C1-C6alkyl, phenyl or benzyl, and r independently is an integer of 1-10.

Afilename are divalent aromatic hydrocarbon radicals of from 6-20 carbon atoms derived by removing two hydrogen atoms from a source of the aromatic cyclic system. Typical allenbyi groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc.

Heterocyclic groups include ring system in which one or more atoms on the ring represent a heteroatom, e.g., nitrogen atom, oxygen and sulfur. Heterocyclic radical comprises 1 to 20 carbon atoms and 1 to 3 heteroatom selected from N, O, P and S. Heterocycle may be a monocycle having 3 to 7 atoms in the ring (2-6 carbon atoms and 1 to 3 heteroatom selected from N, O, P and S), or Bicycle having 7 to 10 atoms in the ring (4-9 carbon atoms and 1-3 heteroatom selected from N, O, P and S), for example, bicyclo-[4,5]-, -[5,5]-, -[5,6]- or[6,6] system. Heterocycles are described in the publications Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), particularly chapters 1, 3, 4, 6, 7, and 9; in the publication "The hemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950, as amended), in particular volumes 13, 14, 16, 19, and 28; and in the publication J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include, but are not limited to, pyridyl, dihydropyridin, tetrahydropyridine (piperidyl), thiazolyl, tetrahydrothiophene, oxidized sulfur-tetrahydrothiophene, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thionaphthene, indole, indoline, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydropyranyl, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6H-l,2,5-thiadiazine, 2H,6H-l,5,2-diazenyl, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine, 2H-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indole, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, chinoxalin, chinazoline, cinnoline, pteridine, 4Ah-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, furutani, phenoxazines, isopropanol, chromanol, imidazolidinyl ureido, imidazolyl, pyrazolidine, pyrazoline, PIP�retinyl, indolinyl, isoindolyl, hinokitiol, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazoles, oxindoles, benzoxazolyl and satanail.

Carbocyclic groups include saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as Bicycle. The monocyclic carbocycle have from 3 to 6 atoms in the ring, and usually 5 or 6 atoms in the ring. Bicyclic carbocycle have from 7 to 12 atoms in the ring, for example, arranged in the form of a bicyclo-[4,5]-, [5,5]-, [5,6]- or [6,6] system, or 9 or 10 atoms in the ring, arranged in the form of bicyclo-[5,6]- or [6,6] system. Examples of monocyclic carbocycles are cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-l-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cycloheptyl and cyclooctyl.

It should be noted that in all representative embodiments, the ADC of formula I, such as compounds II-VI, even if they are not specifically indicated, from 1 to 4 molecules of the drug are linked to an antibody (p=1-4), depending on the number of introduced cysteine residues.

A representative of the extension component is the component of the formula II, derived from maleimido-caproyl (MS), where R17represents -(CH2)5-:

A representative of the extension component is the component of the formula II, derived from maleimido-propanol (Mr), where R17represents -(CH2)2-:

Another representative of the extension component is the component of the formula II, where R17represents -(CH2CH2O)r-CH2-, and r = 2:

Another representative of the extension component is the component of the formula II, where R17represents (CH2)rC(O)NRb(CH2CH2O)r-CH2-, where Rbrepresents N, and each r is 2:

Another representative of the extension component is the component of the formula III, where R17represents -(CH2)5-:

In another embodiment of the invention, the specified extension component is associated with constructed of cysteine-based anti-CD79b antibody by disulfide bonds between the sulfur atom of cysteine antibody and a sulfur atom of the extension component. A representative of the extension component of this variant of the invention shown in the formula IV, where R17Ab-, -W-, -Y-, -D, w and y are defined above.

In esetnod embodiment of the invention, reactive group of the extension component contains the thiol-reactive functional group that can form a bond with a free cysteine thiol of an antibody. Examples of thiol-reactive functional groups include, but are not limited to, maleimide, α-halogenoacetyl, activated esters, such as Succinimidyl, 4-nitrophenolate esters, panafcortelone esters, tetraterpene esters, anhydrides, chlorides, sulphonylchloride, isocyanates and isothiocyanates. A representative of the extension components of this variant of the invention shown in formulas Va and Vb, where R17Ab-, -W-, -Y-, -D, w and y are defined above.

In another embodiment of the invention, the specified linker may be a dendritic linker type for covalent binding more than one molecule of the drug with the antibody through branching multifunctional linker molecules (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) Tetrahedron Letters 43:1987-1990). Dendritic linkers can increase the molar ratio of the drug to the antibody, i.e. when loading, which corresponds to the efficiency of the ADC. Thus, if constructed on the basis of cysteine antibody bears only one reactive thiol group, sistei�and, the many molecules of the drug can be covalently bound through a dendritic linker.

Amino acid component

The linker may contain amino acid residues. Amino acid component (-Ww-), when present, links the antibody (Ab) molecule drug (D) is designed based on the cysteine conjugate "antibody-drug" (ADC) according to the invention.

-Ww- is a dipeptide, Tripeptide, tetrapeptide, Pentapeptide, Hexapeptide, heptapeptide, octapeptide, nonapeptide, Decapeptide, undecapeptide or dodecapeptide component. Amino acid residues constituting the specified amino acid component, are natural remnants, and small amino acids, and unnatural amino acid analogs, such as citrulline. Each of the components-W - is independently has the formula shown below in square brackets, and w stands for an integer from 0 to 12:

,

where R19represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2HE, -CH(Oh)CH3, -CH2CH2SCH3, -CH2CONH2, CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2/sub> )3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(Oh)CH2NH22-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, phenyl, cyclohexyl,

If R19is not hydrogen, the carbon atom attached to R19is chiral. Each carbon atom is attached to R19independently present in (S)- or (R)-configuration or in the form of racemic mixtures. Thus, the amino acid components may be enantiomerically pure, racemic or diastereomeric.

Examples of amino acid components-Ww- is a dipeptide, Tripeptide, tetrapeptide or Pentapeptide. Examples of dipeptides are valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Examples of tripeptides are glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues constituting the linker component, are the natural amino acid residues, and small amino acids, and unnatural amino acid analogs, such as citrulline.

The amino acid component may be enzymatic Ross�captured by one or more enzymes, including tumor-associated protease, with the release of a molecule medicines (D), which, in one embodiment of the invention, after the release undergoes protonation in vivo with the formation of the drug (D). Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by specific enzymes, such as, for example, tumor-associated protease, cathepsin b, C and D, or platinova protease.

The GS spacer component

The GS spacer component (-Y-), when present (y=1 or 2), links an amino acid component (-Ww-) c molecule drug (D), if such amino acid component is present (w=1-12). Alternatively, if the amino acid component is not available, the GS spacer component assigns the extension component of the molecule of the drug. If there are no amino acid component and an extension component (w, y=0), the GS spacer component binds a molecule of the drug with the antibody molecule. The GS spacer components are the components of two General types: smolinerwien and nesmolkayuschee. Nesamierinamais GS spacer component is a component where a portion of the GS spacer component or the entire e�from component remain associated with a molecule of the drug after cleavage, in particular, enzymatic cleavage of the amino acid component from conjugate “antibody-drug” or from the conjugate “drug-linker”. If the ADC containing a glycine-glycine GS spacer component or a glycine GS spacer component, are subjected to enzymatic cleavage by a protease associated with tumor cells, a protease associated with cancer cells, or a protease associated with lymphocytes, a molecule of glycine-glycine-drug" or a molecule of glycine-drug" cleaved Ab-Aa-Ww. In one embodiment of the invention, an independent hydrolysis reaction occurs in the target cells and leads to the cleavage of bonds in the molecule glycine-drug with drug release.

In another embodiment of the invention-YI- is a u-aminobenzeneboronic component (RAV), the phenylene portion of which is substituted with Qmwhere Q represents C1-C8alkyl, -O-(C1-C8alkyl), halogen, nitro or cyano, and m is an integer from 0 to 4.

Representative embodiments nesmolkayuschee GS spacer component (-Y-) is-Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one of its variants the present invention relates to conjugate “drug-�inker” or ADC, lacking the GS spacer component (y=0), or to their pharmaceutically acceptable salt or solvate.

Alternative ADC containing carolinensis GS spacer component may liberate a molecule of D. In one embodiment of the invention,- Y - is a PAB group that is linked to-Ww - via the nitrogen atom of the amino group PAB, and is directly related to the molecule-D via a carbonate, carbamate or ether group, where the ADC has the following representative structure:

,

where Q represents C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-cyano; m is an integer from 0 to 4; and p is 1-4.

Other examples carolinensis spacers include, but are not limited to, aromatic compounds that, by their electronic properties are similar to the RAV group, such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), heterocyclic analogs RAV (US 2005/0256030) beta-glucuronide (WO 2007/011968) and ortho - or para-aminobenzoate. Can spacers be used that undergo cyclization after hydrolysis of the amide bond, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo ring[2.2.1] and bicyclo[2.2.2]-system (Storm et al. (1972 J. Amer. Chem. Soc. 94:5815) and amide 2-aminophenylamino acid (Amsberry, et al. (1990) J. Org. Chem. 55:5867). Examples carolinensis spacers used in the ADC, are amine-containing medications, substituted in position of glycine (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

A representative of the GS spacer components (-YI-) represented by formulas X-XII:

Dendritic linkers

In another embodiment of the invention the linker L may be a dendritic linker type used for covalent binding more than one molecule of the drug with the antibody through branching multifunctional linker molecules (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of the drug to the antibody, i.e., the load, which corresponds to the efficiency of the ADC. Thus, if constructed on the basis of cysteine antibody contains only one reactive thiol group of cysteine, through a dendritic linker can be attached to a large number of molecules of the drug. Representative embodiments of branched dendritic linkers are gendarmerie components, such as 2,6-bis(gidroximetil)-p-cresol and 2,4,6-Tris(gidroximetil)-phenol (WO 2004/01993; Szlai et al. (2003) J. Amer. Chem. Soc. 125:15688-15689; Shamis et al. (2004) J. Amer. Chem. Soc. 126:1726-1731; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499).

In one embodiment of the invention the GS spacer component is a branched bis(gidroximetil)styrene (BHMS), which can be used for the introduction and release of many drugs and has the structure:

,

containing 2-(4-aminobenzylidene)propane-1,3-diology dendrimeric component (WO 2004/043493; de Groot et al. (2003) Angew. Chem. Int. Ed. 42:4490-4494), where Q represents C1-C8alkyl, O-(C1-C8alkyl), -halogen, -nitro or-cyano; m is an integer from 0 to 4, n is 0 or 1; and R is equal to 1-4.

Representative embodiments of the compounds of conjugates of an antibody-drug of formula I are compounds XIIIa (MS), XIIIb (val-cit), XIIIc (MC-val-cit) and XIIId (MC-val-cit-PAB):

Other representative embodiments of the compounds of conjugates of an antibody-drug of formula Ia are compounds XIVa-e:

,

where X represents:

Y represents:

and R independently represents N or C1-C6alkyl, and n is 1-12.

In another embodiment of the invention, the linker has a reactive functional g�the SCP, which contains a nucleophilic group that reacts with an electrophilic group present on the antibody. Suitable electrophilic groups present on the antibody, include, but are not limited to, the carbonyl group of aldehyde and ketone. Heteroatom of a nucleophilic group of a linker can react with an electrophilic group on the antibody and form a covalent bond with the molecule of the antibody. Suitable nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, a hydrazine carboxylate, and originated. The electrophilic group on the antibody provides a suitable site for attachment to the linker.

Usually peptideprophet linkers can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be formed, for example, by the method of synthesis in solution (E. Schroder & K. Lubke (1965) "The Peptides", volume 1, pp 76-136, Academic Press) that is well known to specialists in the field of peptide chemistry. Linker intermediate compounds can be obtained using any combination or sequence of reactions involving the GS spacer, extension and amino acid components. Such GS spacer, extension and amino acid components may contain reactive fu�csinalni group, which, by their nature, are electrophilic, nucleophilic, or free radical. Reactive functional groups include, but are not limited to, a carboxy, a hydroxyl, a pair-nitrophenylarsonic, isothiocyanate, and leaving groups, such as O-mesyl, O-tosyl, -Cl, -Br, -I or maleimide.

For example, the charged substituent such as sulfonate (-SO3-) or ammonium, may increase the solubility of the reactants in water and to facilitate the reaction of the linker reagent with the antibody or with medication, or to facilitate the reaction of Ab-L (intermediate connections “antibody-linker” molecule D or D-L (intermediate connection “drug-linker”) with Ab, depending on the method of synthesis used to obtain the ADC.

Linker reagents

Conjugates of the antibody and auristatin can be obtained using a variety of bifunctional linker reagents such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hex�jiamin), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene).

Conjugates of the antibody-drug" may also be obtained with the use of linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate), and using bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, 1,8-bis-maleimidomethyl (BM(PEO)2), and 1,11-bis-maleimidomethyl (BM(PEO)3), which are commercially available and supplied by Pierce Biotechnology, Inc., ThermoScientific, Rockford, IL, and other suppliers of reagents. Bis-maleimide reagents allow sequentially or simultaneously to attach thiol group in cysteine antibody to a thiol-containing molecule drugs to the label, or linker intermediate connection. In addition maleimide, other functional groups that react with thiol group in cysteine antibody molecule drug, a label, or linker intermediate compound are todatetime, bromoacetamide, vinylpyridine, disulfide, pyridyldithio, isocyanate and isothiocyanate.

Suitable linker reagents can also be obtained from other commercial sources such as Molecular Biosciences Inc. (Boulder, CO), or they can be synthesized in accordance with the procedures described by Toki et al. (2002) J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7:180 to 186; in U.S. patent 6214345; in WO 02/088172; United States patent application 2003130189 and 2003096743; in WO 03/026577; WO 03/043583 and WO 04/032828.

Lengthening the components of the formula (IIIa) can be introduced into a linker by reaction between the following linker reagents with the N-end amino acid component:

,

where n is an integer from 1 to 10, and T represents-H or-SO3Na;

,

where n is an integer from 0 to 3;

Lengthening components can be introduced into a linker by reaction between the following bifunctional reagents with the N-end amino acid component:

,

where X represents Br or I.

Lengthening the components of this formula can also be introduced into a linker by reaction between the following bifunctional reagents with the N-end amino acid component:

Representative dipeptide Lin�Erny reagent valine-citrulline (val-cit or vc), including maleimides extension component and pair-aminobenzeneboronic (PAB) carolinensis the spacer has the structure:

Representative phe-lys(Mtr is mono-4-methoxytrityl)-dipeptide linker reagent comprising maleimide extension component and carolinensis GS spacer RAV component, can be obtained as described in Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60, and has the structure:

Obtaining conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug"

The ADC of formula I may be obtained in several ways with the use of organic synthesis reactions, conditions, and reagents known in the art, including: (1) reaction of cysteine groups constructed on the basis of cysteine antibody with a linker reagent with the formation of intermediate compounds “antibody-linker” Ab-L, via a covalent bond, followed by reaction of interaction with the activated molecule drug D; and (2) reaction of a nucleophilic group of a molecule of the drug with a linker reagent with the formation of the intermediate connection “drug-linker” D-L through a covalent bond, with the subsequent reaction of interaction with cysteine� group antibodies, constructed with cysteine. In the methods of conjugation (1) and (2), to obtain conjugates of an antibody-drug of formula I can be used of different constructed on the basis of cysteine antibody molecule drugs and linkers.

The thiol group of cysteine antibodies are nucleophilic and have the ability to interact with the formation of covalent bonds with electrophilic groups on linker reagents and intermediate compounds “drug-linker”, including (i) active esters such as NHS-esters, HOBt-esters, halogenfree and acid halides; (ii) alkyl - and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group; and (iv) disulfides, including pyridylsulfonyl resulting from sulfide exchange. Nucleophilic groups on the molecule pharmaceuticals include, but are not limited to, amine, thiol, hydroxyl, hydrazide, Aksinya, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group that can react with electrophilic groups on linker units and the linker reagent with the formation of covalent bonds.

Designed on the basis of cysteine antibodies, are capable of forming conjugates with Linke�governmental reagents, can be obtained by treatment with a reducing agent such as DTT (reagent of Cleland, dithiothreitol) or TCEP (hydrochloride Tris(2-carboxyethyl)phosphine; Getz et al. (1999) Anal. Biochem. Vol. 273:73-80; Soltec Ventures, Beverly, MA), followed by re-oxidation reactions for the formation of Mirzayeva and noticablely disulfide bonds (example 5). For example, full-size, constructed on the basis of cysteine monoclonal antibody (thio-Mab), expressed in CHO cells, is subjected to reaction recovery from approximately 50-fold excess of TCEP for 3 hours at 37aboutWith to restore the disulfide bonds in the addition products of cysteine, which can form between the newly introduced cysteine residues and the cysteine present in the culture medium. Restored thio-Mab is diluted and loaded onto HiTrap column S in 10 mm sodium acetate, pH 5, and eluted with PBS containing 0.3 M sodium chloride. Disulfide bonds between cysteine residues present in the parent Mab, then restore using diluted (200 nm) aqueous copper sulfate (CuSO4) at room temperature over night. Alternatively, after reductive cleavage of cysteine adducts, to re-restore noticablely disulfide groups constructed on the basis of cysteine antibodies, can be�ü used in an efficient oxidant such as dehydroascorbic acid (DHAA). Can also be used and other oxidizing agents, i.e., oxidizing agents and oxidation conditions known to specialists. Also effective is the oxidation in air. This stage of mild incomplete re-oxidation with a high degree of reliability contributes to the formation of noticablely disulfides and preservation of the thiol groups of the newly introduced cysteine residues. Then add approximately 10-fold excess of intermediate connection “drug-linker”, for example, MC-vc-PAB-MMAE, mix and leave for about 1 hour at room temperature for the implementation of conjugation with the formation of the conjugate is anti-CD79b antibody-drug”. Then conjugated mixture is subjected to gel filtration, loaded onto HiTrap column S and eluted from this column to remove excess intermediate connection “drug-linker” and other impurities.

Figure 23 illustrates the overall method of obtaining constructed on the basis of cysteine antibodies expressed in cell culture for subsequent conjugation. If the environment for culturing cells contains cysteine, between the newly introduced cysteine amino acid and cysteine to form a disulfide adducts. These cysts�new adducts, marked with circles for a representative thio-Mab (left) figure 23, should be restored with getting designed on the basis of cysteine antibodies that can be used for the subsequent conjugation reaction. Cysteine adducts, presumably, along with various messagevine disulfide bonds, is subjected to reductive cleavage under the action of vosstanovitelya such as TSAR, obtaining reduced forms of the antibody. Messageview disulfide bonds between paired cysteine residues are again formed under conditions of incomplete oxidation, such as treatment with copper sulfate, DHAA, or exposure to atmospheric oxygen. Newly commissioned, designed and unbound cysteine residues become available to react with linker reagents or intermediate compounds “drug-linker”, resulting in formation of conjugates of antibodies according to the invention. Thio-Mab, a murine cell lines of mammals, consists of Cys-adduct, conjugated with an introduced Cys through education-S-S-bonds. Therefore, purified thio-Mab should be subjected to recovery and oxidation as described in example 5, to obtain a reactive thio-Mab. These thio-Mab is used to produce conjugate with maleimide containing �fitotoksicheskie medicines the fluorophores and other markers.

Immunoliposome

Described herein is an anti-CD79b antibody may also be obtained in the form of immunoliposome. "Liposome" is a small vesicles composed of different types of lipids, phospholipids and/or surfactant that can be used to deliver drugs to the mammal. Components of liposomes are usually located so that they form a bilayer, similar to the lipid bilayer in biological membranes. Liposomes containing the antibody are used, known in the art such as the methods described in publication Epstein et al.,Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwang et al.,Proc. Natl Acad. Sci. USA77:4030 (1980); in patents 4485045 and 4544545; and in the application WO97/38731 published October 23, 1997, Liposomes with an increased half-life in blood is described in U.S. patent No. 5013556.

Specifically used liposomes composed of lipids such as phosphatidylcholine, cholesterol and PEG-derivationally phosphatidylethanolamine (PEG-PE), can be obtained by evaporation from a reverse phase. Liposomes extruded through filters with defined pore size to thereby produce liposomes of the desired diameter. Fab'-fragments of the antibodies according to the invention can be anywhereman with liposomes, as described in publications Martin et al.,J. Bol. Chem.257:286-288 (1982), as a result of the reaction of disulfide exchange. The liposome can include, but not necessarily, a chemotherapeutic agent. Cm. Gabizon et al.,J. National Cancer Inst.81(19):1484 (1989).

B.Some methods of obtaining antibodies

1.Screening for anti-CD79b antibodies with the desired properties

Methods of producing antibodies that binds to CD79b polypeptides described above. If desired, can also be selected and other antibodies with defined biological properties.

The growth-inhibitory effect of anti-CD79b antibodies according to the invention can be assessed by methods known in the art, e.g., using cells which Express CD79b polypeptide either endogenously or after transfection CD79b gene. For example, appropriate tumor cell lines and CD79b-transfetsirovannyh cells can be treated with a monoclonal anti-CD79b antibody according to the invention at various concentrations for a few days (e.g., 2-7 days), and colored with crystal violet or MTT or analyzed by some other colorimetric assays. Another method of measuring the level of proliferation may represent a comparison of the uptake of3H-thymidine by the cells treated in the presence or in the absence of anti-CD79b antibodies according to the invention. After IPL treatment�Ki cells gather, and the level of radioactivity incorporated into DNA, counted in a scintillation counter. Appropriate positive control includes processing the selected cell lines to the growth-inhibitory antibody, which is known to inhibit the growth of such cell lines. The inhibition of growth of tumor cells in vivo can be determined by various methods known in the art. Tumor cell line can be a cell line, sverkhekspressiya CD79b polypeptide. In one embodiment of the invention, the anti-CD79b antibody inhibits the proliferation of CD79b-expressing tumor cell in vitro or in vivo by about 25-100%, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, as compared with untreated tumor cells when the antibody concentration of about 0.5 to 30 μg/ml. the Inhibition of growth can be measured when the antibody concentration of about 0.5 to 30 μg/ml or about 0.5 nm to 200 nm in cell culture, where the specified growth inhibition is determined 1-10 days after treatment of tumor cells with antibody. The antibody has a growth-inhibitory effect in vivo, if the introduction of anti-CD79b antibody in amounts of from about 1 μg/ml to 100 mg/kg body weight results in reduction in tumor size or a reduction in the level of proliferation of tumor cells over a period of time�and from about 5 days to 3 months and preferably for a time period of about 5 to 30 days after the first administration of the antibody.

For the selection of anti-CD79b antibody that induces cell death, can be estimated a loss of membrane integrity, as it was found, for example, the uptake of propidium iodide (PI), trypan blue or 7AAD compared with the control. Analysis of PI uptake may be achieved in the absence of complement and immune effector cells. Tumor cells expressing CD79b polypeptide incubated either with medium or with medium containing the appropriate anti-CD79b antibody (e.g., about 10 μg/ml). Cells were incubated for 3 days. After each treatment the cells were washed and divided into aliquots in test tubes (35 mm, 12×75) with a tight fitting lid (1 ml per tube, 3 tubes per treatment group) for removal of cell clusters. Then in a test-tube add PI (10 μg/ml). Samples can be analyzed on a flow cytometer FACSCAN® and using a computer program FACSCONVERT® CellQuest (Becton Dickinson). Anti-CD79b antibodies that induce statistically significant levels of cell death as determined by PI uptake may be selected as an anti-CD79b antibody inducing cell death.

For the screening of antibodies that bind to the epitope on CD79b polypeptide associated with the performance�against the interest of the antibody, can be carried out routine analysis on cross-blocking, such as analysis, described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This analysis can be carried out in order to determine whether associated antibody test with the same site or epitope that binds a known anti-CD79b antibody. Alternative or additionally can be carried out mapping of epitopes known methods. For example, the sequence of the antibody may be subjected to mutagenesis, such as alanine-scanning mutagenesis to identify contact residues. The mutant antibody is first tested for binding to a polyclonal antibody to guarantee a "correct" installation. In a different method, peptides corresponding to different regions of the CD79b polypeptide, can be used in assays for competitive binding with the test antibodies or with a test antibody and an antibody that binds to a characterized or known epitope.

2. Some methods of screening libraries

Anti-CD79b antibody according to the invention can be obtained using combinatorial libraries to screen for antibodies with the desired activity or activities need. For example, experts know a number of methods for phage libraries�about submission and screening such libraries for antibodies, having desirable binding properties. Such methods are mainly described in the publication Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O'brien et al., ed., Human Press, Totowa, NJ), and some of their variants in the publication of Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

In principle, the synthetic clones of antibodies are selected by screening phage libraries containing phage that represents the various fragments of the variable regions of an antibody (Fv) attached to the protein shell of the phage. Such phage libraries are being panning using affinity chromatography against the desired antigen. Clones expressing Fv-fragments, is able to communicate with the desired antigen, is subjected to adsorption on the antigen and, thus, their separation from nesvezhije clones in the library. Then communicating clones eluted from the antigen, after which the number of these clones may be increased by additional cycles of adsorption/elution of the antigen. Any of the anti-CD79b antibodies according to the invention can be obtained using the appropriate procedure screening antigen for the selection of interest on phage clone with the subsequent construction of a full-sized clone of anti-CD79b antibodies using the Fv sequences derived from interest phage clone, and appropriate sequences of the constant region (Fc), described by Kabat et al., Sequeces of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

In some embodiments of the invention antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one of which is located in the light chain (VL) and the other in the heavy chain (VH) and variable in these areas, there are three hypervariable loops (HVR) or complementarity-determining region (CDR). The variable domains can be functionally presented on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which each of these variable domains attached to a constant domain and undergoes non-covalent interaction, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Used here scFv-encoding phage clones and Fab encoding phage clones, in General, are called "fagbemi Fv-clones" or "Fv clones".

The sets of genes VH and VL can be separately cloned by polymerase chain reaction (PCR) and subjected to non-specific recombination in phage libraries, and then they can be analyzed for antigen-binding clones as described in the publication of Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources allow to obtain antibodies with high affinal�Yu to the immunogen, and it does not require the procedure of constructing a hybrid. Alternative can be cloned set of human wild-type antibodies for one source of human antibodies against noautoindent, as well as a wide range of autoantigens, without resorting to any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). And finally, the original library can also be synthesized by cloning paranirvana segments V-gene from stem cells, and using PCR primers containing randomizearray sequence encoding highly variable CDR3-region, in order to achieve rearrangement in vitro as described by Hoogenboom &Winter, J. Mol. Biol., 227:381-388 (1992).

In some embodiments of the invention, to represent fragments of antibodies by their attachment to minor shell protein pIII, using filamentous phage. Fragments of antibodies can be presented in the form of single-chain Fv fragments, in which the domains VH and VL connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or as Fab fragments, in which one chain is attached to pIII and the other is secreted into the bacterial periplasm of the host cell, where the Assembly structure of the protein shell of the Fab, which is about�obrazuetsa on the surface of phage by replacing some of the envelope protein of the wild type, for example, as described in the publication Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

Typically, the nucleic acid containing genes that encode fragments of antibodies obtained from immune cells taken from humans or animals. If you must obtain a library consisting mainly of clones of anti-CD79b antibodies, individual immunize CD79b for helping him humoral response, then, to construct the library, the isolated spleen cells and/or circulating b cells not belonging to the population of peripheral blood lymphocytes (pbls). In a preferred embodiment of the invention a library of gene fragments of human antibodies, clones containing predominantly anti-CD79b antibody, is obtained by means of the formation of the response in the form of the production of anti-CD79b antibodies in transgenic mice bearing an array of functional human immunoglobulin genes (and not containing the functional system of the production of endogenous antibodies), where such CD79b-immunization will lead to the formation of b cells producing human antibodies against CD79b. Generation of transgenic mice producing human antibody, described below.

Further enrichment of the cell population reactive anti-CD79b antibodies can be achieved by using a suitable screening procedure for the apportionment�of b-cells, expressing CD79b-specific membrane-bound antibody, e.g., by separation of cells using CD79b-affinity chromatography or adsorption of cells to CD79b-labeled fluorochromes, and subsequent cell sorting with activation of fluorescence (FACS).

The alternative use of spleen cells and/or b cells or other pbls taken from unimmunized donor provides a better representation of the possible antibody repertoire, and also allows you to design a library of antibodies in animals of any kind (human or other animal) who CD79b is not antigenic. To create the library, including the design of antibody genes in vitro, the individual taking stem cells and isolated nucleic acids that encode paranirvana segments of antibody genes. Interest immune cells can be isolated from animals of different species such as human, mice, rats, lagomorphs, wolves, dogs, cats, pigs, cows, horses, birds, etc.

Nucleic acid encoding the gene segments of variable regions of antibodies (including VH - and VL segments), is isolated from the interest of cells and amplificateur. In the case of libraries rearranging genes VH and VL, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed in-house�m polymerase chain reaction (PCR) using the primers the respective 5'- and 3'-ends rearranging genes VH and VL described in the publication of Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), and thereby obtain different sets of V-genes for expression. V-genes may be amplified from cDNA and genomic DNA using reverse primers at the 5'end of the exon encoding the Mature V-domain and direct primers derived from the J-segment as described in the publication of Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989). However, for amplification of cDNA reverse primers can also be obtained on the basis of leader exon as described in Jones et al., Biotechnol., 9:88-89 (1991), and direct primers can be introduced into the constant region, as described in the publication Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarity can be obtained with the degenerate primers described in the publication of Orlandi et al. (1989) or Sastry et al. (1989). In some embodiments of the invention a variety of libraries to maximize the use of PCR primers targeted to each family of V-genes for amplification of all existing arrangements VH and VL that are present in the sample nucleic acid immune cells, for example, by the method described in the publication Marks et al., J. Mol. Biol., 222:581-597 (1991) or the method described in the publication Orum et al., Nucl Acids Res., 21:4491-4498 (1993). To clone the amplified DNA into expression vectors, PCR-p�aimer can be entered rare restriction sites as a label at one end, as described by Orlandi et al. (1989), or such introduction can be carried out by PCR amplification using a labeled primer as described by Clackson et al., Nature, 352:624-628 (1991).

Sets synthetically rearanging V genes can be obtained in vitro from V-gene segments. Most segments of human VH genes were cloned and sequenced (as reported in the publication of the Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and then mapped (as reported in the publication Matsuda et al., Nature Genet., 3:88-94 (1993); these cloned segments (including all the major conformations of the loop H1 and H2) can be used to obtain different sets of VH genes using PCR primers encoding H3 loop with different sequences and different lengths, as described in the publication Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Can be also obtained sets VH, having the diversity of sequences, centered in a long H3 loop of a single length as described in the publication Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Were cloned and sequenced human segments are Vκ and Vλ (as reported in the publication of Williams & Winter, Eur. J. Immunol., 23:1456-1461 (1993)), and such segments can be used to generate synthetic sets of light chains. Sets synthesized V genes derived from the folded structures of a number of VH and VL, and L3 and H3 of different lengths, will code�the presence of antibodies with significantly varying structure. After amplification of the coding DNA of the gene V, gene segments V germ line can be rearrangeable in vitro methods described in the publication Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

Sets of fragments of antibodies can be constructed by combining the sets of genes VH and VL in several ways. Each set can be created in different vectors, and these vectors are subjected to recombination in vitro, for example, as described in publications Hogrefe et al., Gene, 128:119-126 (1993), or in vivo by combinatorial infection, e.g., the loxP system described in the publication Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). In the method of recombination in vivo use of double-stranded nature of the Fab-fragments to eliminate restrictions on the size of the library and associated with the efficiency of transformation of E. coli. The original sets of VH and VL cloned separately, one into fahmida and the other into a phage vector. Then combine two libraries by infection with bacteria containing fahmida, phage, so that each cage contained various combinations, and the library size was limited only by the number of cells present (about 1012clones). Both vectors contain the signals of recombination in vivo, resulting in the genes of VH and VL undergo recombination with the formation of a single replicon together and packaged into phage virions. These huge libraries represent b�lshoe the number of different antibodies with high affinity (K d-1about 10-8M).

Alternative such sets can be sequentially cloned in the same vector, e.g. as described in the publication Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or they can be assembled together by PCR and then cloned, for example, as described in the publication Clackson et al., Nature, 352:624-628 (1991). PCR Assembly can also be implemented to attach the DNA of VH and VL to DNA encoding a flexible peptide spacer, with the formation of sets of single-chain Fv (scFv). In yet another method "PCR Assembly in cells" is used to group genes VH and VL in lymphocytes by PCR, followed by cloning of sets of linked genes as described in the publication Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

Antibodies produced source libraries (natural or synthetic) may have a low affinity (Kd-1from about 106up to 107M-1), but affinity maturation can also be modelled in vitro by constructing and re-selection from secondary libraries as described in the publication of Winter et al. (1994), see above. For example, mutation can be introduced accidentally in vitro using the polymerase reaction with a probability of error (Leung et al., Technique, 1:11-15 (1989)) in the method described by Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or the method described by Gram et al., Proc. Natl. Acad. Sci USA, 89:3576-3580 (1992. In addition, affinity maturation can be achieved by random mutation of one or more CDRs, for example, by using PCR conducted using primers carrying randomizearray sequence spanning the CDR of interest in individual Fv clones and screening of clones with higher affinity. In the application WO 9607754 (published March 14, 1996) describes a method for inducing mutagenesis in a hypervariable region light chain immunoglobulin with obtaining a library of light chain genes. Another effective method is the recombination of VH domains or VL, selected using phage presentation, with sets of natural variants of the V-domain, isolated from unimmunized donors, and screening for higher affinity, carried out in several rounds of the permutation circuits, as described in publications Marks et al., Biotechnol., 10:779-783 (1992). This method allows to produce the antibodies and fragments of antibodies with appendectomy about 10-9M or less.

Screening libraries can be accomplished by various methods known to specialists. For example, CD79b can be used to cover the wells of adsorption plates, or expressed on the cells of the host that is attached to the adsorption tablets, or it can be used for cell sorting, or �anywhereman with Biotin for capture on spheres coated with streptavidin, or it can be used in any other method of panning phage libraries of the view.

Samples of phage libraries are contacted with immobilized CD79b in conditions suitable for binding at least part of the phage particles with the adsorbent. Typically, to simulate physiological conditions, select the appropriate pH, ionic strength, temperature, etc. Phages associated with the solid phase, washed, and then eluted with acid, for example as described in the publication Barbas et al., Proc. Natl. Acad. Sci USA, 88:7978-7982 (1991), or by alkali, e.g. as described in publications Marks et al., J. Mol. Biol., 222:581-597 (1991), or by competitive binding to CD79b antigen, for example, in accordance with the procedure similar to the procedure carried out in the method of competitive binding to the antigen, as described in the publication Clackson et al., Nature, 352:624-628 (1991). Phages can be subjected 20-1000-fold enrichment in a single round of selection. In addition, the enriched phage can be grown in bacterial culture and subjected to additional rounds of selection.

The effectiveness of selection depends on many factors, including the kinetics of dissociation during washing, and on the ability of many antibody fragments simultaneously to contact with the antigen on the same phage. Antibodies with fast dissociation kinetics (and weak affinity tie�tion) can be fixed due to short-term leaching, the use of multivalent phage performance and high-density coating antigen on the solid phase. This high density not only stabilizes the phage due to multivalent interactions, but also conducive to the re-binding of the phage, which is dissociatively. The selection of antibodies with low dissociation kinetics and high affinity of binding) can be improved with longer leaching and the use of monovalent phage ideas described in the publication Bass et al., Proteins, 8:309-314 (1990) and in WO 92/09690, and also due to the low density coating antigen, as described in publications Marks et al., Biotechnol., 10:779-783 (1992).

May be the selection between fagbemi antibodies with different affiniscape, even with slightly different affinity to CD79b. However, random mutation of a selected antibody (e.g., vnesennya in some of the above-described methods of affinity maturation) is likely to lead to the formation of many mutants, most of which will be contacted with the antigen, and some of them with even greater affinity. With a limited level CD79b rare phage with high affinity can withstand such competition. To save all mutants with higher affinity, phages can be incubated with isbit�Ohm biotinylating CD79b, but this biotinylating CD79b should have a lower molar concentration than the molar affinity constant target for CD79b. Then the phages with high affinity binding can be captured by paramagnetic spheres coated with streptavidin. Such "equilibrium capture" allows the selection of antibodies based on their binding affinity with the sensitivity, which allows a large excess of phages with lower affinity to isolate mutant clones, which is only two times higher affinity. The conditions used for washing phages associated with the solid phase, can also be modified to identify their differences on the basis of the kinetics of dissociation.

Clones of anti-CD79b antibodies can be selected according to their activity. In certain embodiments, the present invention relates to anti-CD79b antibodies that bind to living cells expressing CD79b. In one of its variants the present invention relates to anti-CD79b antibodies that block ligand binding with CD79b CD79b, but do not block ligand binding CD79b with a second protein. Fv clones corresponding to such anti-CD79b antibodies, can be selected by: (1) selection of clones of anti-CD79b antibodies of ragovoy library, as described above, and, but not necessarily, amplification of the selected population of phage clones by culturing the�Oh population in a suitable bacterial host; (2) select CD79b and a second protein against which it is necessary to produce a blocking and non-blocking activity, respectively; (3) adsorption of phage clones of anti-CD79b antibodies to immobilized CD79b; (4) the use of an excess of the second protein for the elution of any undesired clones that recognize CD79b-binding determinants which overlap with the binding determinants of the second protein or have a common binding determinants; and (5) elution of the clones which remain adsorbed after carrying out stage (4). Clones with the desired blocking/non-blocking properties, may also be, but not necessarily enriched by conducting one or more re-selection procedures described in this application.

DNA encoding produced by hybridoma monoclonal antibodies or Fv phage clones representation according to the invention, can be easily isolated and sequenced in accordance with standard procedures (e.g., using oligonucleotide primers that are able to specifically amplificatoare of interest encoding the heavy and light chains from hybridomas or ragovoy DNA-matrix). After isolation of this DNA can be integrated into expression vectors, which are then transferout in the host cell, such as E. coli cells, simian TC�TCI COS cells Chinese hamster ovary (Cho) cells or myeloma cells that otherwise do not produce the protein immunoglobulin, resulting in these recombinant cells-the owners are synthesized the desired monoclonal antibodies. Discussion recombinant expression of an antibody-coding DNA in bacteria can be found in review articles Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

DNA encoding the Fv clones according to the invention, can be combined with known DNA sequences encoding the constant region of the heavy chain and/or light chain (e.g., the appropriate DNA sequence may be obtained as described in the publication Kabat et al., see above), with the formation of clones encoding the full-sized heavy and/or light chains or their fragments. It should be noted that this purpose can be used a constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region may be derived from humans or animals of any kinds. In the definition used here, the term "chimeric" and "hybrid" antibody includes Fv-clone, derived from DNA variable domain of an animal of one species (e.g. human), which was then attached to the DNA of a constant region of an animal of another species with the formation of the coding(them) a sequence�(s) "hybrid" full-size heavy chain and/or light chain. In some embodiments of the invention Fv clone derived from human DNA variable regions, attached to the DNA of a human constant region with the formation of the encoding(s) sequence(s) for all full-sized human or non-heavy and/or light chains.

DNA encoding anti-CD79b antibody, derived from the hybridomas, can also be modified, for example, by replacement of homologous murine sequences derived from hybrid clone, the coding sequence for the human constant domains of the heavy and light chain (e.g. as in the method described by Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The DNA that encodes the antibody, derived from the hybridomas or Fv-clone, or a fragment thereof, can also be modified by covalent binding of the immunoglobulin coding sequence with full-size sequence that encodes nimmanahaeminda polypeptide, or a part of it. Thus can be obtained a "chimeric" or "hybrid" antibodies having the binding specificity of antibodies, derived from Fv-clone or a hybrid clone according to the invention.

C.Antibody-dependent mediated by the enzyme Pro-drug therapy(ADEPT)

Antibodies according to the invention can also be used in ADEPT in �IDA antibodies, conjugated with a prodrug-activating enzyme which converts a prodrug (e.g., peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. patent No. 4975278.

Enzyme component immunoconjugate suitable for ADEPT includes any enzyme capable of acting on a prodrug, namely, to convert the prodrug to the more active cytotoxic form.

Enzymes that can be used in the method according to the invention, include, but are not limited to, alkaline phosphatase, which can be used for converting phosphate-containing prodrug into the free drug; arylsulfatase, which can be used for converting sulfate-containing prodrug into the free drug; sitoindosides, which can be used for converting non-toxic 5-fertilizin in anti-cancer drug, namely 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which can be used for converting peptide-containing prodrug into free drugs; D-alanismorissette, which can be used to develop concept design for�cluding prodrug, containing D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase, which can be used for converting glycosylated prodrug into the free drug; β-lactamase, which can be used for the conversion of medicines, derivatizing β-lactams, into free drugs; and penicillin-amidase, such as penicillin V amidase or penicillin G-amidase, which can be used for the conversion of medicines, derivatizing of nitrogen atoms of amine phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Alternative antibodies with enzymatic activity, also known in the art as "abzyme", can be used for the conversion of a prodrug according to the invention into free active drugs (see, e.g., Massey,Nature328:457-458 (1987)). Conjugates of an antibody-Abim" can be obtained as described in the present application, for the delivery of abzyme in the population of tumor cells.

The enzymes according to the invention can be covalently attached to anti-CD79b antibody by methods known in the art, for example, using heterobifunctional cross-crosslinking reagents discussed above. Alternate�UPE hybrid proteins containing at least the antigen-binding region of the antibody according to the invention, associated with at least a functionally active portion of an enzyme according to the invention, can be constructed by methods of recombinant DNA, known in the art (see, e.g., Neuberger et al.,Nature312:604-608 (1984).

D. Anti-CD79b antibody

In addition to those described here anti-CD79b antibody is also considered obtaining variants of anti-CD79b antibodies. Options anti-CD79b antibodies can be obtained by introducing appropriate nucleotide modifications in the encoding DNA and/or by synthesis of the desired antibody or polypeptide. It should be noted that amino acid modifications can affect post-translational processes of the anti-CD79b antibody, such as changing the number or position of glycosylation sites or changing membrane-zakalivayuschie properties.

The changes described here in the anti-CD79b antibody may be made, for example, with the use of any methods and guidelines for making conservative and non-conservative substitutions are described, for example, in U.S. patent No. 5364934. These modifications may be substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide, resulting in a change in amino acid sequence compared with the sequence of natural antibodies or poly�of eptica. Such a modification is, but not necessarily, replacing at least one amino acid residue with any other amino acid in one or more domains of an anti-CD79b antibody. In order to determine whether to integrate, replace, or deleteroute acid balance without any negative impact on the desired activity of the antibody may be a comparison of the sequences of the anti-CD79b antibody with the sequence of homologous known protein molecules to minimize the number of modifications of the amino acid sequence deposited in a region with high homology. Amino acid substitutions can be the result of replacing one amino acid by another amino acid having similar structural and/or chemical properties, such as replacement of leucine by serine, i.e., conservative amino acid substitutions. Insertions or deletions may, but need not, be in the range of from about 1 to 5 amino acids. Allowable modifications can be identified through the systematic introduction of insertions, deletions or substitutions of amino acids in the sequence and testing the obtained variants on the activity of detectable full-length or Mature native sequence.

The present invention also relates to fragments of the anti-CD79b antibody. Such fragments may be truncated�t N-end or C-end, or they may not contain internal residues, for example, compared with full-sized native antibody or protein. In certain fragments lack amino acid residues that do not play an important role in the desired biological activity of the anti-CD79b antibody.

Fragments of the anti-CD79b antibody may be obtained by any of various standard methods. Desired peptide fragments may be obtained by chemical synthesis. An alternative method involves the production of antibodies or fragments of the polypeptide by enzymatic digestion, e.g., by treating the protein with an enzyme, which is known cleaves proteins at sites defined by particular amino acid residues, or by hydrolysis of DNA suitable restricteduse enzymes and make your selection. Another suitable method involves the extraction and the amplification of the DNA fragment encoding the desired fragment of the antibody or polypeptide with a polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the DNA fragment used in PCR as 5'- and 3'-primers. Preferably, the fragments of anti-CD79b antibodies possess at least one biological and/or immunological activity similar to the activity described herein for the native anti-CD79b antibody.

In particular mariantonietta interest of the conservative substitutions provided in table 8 under the heading of "Preferred substitutions". If such substitutions result in a change in biological activity, then can be introduced more significant replacement, referred to in table 8 of "representative substitution" or as described below in the section belonging to the class of amino acids, and then the resulting products can be skanirovana.

Table 8
Initial residuesRepresentative substitutionsPreferred replacement
Ala (A)Val; Leu; IleVal
Arg (R)Lys; Gln; AsnLys
Asn (N)Gln; His; Lys; ArgGln
Asp (D)GluGlu
Cys (C)SerSer
Gln (Q)AsnAsn
Glu (E)AspAsp
Gly (G)Pro; Ala Ala
A His (H)Asn; Gln; Lys; ArgArg
Ile (I)Leu; Val; Met; Ala;
Phe; norleucine
Leu
Leu (L)Norleucine; Ile; Val;
Met; Ala; Phe
Ile
Lys (K)Arg; Gln; AsnArg
Met (M)Leu; Phe; IleLeu
Phe (F)Leu; Val; Ile; Ala; TyrLeu
Pro (P)AlaAla
Ser (S)ThrThr
Thr (T)SerSer
Trp (W)Tyr; PheTyr
Tyr (Y)Trp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe;
Ala; norleucine
Leu

Significant changes�termination of function or immunological properties of anti-CD79b antibody are accomplished by selecting substitutions, which differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (C) the volume of the side chain. Natural residues are divided into the following groups according to common properties of the side chains:

(1) hydrophobic residues: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic residues: cys, ser, thr;

(3) acidic residues: asp, glu;

(4) basic residues: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic residues: trp, tyr, phe.

Non-conservative substitutions lead to the substitution of a member of one of these classes with a member of another class. Such substitutions can also be introduced in the field of conservative substitutions, or, more preferably, into the remaining (non-conservative) area.

Such modifications can be made by methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine-scanning mutagenesis and PCR mutagenesis. To produce the variant DNA of the anti-CD79b antibody, cloned DNA can be subjected to site-directed mutagenesis [Carter et al.,Nucl. Acids Res.,13:4331 (1986); Zoller et al.,Nucl. Acids Res.,10:6487 (1987)], cluster mutagenesis [Wells et al.,Gene,34:315 (1985)], restriction Mut�Genesis [Wells et al., Philos. Trans. R. Soc. London SerA,317:415 (1986)] mutagenesis or by other methods.

To identify one or more amino acids throughout the sequence can also be carried out with a scanning amino acid analysis. The preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids are alanine, glycine, serine, and cysteine. Typically a preferred scanning amino acid for this group is alanine, because it eliminates the side chain beyond the beta-carbon and is, obviously, but less likely alters the conformation of the main chain of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also preferred as it represents the most common amino acid. In addition, it is often present in both the hidden and open positions [Creighton,The Proteins, (W. H. Freeman & Co., N. Y.); Chothia,J. Mol. Biol.,150:1 (1976)]. If alanine substitution does not give adequate amounts of variant, it can be used esoteric amino acid.

To increase resistance of the molecule to oxidation and to prevent the formation of undesirable cross-linkages can be replaced with any cysteine residue not involved in maintaining the "correct" conformation of the anti-CD79b antibody, and usually Taco� replacement is the replacement of cysteine by serine. Conversely, to improve its stability (particularly where the antibody is a fragment such as an Fv fragment) for anti-CD79b antibody may be added(s) cysteine(s) relationship(s).

Especially preferred replacement is the replacement of one or more residues of the hypervariable region of the parent antibody (e.g. a humanized or human antibody). In General, the obtained(s) option(s) chosen one(s) for further development(ut) to contribute to improved biological properties compared to the properties of the parent antibody from which they originate. Standard way of generating such variants with substitutions involves affinity maturation using the method of phage view. Several sites hypervariable region (e.g., 6-7 sites) are subjected to mutation to generate all possible amino substitutions at each site. The thus obtained variants of the antibodies can be provided on the particles of filamentous phage in the form of monovalent hybrids with the product of gene III, Packed in every particle of phage M13. Then the options presented on the phage, sceneroot on their biological activity (e.g. binding affinity) as described in the present application. To identify hypervariable sites about�lusty, are candidates for modification, can be carried out alanine-scanning mutagenesis, which is used to identify the remains of the hypervariable region, playing an important role in binding to the antigen. Alternative or additionally, it may be beneficial to analyze a crystal structure of the complex antigen-antibody to identify the contact sites between the antibody and CD79b polypeptide. Such contact residues and neighboring residues are candidates for replacement, carried out in accordance with methods developed here. After receipt of such options panel these variants is subjected to screening as described in the present application, and antibodies that detect superior properties in one or more relevant assays may be selected for further study.

Nucleic acid molecules encoding amino acid sequence variants of the anti-CD79b antibody, receive various methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of the natural amino acid sequence variants) or via mediated by an oligonucleotide (or site-directed) mutagenesis, PCR mutagenesis, and clustered mutagenesis previously received options or named�modied version of the anti-CD79b antibody.

E.Modification of anti-CD79b antibodies

Covalent modifications of anti-CD79b antibodies included in the scope of the present invention. One type of covalent modification is the reaction of the desired amino acid residues of an anti-CD79b antibody with organic derivatizing agent capable of reacting with selected side chains of the N - or C - terminal residues of the anti-CD79b antibody. The derivatization using bifunctional agents can be implemented, for example, to conduct cross-binding of anti-CD79b antibody with an insoluble matrix, carrier, or surface used in the method of purification of anti-CD79b antibodies and Vice versa. The most commonly used cross-linking agents are, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, N-hydroxysuccinimide, for example, esters formed 4-azidoaniline acid; homobifunctional imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester), bifunctional maleimide, such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propionamide.

Other modifications are desametasone glutaminergic and asparaginyl residues relevant to glutamine and aspartyl residues, respectively; hydro�solirovanie Proline and lysine, phosphorylation of hydroxyl groups merilnyh or traveling residues, methylation of the α-amino groups of the side chains of lysine, arginine and histidine [T. E. Creighton,Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl groups.

Another type of covalent modification of anti-CD79b antibodies included in the scope of the present invention, includes a change in the nature of glycosylation of the native antibody or polypeptide. The term "the changing nature of native glycosylation" as used in the description of the present invention, means a deletion of one or more carbohydrate groups present in the native sequence anti-CD79b antibody (either by removing the hidden sites of glycosylation, or by removing helicoiling residues chemical and/or enzymatic techniques) and/or adding one or more glycosylation sites that are not present in the native sequence anti-CD79b antibody. In addition, the term includes qualitative changes in the nature of glycosylation of the native proteins, leading to changes in the nature and quantity present different carbohydrate groups.

Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked glycosyl�the stripes mean the accession of carbohydrate groups to the side chain of aspartic residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X means any amino acid except Proline, are the recognition sequence for enzymatic joining of the carbohydrate moiety to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide that contributes to the creation of a potential glycosylation site. O-linked glycosylation refers to the accession of one of the sugars, such as N-acetylgalactosamine, galactose or xylose, to hydroxynicotinate mainly to serina or threonine, although they may also be 5-hydroxyproline or 5-hydroxylysine.

The accession of glycosylation sites to the anti-CD79b antibody is usually carried out by such modifications of the amino acid sequence in which this amino acid sequence will contain one or more of the above Tripeptide sequences (for sites of N-linked glycosylation). This modification can also be implemented by adding to the sequence of the original anti-CD79b antibodies of one or more serine or treoninove residues or their substitutions in a sequence of the original antibody (for sites of O-linked glycosylation). Amino acid� sequence anti-CD79b antibody may be, but not necessarily, changed through modifications at the DNA level, particularly by mutating the DNA encoding anti-CD79b antibody, pre-selected basis, the result of which will form the codons which are piped into the desired amino acids.

Another way of increasing the number of carbohydrate molecules on anti-CD79b antibody is by chemical or enzymatic joining of glycosides to the polypeptide. Such methods are described in the literature, for example in the application WO 87/05330, published 11 September 1987, and publication in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate groups present on the anti-CD79b antibody may be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Methods of chemical deglycosylation known in the art and described, for example, in the publication Hakimuddin, et al.,Arch. Biochem. Biophys.,259:52 (1987) and by Edge et al.,Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate molecules to the polypeptides can be accomplished using a variety of endo - and ectoparasites, as described in the publication Thotakura et al.,Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of anti-CD79b antibody comprises linking the antibody to one of the various mo�Ecol non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene, as described in U.S. patents№№ 4640835, 4496689, 4301144, 4670417, 4791192 or 4179337. The antibody may also be enclosed in microcapsules prepared, for example, methods of coacervation or interfacial polymerization (for example, in hydroxymethylcellulose or gelatin microcapsule and polymethylmethacrylate microcapsule, respectively), in systems for the delivery of colloidal medicines (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. This technique is described inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

Anti-CD79b antibody according to the invention can also be modified with the formation of chimeric molecules containing an anti-CD79b antibody associated with another a heterologous polypeptide or with a different amino acid sequence.

In one embodiment of the invention, such a chimeric molecule contains a hybrid anti-CD79b antibody with a polypeptide label, ensure the availability of the epitope, which can be selectively contacted by the antibody against the tag. Epitope label is usually located at the amino - or carboxy-end of the anti-CD79b antibody. The presence of such labeled epitope forms of anti-CD79b antibody may be detected using antibodies against polypep�IDA-label. In addition, the presence of the epitope tag allows for easy cleaning anti-CD79b antibody by the method of affinity purification using an anti-CD79b antibodies against the tag or affine matrix of another type, which binds to epitope tag. Different polypeptides-labels and their respective antibodies are well known in the art. Examples are polyhistidine tag (poly-his) tag or poly-histidine-glycine (poly-his-gly); a polypeptide tag HA of influenza virus and an antibody against the polypeptide, 12CA5 [Field et al.,Mol. Cell. Biol.,8:2159-2165 (1988)], c-myc-tag antibodies against this tag, 8F9, 3C7, 6E10, G4, B7 and 9E10 [Evan et al.,Molecular and Cellular Biology,5:3610-3616 (1985)]; and the label "glycoprotein D (gD) of herpes simplex virus and antibody against such label [Paborsky et al.,Protein Engineering,3(6):547-553 (1990)]. Other polypeptide markers are Flag-peptide [Hopp et al.,BioTechnology,6:1204-1210 (1988)]; the peptide epitope KT3 [Martin et al.,Science,255:192-194 (1992)]; the peptide epitope of α-tubulin [Skinner et al.,J. Biol. Chem.,266:15163-15166 (1991)]; and the label "peptide protein 10, encoded by the gene T7" [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment of the invention, the chimeric molecule can contain a hybrid of "anti-CD79b antibody-an immunoglobulin or a particular region of an immunoglobulin". In the case of a bivalent form of the chimeric molecule (also referred to as "immunoadhesin"), died�ID can be attached to the Fc-region of IgG molecules. IgG-hybrids preferably include replacing at least one variable region in the Ig molecule in a soluble form (with deletionism or an inactivated transmembrane domain) anti-CD79b antibody. In a particularly preferred embodiment of the invention, the hybrid immunoglobulin comprises a hinge region, CH2and CH3or hinge region, CH1, CH2and CH3, IgG1 molecules. Description of the production of hybrid antibodies can also be found in U.S. patent No. 5428130, issued June 27, 1995

F. Receiving anti-CD79b antibodies

As described below, mainly producing anti-CD79b antibody by culturing cells transformed or transfected with a vector containing nucleic acid encoding an anti-CD79b antibody. It is envisaged that for obtaining anti-CD79b antibody may be applied in alternative methods, well known in the art. For example, the corresponding amino acid sequence or part thereof can be produced by direct peptide synthesis on solid phase [see for example, Stewart et al.,Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, CA (1969); Merrifield,J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be carried out manually or automatically. Automated synthesis may be accomplished, for example, the peptide sintezator�e Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturers instructions. The various parts of the anti-CD79b antibody may be chemically synthesized separately and in combination with chemical or enzymatic methods to obtain the desired anti-CD79b antibody.

1.The isolation of DNA encoding anti-CD79b antibody

DNA encoding anti-CD79b antibody may be obtained from a cDNA library isolated from the tissue that is suspected to contain mRNA of anti-CD79b antibodies and expresses such mRNA detektiruya level. In line with this, human DNA-anti-CD79b antibodies can be easily obtained from a cDNA library isolated from human tissue. The gene encoding anti-CD79b antibody may also be obtained from a genomic library or by application of known methods of synthesis (e.g., automated nucleic acid synthesis).

Libraries can be skanirovana using probes (such as oligonucleotides comprising at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by this gene. The screening of cDNA or genomic library with the selected probe may be carried out in accordance with standard procedures, as in the procedure described in publications Sambrook et al.,Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Labortory Press, 1989). An alternative method of isolation of the gene encoding anti-CD79b antibody is PCR technology [Sambrook et al., supra; Dieffenbach et al.,PCR Primer: A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1995)].

Methods of screening cDNA libraries are well known in the art. Oligonucleotide sequences selected as probes should be of a length sufficient and sufficiently well-defined to minimize false-positive results. The oligonucleotide is preferably labeled so that they can be detected after hybridization to DNA in the library being screened. Labelling methods well known in the art and include the use of radioactive labels, such as32P-labeled ATP, biotinylation or enzymatic labeling. The hybridization conditions, including moderate and high rigidity described in Sambrook et al., see above.

Sequences identified in such methods of screening libraries, can be subjected to comparison and alignment with other known sequences deposited and available in public databases such as GenBank or other private databases of sequences. The sequence identity (at either the amino acids or nucleotides) in defined regions of the molecule or across the full-size posledovatelnostyu to be determined by methods known in the art and described in the present application.

Nucleic acid having protein-encoding sequence, can be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence, first described in the present application, and if necessary, in accordance with the standard procedures of elongation of the primers described in Sambrook et al., supra, to detect precursors and processing of intermediate forms of mRNA that may not be subjected to reverse transcription into cDNA.

2.The selection and transformation of host cells

The host cell transferout or transform using the here described expression or cloning vectors for the production of anti-CD79b antibodies, and then cultured in a standard culture medium, modified, if necessary, for inducing promoters, selecting transformants, or amplification of genes encoding the desired sequences. Culturing conditions, such as environment, temperature, pH, etc. can be selected by the technician without undue experimentation. In General, principles, protocols, and practical methods of maximizing the productivity of cell cultures can be found in the publication ofMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press,1991) and Sambrook et al., see above.

Methods of transfection and transformation of eukaryotic prokaryotic cells, comprising the introduction of DNA into a host cell to replicate DNA either as an extrachromosomal or chromosomal integrator, known to the average skilled in the art, for example, such methods are methods that are mediated CaCl2, CaPO4liposomes, methods using polyethylene glycol/DMSO and electroporation. Depending on the host cells, transformation is carried out by standard methods suitable for such host cells. In the case of the prokaryotes usually spend processing calcium, namely calcium chloride, as described in Sambrook et al., see above, or electroporation. Infection with Agrobacterium tumefaciens is carried out for the purposes of transformation of certain plant cells, as described in the publication of Shaw et al.,Gene,23:315 (1983) and in the application WO 89/05859, published June 29, 1989 In the case of mammalian cells that do not contain cell walls, can be applied the method of precipitation of calcium phosphate described by Graham and van der Eb,Virology,52:456-457 (1978). General aspects transfection systems host cells of mammals is described in U.S. patent No. 4399216. Transformation into yeast are typically carried out by the method described by Van Solingen et al.,J. Bact.,130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA) ,76:3829 (1979). However, for the introduction of DNA into cells can be subjected to other methods, such as microinjection into the nucleus, electroporation, fusion of bacterial protoplasts with intact cells, or methods with the use of polycations, e.g., polybrene, polyamidine. Various methods of transformation of mammalian cells can be found in publications Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Appropriate cell hosts for cloning or expression of the DNA in the vectors are prokaryotic cells, yeast cells, or higher eukaryotic cells.

a. Prokaryotic cells-owners

Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31446); E. coli X1776 (ATCC 31537); E. coli strain W3110 (ATCC 27325) and K5 772 (ATCC 53635). Other suitable prokaryotic cells-owners are Enterobacteriaceae such as Escherichia, for example. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. Bacillus licheniformis (e.g., B. Bacillus licheniformis 41P described in DD 266710, published 12 April 1989), Pseudomonas such as P. aerugiNOa, Rhizobia, Viteoscilla, Paracoccus and Streptomyces. These examples are only illustrative, but not limiting purposes. One of the most preferred owners or parent host cells is a strain W3110, because it is most often used as a strain-host for the fermentation of recombinant DNA products. The host cell preferably secrete minimal amounts of proteolytic enzymes. For example, strain W3110 (Bachmann,Cellular and Molecular Biologyvol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC, Deposit No. 27325) can be modified so that it had a genetic mutation in the genes encoding proteins that are endogenous to the host, where examples of such hosts are E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kanr; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a deletion mutation degP and which is not resistant to kanamycin; E. coli W3110 strain 33D3 having genotype W3110 ∆fhuA (∆tonA) ptr3 lac Iq lacL8 ∆ompT∆(nmpc-fepE) degP41 kanR(U.S. patent No. 5639635) and the E. coli strain having mutant periplasmic protease and is described in U.S. patent No. 4946783, issued August 7, 1990 Suitably�mi are also other strains and derivatives, such as E. coli 294 (ATCC 31446), E. coli B, E. coliλ1776 (ATCC 31537) and E. coli RV308(ATCC 31608). These examples are only for illustrative purposes and are not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes, known in the art and described, for example, Bass et al.,Proteins,8:309-314 (1990). Generally speaking, the appropriate bacteria should be selected based on replenishement replicon in the cells of bacteria. For example, cells of the species E. coli, Serratia, or Salmonella can be used as the owners, if the delivery of the replicon are well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and optionally in a cell culture can be introduced additional protease inhibitors. Alternative suitable methods of cloning in vitro are, for example, PCR or other polymerase reactions of nucleic acids.

Full-size antibody, fragments of antibodies and hybrid proteins antibodies can be produced in bacteria, and in particular, if there is no need for glycosylation and effector functions of the Fc, for example, if therapeutic antibody anywhereman with a cytotoxic agent (e.g., toxin), and he immunoconjugate is an effective�actions for destruction of tumor cells. Full-size antibodies have a longer half-life in the bloodstream. Production in E. coli is a more rapid and less costly method. Expression of fragments of the antibodies and polypeptides in bacteria is illustrated, for example, in U.S. patent No. 5648237 (Carter et. al.), in U.S. patent No. 5789199 (Joly et al.), and in U.S. patent No. 5840523 (Simmons et al.), where the described sequence of the region of translation initiation (TIR) and signal sequences for optimizing expression and secretion, these patents are introduced into the present description by reference. After expression, the antibody is isolated from cell paste of E. coli in the soluble fraction, and the antibody may be purified, for example, on a column with protein A or G-protein, depending on the isotype. Final cleaning can be carried out in a manner similar to the method of purification of antibodies expressed in cells SNO.

b. Eukaryotic cells-owners

In addition to prokaryotes, suitable hosts for cloning or expression vectors encoding anti-CD79b antibody, are eukaryotic microbes such as filamentous fungi. The most commonly used lower eukaryotic microorganism-host are Saccharomyces cerevisiae. Other microorganisms are Schizosaccharomyces pombe (Beach and Nurse,Nature, 290:140 [1981]; EP 139383, published 2 may 1985); cells-the owners�VA Kluyveromyces (U.S. patent No. 4943529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al.,J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC price has been 56500), K. drosophilarum (ATCC 36906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244234); Neurospora crassa (Case et al.,Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357, published 10 January 1991), and cells of Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al.,Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes,EMBO J., 4:475-479 [1985]). Suitable include, but are not limited to, methylotrophy yeast, for example, yeast is able to grow on methanol selected from the genus consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that belong to this class of yeasts may be found in the publication C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

The host cell suitable for the expression and glycosylation of the anti-CD79b antibody may be derived from multicellular organisms. Examples of invertebrate cells include insect cells such as cells of Drosophila S2 and Spodoptera Sf9, as well as cells raste�rd, such as cell cultures of cotton, corn, potato, soybean, Petunia, tomato and tobacco. Have been identified various baculovirus strains and variants and corresponding permissive cells-the hosts are insects that occur from such owners as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A number of viral strains for transfection is publicly available, for example, a variant L-1 Autographa californica NPV and the strain Bm-5 Bombyx mori NPV, and such viruses may be used as a virus according to the invention, particularly for transfection of cells Spodoptera frugiperda.

However, of most interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable cell lines of mammalian hosts cells are monkey kidney CV1 transformed by SV40 virus (COS-7, ATCC CRL 1651); the cell line of human embryonic kidney (293 cells or 293, sublimirovanny for growth in suspension culture, Graham et al.,J. Gen Virol.36:59 (1977)); the kidney cells baby hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/DHFR (CHO, Urlaub et al.,Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse Sertoli cells (TM4, Mather,Biol. Reprod.23:243-251 (1980)); kidney cells of monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); cells CT�enemy human cervical (HELA, ATCC CCL 2); cells of the kidneys of dogs (MDCK, ATCC CCL 34); liver cells of laboratory rats Buffalo (BRL 3A, ATCC CRL 1442); cells of the human lung (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor cells of mouse breast cancer (MMT 060562, ATCC CCL51); cell TRI (Mather et al.,Annals N. Y. Acad. Sci.383:44-68 (1982)); the cells, MRC 5; FS4 cells; and the cell line human hepatoma (Hep G2).

The host cell is transformed above expressing or cloning vectors for the production of anti-CD79b antibodies and cultured in standard culture medium, modified, if necessary, for inducing promoters, selecting transformants, or amplification of genes encoding the desired sequences.

3.The selection and use of replicating vectors

For recombinant production of an antibody according to the invention, nucleic acid (e.g., cDNA or genomic DNA) encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. The DNA that encodes the antibody, can be easily isolated and sequenced in accordance with standard procedures (e.g., by using oligonucleotide probes that are able to specifically communicate with the genes encoding the heavy and light chain antibodies). For this purpose many are suitable vectors. The choice of vector� partly depends on the host cell. Basically, the preferred cells of the host are either prokaryotic cells or eukaryotic cells (typically mammalian cells).

This vector can be, for example, obtained in the form of plasmids, Comedy, viral particle, or phage. The appropriate nucleic acid sequence may be incorporated into the vector in accordance with a variety of treatments. Basically, DNA is inserted into the corresponding(s) site(s) restricteduse the restriction enzyme by methods known in the art. Vector components generally include, but are not limited to, one or more signal sequences, origin of replication, one or more marker genes, enhancer element, a promoter and termination sequence transcription. Construction of suitable vectors containing one or more of the components is carried out by standard methods of ligation, known to specialists.

CD79b can be produced recombinante not only by the direct method, but also in the form of a hybrid polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-Terminus of the Mature protein or polypeptide. In General terms, the specified signal sequence can be comp�component of the vector, any such sequence can be a part of DNA encoding anti-CD79b antibody, which is inserted into this vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II. For secretion in yeast signal sequence may be, for example, a leader sequence of yeast invertase leader sequence of alpha-factor (including the leader sequence of α-factor of Saccharomyces and Kluyveromyces, and the second of these sequences is described in U.S. patent No. 5010182) or leader sequence of acid phosphatase, leader sequence of the C. albicans glucoamylase (patent EP 362179 published 4 April 1990), or signal sequence, as described in the application WO 90/13646 published on 15 November 1990, When overexpressed in mammalian cells, to direct secretion of the protein can be used signal sequence mammals, such as signal sequences, derived from secreted polypeptides of the same species or related species, as well as viral secretory leader sequence.

and. Prokaryotic cells-owners

Polynucleotide seq�to the dedication, encoding polypeptide components of the antibody according to the invention, can be obtained by standard recombinant methods. The desired polynucleotide sequences can be isolated and sequenced from the antibody-producing cells, such as hybrid cells. Alternative polynucleotides can be synthesized using a nucleotide synthesizer or PCR methods. After obtaining the sequences encoding the polypeptides, the sequences are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. For the purposes of the present invention can be used by many available and well known vectors. The selection of an appropriate vector depends mainly on the size of nucleic acids, embedded in the specified vector and the particular host cell transformed with this vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with specific host cell in which it resides.

Basically, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used together with the same x�zaebali. Such expressing and cloning vectors contain a nucleic acid sequence that promote replication of the vector in one or more selected cells of the host, as well as sequence markers that allow phenotypic selection in transformed cells. Such sequences of various bacteria, yeasts and viruses are well known. Origin of replication from the plasmid pBR322 and contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and also allows for easy identification of transformed cells is suitable for most gram-negative bacteria; origin of the 2μ plasmid is suitable for yeast, and various viral oridzhiny (SV40, polyomavirus, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain promoters that can be used by the microbial organism for expression of endogenous proteins, or they can be modified to include such promoters. Examples of pBR322 derivatives used for expression of particular antibodies, are described in detail in U.S. patent No. 5648237, Carter et al.

In addition, phage vectors containing replicon and regulatory PEFC�lovatelli, compatible with the microorganism-host, can be used as transforming vectors in combination with these hosts. For example, bacteriophage such as λGEMTM-11 may be used to obtain a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

Investigating the expression vector according to the invention may contain two or more pairs of the promoter-cistron encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence, localized above (from 5'-end) from cistron, modulating its expression. Prokaryotic promoters are typically divided into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of cistron under its control, in response to changes in the cultivation conditions, for example, in the presence or in the absence of nutrients or when the temperature changes.

The majority of promoters recognized by a variety of potential cells-owners, well known in the art. The selected promoter may be functionally attached to castronno DNA encoding the light or heavy chain by separating the promoter from the DNA source for�the case of hydrolysis restricteduse enzymes and embedding the selected promoter sequence into the vector according to the invention. Native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of target genes. In some embodiments of the invention are heterologous promoters, as they, in comparison with the native promoter for the desired polypeptides can significantly increase the level of transcription and increase yields of expressed gene is a target.

Promoters recognized by a variety of potential cells-owners, well known in the art. Promoters suitable for use in prokaryotic hosts include the PhoA promoter, promoter of the system β-galactosi and lactose [Chang et al.,Nature, 275:615 (1978); Goeddel et al.,Nature, 281:544 (1979)], the promoter system, the alkaline phosphatase, and the tryptophan (trp) [Goeddel,Nucl Acids Res., 8:4057 (1980); EP 36776], and hybrid promoters such as the tac promoter [deBoer et al.,Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)] or the trc promoter. The promoters used in bacterial systems can also contain a sequence of Shine-Dalgarno (S. D.), functionally connected to the DNA encoding anti-CD79b antibody. However, it can also be used and other promoters that are functional in bacteria (for example, other known bacterial or phage promoters). Nucleotide sequences of these promoters were �published, that makes it easier for the experts in this field to carry out their functional ligation with cisternae, encoding the desired light and heavy chains (Siebenlist et al. (1980) Cell 20:269) using linkers or adapters to deliver all of the required restriction sites.

In one aspect of the invention, each of cistron in the recombinant vector contains a component of the secretory signal sequence regulating translocation of expressed polypeptides across a membrane. In General terms, the specified signal sequence may be a component of a vector or a sequence can be a portion of the desired DNA-polypeptide embedded in this vector. The signal sequence selected for the purpose of carrying out the invention must be a sequence recognized and processarea (i.e. contain no cleavable signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and do not processorbased signal sequences that are native to the heterologous polypeptides, the signal sequence is replaced prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin� II (STII), LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistronic gene expression system are the signal sequence STII or their variants.

In another aspect of the invention, the production of immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore in this case does not require the presence of secreting signaling sequences in each cistron. In accordance with this, the light and heavy chain immunoglobulin expressed and subjected to the stacking and Assembly of a functional immunoglobulin in the cytoplasm. Some strains of the hosts (e.g., trxB-strains of E. coli) have the appropriate conditions in the cytoplasm, which favor the formation of disulfide bonds, and thereby contribute to the proper installation and Assembly of subunits expressed protein. Proba & Pluckthun, Gene, 159:203 (1995).

The present invention relates to gene-expression system in which the quantitative relationship expressed polypeptide components can be modulated to maximize the yield of secreted and properly assembled antibody according to the invention. Such a modulation is performed at least partially by means of the simultaneous modulation of the activity broadcast polypep�risk hedging component.

One method of modulation of translational activity are described in U.S. patent 5840523 Simmons et al. This method uses the options field of translation initiation (TIR) in castrone. For this TIR can be created by a series of variants of the amino acid sequence or nucleic acid sequence with a range of translational activities that makes it possible to adjust this factor to achieve the desired level of expression of a particular circuit. Options TIR can be produced by standard methods of mutagenesis, resulting in the codon can be altered, which may modify amino acid sequence, are preferred although silent mutation in the nucleotide sequence. Modifications in TIR can be, for example, changes in the number of sequences Shine-Dalgarno or the distance between them, as well as changes in the signal sequence. One way of obtaining a mutant signal sequences is to create a "Bank of codons in the early coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., such modifications are silent). This can be achieved by replacing the third nucleotide of each codon, and replacement of some amino acids, such as Le�Qing, serine and arginine, in many first and second positions, which may create some difficulties in obtaining such a Bank. This method of mutagenesis is described in detail in the publication Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

A set of vectors is preferably generated using the TIR activity for each cistron. This limited set provides the ability to compare the expression levels of each circuit, and also outputs the desired product antibodies at different combinations of TIR-activity. TIR-activity can be determined by quantifying the expression levels of a gene-reporter as described in detail in U.S. patent No. 5840523 Simmons et al. Based on the comparison of translational activities, may be chosen, the individual TIR, which can be combined in the construction of expression vectors according to the invention.

b. Eukaryotic cells-owners

Components of vectors are typically, but not limited to, one or more of the following components: a signal sequence, origin of replication, one or more marker genes, enhancer element, a promoter and termination sequence transcription.

(i) signal Component sequence

The vectors used in eukaryotic cells-the owners, may also contain signal posledovatelno�ü or other polypeptide, having a specific cleavage site at the N-Terminus of the Mature protein, or of interest polypeptide. Selected heterologous signal sequence is preferably a sequence that is recognized and processinputs (i.e. cleaved by a signal peptidase) by the host cell. For expression in mammalian cells are the signal sequence mammals, as well as viral secretory leader sequence, for example, the signal sequence of the gD of herpes simplex virus.

The DNA for such area predecessor be ligated with the antibody-coding DNA with preservation of the reading frame.

(2) the origin of replication

Usually a component of the origin of replication is not necessary for gene-expression vectors mammals. For example, the origin of SV40 replication is typically used only because it contains the early promoter.

(3) The selective gene

Expression and cloning vectors usually contain a selective gene, also known as selective marker. Usually selective genes encode proteins that (a) reported resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) compensate for the deficit caused by auxotrophies, if necessary, silt� (C) ensure the delivery of important nutrients which do not come from complex media, e.g. the gene encoding D-alanine-racemase for Bacilli.

One example of a selection scheme is the use of drugs that stop the growth of the host cell. Cells that were successfully transformed with a heterologous gene produce a protein that tells a resistance to the drug and thereby contributing to the survival of these cells in the selective medium. For such dominant selection can be used, for example, drugs such as neomycin, mycophenolic acid and hygromycin.

Examples of suitable selective markers for mammalian cells are markers that identify cells that are able to incorporate nucleic acid encoding an anti-CD79b antibody, such as DHFR genes, timedancing, metallothionein-I and II, preferably genes encoding metallothionein primates, adenosine-deaminase, ornithine-decarboxylase, etc. In the case of DHFR wild-type a suitable host cell is a CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096), and this cell line was obtained and multiplied, as described in the publication Urlaub et al.,Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with selective DHFR gene, first identified by culturing all t�of informants in a culture medium, containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternative cell owners (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibody, protein DHFR wild-type and the other a selective marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by culturing cells in medium containing the agent for the selection, carried out with a selective marker such as aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

Suitable selective gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al.,Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al.,Gene, 10:157 (1980)]. Gene trp1 is a selective marker for a mutant yeast strain that cannot grow in the presence of tryptophan, for example, a strain deposited with the ATCC No. 44076 or RER-1 [Jones,Genetics, 85:12 (1977)].

(4) the promoter Component

Expression and cloning vectors usually contain a promoter functionally attached to a nucleic acid sequence encoding an anti-CD79b antibody, to direct mRNA synthesis. Promoters recognized by a variety of potential cells-owners, well-known specialist�M.

In fact, all the genes of eukaryotes have at-rich region, localized approximately in the area of 25-30 nucleotides from above the site of initiation of transcription. Another such sequence, localized approximately in the area of 70-80 nucleotides from above the site of initiation of transcription of many genes, is the area CNCAAT, where N can mean any nucleotide. The 3'-end of most eukaryotic genes is the sequence of AATAAA, which can serve as a signal for attachment of poly A-tail to the 3'-end of the coding sequence. All of these sequences can be properly built into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in yeast cells-the owners are the promoters for 3-phosphoglycerate-kinase [Hitzeman et al.,J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland,Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate-mu'taz, pyruvate kinase complex, triazolopyrimidine, phosphoglucomutase and glucokinase.

Other yeast promoters, which are inducible PR�motors, having the additional advantage lies in their ability to regulate transcription in certain culture conditions, are the promoter region of genes alcohol dehydrogenase 2, sociogram C, acid phosphatase, hydrolytic enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast, is also described in EP 73657.

Transcription of anti-CD79b antibodies of the vectors in the cells of the host mammal is regulated, for example, by promoters obtained from the genomes of viruses such as virus polyoma, the smallpox virus in poultry (UK 2211504 published 5 July 1989), adenovirus (such as adenovirus 2), the vaccinia papilloma, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis b virus and most preferably simian virus 40 (SV40); by heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter; and by the promoters of heat shock proteins, provided such promoters are compatible with the systems of host cells.

Early and late promoters of SV40 virus are usually obtained in the form of an SV40 restriction fragment that also contains the origin of replication of SV40 virus. Pretani PR�engine of the human cytomegalovirus is usually obtained as a HindIII restriction fragment E. The system of expression of the DNA in the cells of the host mammal derived from bovine papillomavirus, is used as the vector described in U.S. patent No. 4419446. Modification of the system described in U.S. patent No. 4601978. Expression of cDNA of human β-interferon in mouse cells under the control of the promoter timedancing herpes simplex virus is also described in the publication Reyers et al., Nature 297:598-601 (1982). Alternatively, as the promoter can be used by the rous sarcoma virus, having long terminal repeat.

(5) Component enhancer

Transcription of DNA encoding anti-CD79b antibody, in higher eukaryotes can be increased by incorporating into the vector enhancer sequence. The enhancers are CIS-acting elements of DNA, usually about from 10 to 300 BP, which, acting on the promoter, increase its activity in the initiation of transcription. Currently there are many enhancer sequences derived from mammalian genes (globin genes, elastase, albumin, α-fetoprotein and insulin). However, commonly used enhancer of eukaryotic cell virus. An example is the enhancer of SV40 virus localized in the late region of the replication origin (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyomavirus localized in late on�region of origin of replication and adenovirus enhancers. Cm. also Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for activation of eukaryotic promoters. Enhancer can be playserver into the vector in the 5'- or 3'-polozhenii in relation to a sequence encoding an anti-CD79b antibody, but preferably, it was in the 5'-position from the promoter.

(6) The site of termination of transcription

Expression vectors used in eukaryotic cells-the masters (in yeast, fungi, insects, plants, animals, human, or nucleated cells originating from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are usually located from the 5'-end, and sometimes the 3'-end untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed in the form polyadenylated fragments in netransliruemoi part of the mRNA encoding anti-CD79b antibody. One of the suitable components of the transcription termination region is polyadenylation bovine growth hormone. Cm. description of gene expression vectors in WO 94/11026 and in the present application.

Other methods, vectors, and the host cell suitable for adaptation to the synthesis of anti-CD79b and�of tetela in recombinant vertebrate cell cultures, described in the publication Gething et al.,Nature,293:620-625 (1981); Mantei et al.,Nature,281:40-46 (1979); in EP and EP 117060 117058.

4.Culturing host cells

The host cell used for the production of anti-CD79b antibodies according to the invention, can be cultured in a variety of environments.

and. Prokaryotic cells-owners

Prokaryotic cells used for the production of polypeptides according to the invention, cultured in a medium, known in the art and suitable for culture of the selected host cells. Examples of a suitable environment are broth, Luria (LB) with the necessary nutritional supplements. In some embodiments of the invention, such an environment also provides a means for selection, selected on the basis of constructing expression vector, which promote selective growth of prokaryotic cells containing the expression vector. For example, in an environment for culturing cells expressing the gene of resistance to ampicillin, add ampicillin.

In addition to sources of carbon, nitrogen and inorganic phosphate in the medium may also include any appropriate additives in appropriate concentrations introduced alone or in admixture with other additives or environment, such as a complex nitrogen source. The culture medium may, but need not, contain� one or more reducing agents, selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic cells-the hosts are cultivated at appropriate temperatures. For cultivation of E. coli, the preferred temperature is, for example, temperatures from about 20°C to 39°C, more preferably from about 25°C to 37°C, and more preferably at about 30°C. the pH of the medium can vary from about 5 to 9, depending mainly on the host organism. For E. coli pH is preferably about between 6.8 to 7.4, and more preferably approximately 7,0.

If the expression vector according to the invention is used inducible promoter, the protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the invention for regulating transcription of the polypeptides used are PhoA promoters. Accordingly, the transformed cell hosts are cultivated in the medium for induction with a limited content of phosphate. Preferred environment with limited content of phosphate is Wednesday S. R. A. R. (see, e.g., Simmons et al., J. Immunol. Methods (2002) 263:133-147). In combination with the used vector design may be used and various other inducers, known to specialists.

In one variation�tov the invention, expressed polypeptides according to the invention are secreted into the periplasm of host cells and can be isolated from this periplasm. Protein expression, mainly carried out by disruption of a microorganism, usually by methods such as osmotic shock, sonication or lysis. After disruptio cells, cell debris or whole cells can be removed by centrifugation or filtration. These proteins can be further purified, for example, using chromatography on affinity resin. Alternative proteins can be transported in the cellular environment and to separate from her. Cells can be removed from the culture, and the culture supernatant may be subjected to filtration and concentration to additional purification of the obtained proteins. Expressed polypeptides can then be isolated and identified by well-known methods such as polyacrylamide gel electrophoresis (page) and Western blot analysis.

In one aspect of the invention, the antibodies produced in large quantity by fermentation. To obtain recombinant proteins can be carried out various large-scale fermentation procedure in culture with nourishment. Large-scale fermentation is carried out in a fermenter with a capacity of at least 1000 liters, and preferably from about 1,000 to 100,000 liters�spectra. Such fermenters equipped with a paddle stirrer for uniform distribution of oxygen and trace elements, and especially glucose (the preferred source of carbon/energy). The term “laboratory fermentation”, in General terms, means the fermentation in the fermenter with a volume of no more than about 100 liters, and this amount can vary from about 1 liter to 100 liters.

During the fermentation process, induction of protein expression is typically initiated after the cells are cultured in suitable conditions, will achieve the desired density, for example, OD550of approximately 180-220, i.e. early stationary phase. In combination with the used vector design may be used and various other inducers, known in the art and described above. Before induction, the cells can be cultured for a shorter period of time. Cells are usually induced for about 12-50 hours, although the induction time can be increased or decreased.

To increase the yield and to improve the quality of polypeptides according to the invention can be modified by different conditions of fermentation. So, for example, to ensure the “correct” Assembly and laying of secreted polypeptides antibodies can be used for more vectors in which verhexte�this protein chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptideprophet - CIS,TRANS-isomerase with chaperone activity), and which are intended for co-transformation of prokaryotic host cells. It was demonstrated that proteins-chaperones facilitate proper stacking and solubility of heterologous proteins produced in bacterial cells-hosts. Chen et al. (1999) J. Bio. Chem. 274:19601-19605; Georgiou et al., U.S. patent No. 6083715; Georgiou et al., U.S. patent No. 6027888; Bothmann & Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm & Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (and in particular, proteins that are proteoliticeski sensitive), in the present invention can be used by some strains are hosts deficient in proteolytic enzymes. For example, strains of host cells can be modified for the introduction of genetic(s) mutation(s) in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and combinations thereof. Some deficient in the protease strains of E. coli are available and are described, for example, Joly et al. (1998), supra; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment of the invention, the E. coli strains deficient in p�teoriticheski enzymes and transformed with plasmids sverkhekspressiya one or more proteins-chaperones, are used as host cells in the expression system according to the invention.

b. Eukaryotic cells-owners

Commercially available media suitable for culturing the host cells are environment, such as environment of Hams F10 (Sigma), minimal maintenance medium ((MEM)(Sigma), RPMI-1640 (Sigma) and modified according to the method of Dulbecco Wednesday Needle (DMEM), Sigma). In addition, as a culture medium for culturing the host cells may be used in any environment that is described in the publication Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), in U.S. patents №№ 4767704, 4657866, 4927762, 4560655 or 5122469; in WO 90103430; in WO 87/00195 or in U.S. patent Re. No. 30985. In any of these environments can be added, if necessary, hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride and calcium phosphate and magnesium), buffers (such as HEPES), nucleotides (such as adenosin and thymidine), antibiotics (such as a drug gentamicinTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar doses), and glucose or an equivalent energy source. May also include any other necessary additives in suitable�ing concentrations known to specialists in this field. Culturing conditions, such as temperature, pH, etc., similar to the conditions previously used for the expression of selected host cells, and known to the average expert in this field.

5. Detection of amplification/expression gene

Amplification and/or gene expression can be measured in a sample directly, for example, using southern blotting, Northern blotting to quantify the transcription of mRNA [Thomas,Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization using appropriately labeled probe based on the sequence described here. Alternatively can be used for antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes DNA-RNA or DNA duplexes-protein. The antibodies, in turn, can be tagged, and can be analyzed, in which the duplex is attached to the surface so that after the formation of duplex on the surface, it was possible to detect the presence of antibodies associated with the duplex.

Alternative gene expression can be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and analysis of cell culture or physiological LM�bones for the direct and quantitative assessment of the expression of the gene product. Antibody suitable for immunohistochemical staining and/or analysis of samples of physiological liquids, can be monoclonal or polyclonal and can be obtained from any mammal. Basically can be derived antibodies against native sequence CD79b polypeptide or against a synthetic peptide derived from these DNA sequences, or against exogenous sequence attached to DNA and CD79b encoding epitope-specific antibodies.

6.Purification of anti-CD79b antibodies

Forms of anti-CD79b antibodies can be isolated from culture medium or from lysates of the host cells. If antibodies are membrane-bound, they can be isolated from the membrane using a suitable detergent solution (e.g. Triton X-100) or by enzymatic cleavage. Cells used for expression of the anti-CD79b antibody may be subjected to disruption various physical or chemical methods such as the conduction cycle of freezing and thawing, sonication, mechanical disruptive or the use of agents for cell lysis.

It may be desirable cleaning anti-CD79b antibodies from recombinant cell proteins or polypeptides. Suitable methods of purification methods are as follows: f�incorporation of the ion-exchange column, the ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing, electrophoresis in LTOs-page, precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; purification on a column with protein a-separate to remove contaminants such as IgG; and purification on columns that form chelate complexes with metal-bound labeled epitope forms of anti-CD79b antibodies. Can be applied to various protein purification methods, and such methods are known in the art and described, for example, Deutscher,Methods in Enzymology, 182 (1990); Scopes,Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Selection stage(s) of cleaning depends on the nature of the method used in producing and produced specifically anti-CD79b antibody.

Using recombinant DNA techniques, the antibody can be produced intracellularly, or it can be directly secreted into the medium. If in the first stage antibody is produced inside the cells, cellular debris, or the host cell or lysed fragments, is removed, e.g., by centrifugation or ultrafiltration. In publications Carter et al.,Bio/Technology10:163-167 (1992) describe a procedure for selection of antibodies that are secreted into the periplasmic space of E. coli. �short, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl (PMSF) over about 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, usually first concentrated supernatants obtained from such expression systems, using commercially available filter for concentrating proteins, for example, devices for ultrafiltration Amicon or Millipore Pellicon®. For inhibition of proteolysis in any of the previous stages can be used a protease inhibitor such as PMSF, and to prevent the propagation of accidental insertion of the impurity of microorganisms can be used antibiotics.

The composition of the antibodies obtained from cells can be purified, for example, using chromatography on hydroxiapatite, gel electrophoresis, dialysis, and affinity chromatography, with the preferred method of purification is affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc-domain of an immunoglobulin that is present in this antibody. Protein A can be used for purification of the antibody containing the heavy chain γ1, γ2, or γ4 human immunoglobulin (Lindmark et al.,J. Immunol. Meth.62:1-13 (1983)). For all mouse isotypes and for human γ3 chain Ryoko�indoesia G-protein (Guss et al., EMBOJ. 5:15671575 (1986)). As the matrix is associated with an affine ligand, is used most often agarose, but can be used and other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene, provide higher flow rates and reduce processing times than can be achieved using agarose. If the antibody contains a domain WithN3, it is possible to clean it can be used resin Bakerbond ABXTM(J. T. Baker, Phillipsburg, N. J.). Depending on secreted antibodies can also be used and other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin-sepharoseTM, chromatography on anyone - or cation-exchange resin (for example, on a column poliasparaginovaya acid), chromatofocusing, electrophoresis in LTOs-page and precipitation with ammonium sulfate.

After carrying out any(s) advance(s) stage(s) of purification, a mixture containing interest is an antibody and impurities, can be subjected to hydrophobic chromatography at low pH using elution buffer at pH of about 2.5 to 4.5, and preferably at low salt concentrations (e.g.,�Erno 0-0. 25 M).

G. Pharmaceutical composition

Conjugates of the antibody-drug" (ADC) according to the invention can be administered by any method suitable for the treatment of this condition. ADC is typically administered parenterally, i.e. by injection, podgorna, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

In one embodiment of the invention, for the treatment of cancer, conjugate "antibody-drug" administered by intravenous infusion. Dose, administered by infusion, is in the range from about 1 μg/m2to about 10000 μg/m2per dose, and is usually administered once a week, and only can be administered one, two, three or four doses. Alternative such a dose from about 1 μg/m2up to 1000 mg/m2from about 1 μg/m2to 800 g/m2from about 1 μg/m2up to 600 µg/m2from about 1 μg/m2to 400 g/m2from about 10 μg/m2to 500 μg/m2from about 10 μg/m2up to 300 g/m2from about 10 μg/m2to 200 µg/m2and from about 1 μg/m2to 200 µg/m2. To eliminate or ameliorate symptoms of the disease the dose may be administered once a day, once a week, several times a week but not every day; several times a month, but not every day; a few �AZ in a month but not every week; once a month or periodically. The introduction can be carried out during any of these intervals, until, until you achieved the reduction of a tumor or reducing the symptoms of lymphoma and leukemia exposed to treatment. The introduction can be continued after remission or attenuation of disease symptoms, if during such continuous introduction of remission or reduced symptoms still continue.

The present invention also relates to a method of treatment of an autoimmune disease, where the method comprises administering to a patient suffering from an autoimmune disease, a therapeutically effective amount of the conjugate "humanized MA79b antibody-drug" according to any of the previous options. In preferred embodiments of the invention, the antibody is administered intravenously or subcutaneously. The specified conjugate "antibody-drug" administered intravenously at a dose of approximately from 1 μg/m2up to 100 mg/m2per dose and in a specific embodiment of the invention is from 1 mg/m2to about 500 μg/m2. To eliminate or ameliorate symptoms of the disease the dose may be administered once a day, once a week, several times a week but not every day; several times a month, but not ka�every day; a few times a month but not every week; once a month or periodically. The introduction can be carried out during any of these intervals, until, until will be achieved by eliminating or reducing the symptoms of the autoimmune disease subject to treatment. The introduction can be continued after remission or attenuation of disease symptoms, if during such continuous introduction of such remission or reduced symptoms still continues.

The present invention also relates to a method of treatment of b-cell disorder, comprising administering to a patient suffering from b-cell disorders such as b-cell-proliferative disorder (including, but not limited to, lymphoma and leukemia) or an autoimmune disease, a therapeutically effective amount of a humanized MA79b antibody in accordance with any of the previous variants, where the specified antibody is not conjugated with a cytotoxic molecule or apparently detected molecule. The specified antibody is usually administered in a dose from about 1 μg/m2up to 1000 mg/m2.

In one of its aspects the present invention relates to pharmaceutical compositions containing at least one anti-CD79b antibody and/or at least one immunoconjugate and/or �of at least one conjugate is anti-CD79b antibody-drug according to the invention. In some embodiments of the invention, the pharmaceutical preparation contains (1) an antibody according to the invention and/or its immunoconjugate and (2) a pharmaceutically acceptable carrier. In some embodiments of the invention the pharmaceutical composition comprises (1) an antibody according to the invention and/or its immunoconjugate and, optionally, (2) at least one additional therapeutic agent. Additional therapeutic agents include, but are not limited to, the facilities described below. ADC is typically administered parenterally, by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

Therapeutic compositions comprising anti-CD79b antibody or immunoconjugate with CD79b for use in accordance with the present invention receive in a convenient storage forms by mixing the antibody or immunoconjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), to obtain lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers in the dosages and concentrations must be non-toxic to recipients, and they are buffers, such as acetate, Tris, phosphate, citrate and other organic� acid; antioxidants, including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; hexamethonium chloride; benzalkonium chloride; chloride benzathine; finaliser, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (having approximately less than 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; hepatoblastoma agents such as EDTA; agents that give the correct tonicity such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as Polysorbate; soleobrazutaya counterions such as sodium; metal complexes (e.g., complexes of Zn-protein); and/or nonionic surfactants such as tweenTMthe pluronicTMor polyethylene glycol (PEG). The pharmaceutical composition used for administration in vivo, typically must be sterile. This can be easily accomplished by filtration through sterile filtration membranes.

The active ingredients may also be enclosed in a microcapsule obtained, for example, methods of coacervation or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin microcapsule and polymethylmethacrylate microcapsule, respectively, in systems for the delivery of colloidal medicines (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. This technique is described inRemington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Can be obtained the drugs prolonged release. Fittingly�mi examples of prolonged release preparations are semi-permeable matrices of solid hydrophobic polymers, containing the immunoglobulin according to the invention, where the above matrices are in the form of finished articles, e.g. films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate) or polyvinyl alcohol), polylactic acid called PLA (U.S. patent No. 3773919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-biodegradable copolymer of ethylene-vinyl acetate, degradable copolymers of lactic acid-glycolic acid such as the LUPRON DEPOTTM(microspheres for injection, consisting of a copolymer of lactic acid-glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as a copolymer of ethylene and vinyl acetate and a copolymer of lactic acid-glycolic acid that can release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long period of time, they can denaturirate or aggregated under the influence of moisture at 37aboutWith that leads to loss of biological activity and possible changes in immunogenicity. For stabilization depending on the mechanism, may be a rational strategy. For example, if it was discovered�, what is the mechanism of aggregation is the formation of intermolecular S-S-ties through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, the regulation of moisture content using appropriate additives, and producing concrete compositions based on the polymer matrix.

The antibody may be obtained in any suitable form for delivery to the desired cell/tissue. So, for example, antibodies can be prepared in the form of immunoliposome. "Liposome" is a small vesicles composed of different types of lipids, phospholipids and/or surfactant that can be used to deliver drugs to the mammal. Components of liposomes are usually located so that they form a bilayer, similar to the lipid bilayer in biological membranes. Liposomes containing the antibody are used, known in the art such as the methods described in publication Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); in patents 4485045 and 4544545; and in the application WO97/38731 published October 23, 1997, Liposomes with an increased half-life in blood is described in U.S. patent No. 5013556.

Specifically used liposomes composed of lipids such as phosphate�milholin, cholesterol and PEG-derivationally phosphatidylethanolamine (PEG-PE), can be obtained by evaporation from a reverse phase. Liposomes extruded through filters with defined pore size to thereby produce liposomes of the desired diameter. Fab'-fragments of the antibodies according to the invention can be anywhereman with liposomes, as described in publications Martin et al., J. Biol. Chem. 257:286-288 (1982), as a result of the reaction of disulfide exchange. The liposome can include, but not necessarily, a chemotherapeutic agent. Cm. Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

The pharmaceutical composition used for administration in vivo, typically must be sterile. This can be easily accomplished by filtration through sterile filtration membranes.

H.Treatment is anti-CD79b antibodies

To determine the expression of CD79b in cancer the disease can be done a variety of detection assays. In one embodiment of the invention, overexpression of a CD79b polypeptide can be assessed using immunohistochemical analysis (IHC). Drenched in paraffin sections of tissue obtained by biopsy of the tumor can be subjected to IHC analysis and evaluated for intensity of staining of protein CD79b in accordance with the following criteria:

Evaluation 0 - no staining is staining of the membranes at less than 10% of�Oholibah cells.

Evaluation 1+ - very faint/barely perceptible membrane staining, detected in more than 10% of tumor cells. These cells are stained only in part of their membrane.

Assessment 2+ - weak or moderate staining of the entire membrane is observed in more than 10% of tumor cells.

Grade 3+ - moderate or strong staining of the entire membrane is observed in more than 10% of tumor cells.

Tumors were analyzed for sverkhekspressiya CD79b and who received 0 or 1+, can be described as tumors, which is not observed overexpression CD79b, and the tumors with estimates of 2+ or 3+ can be characterized as tumors, sverkhekspressiya CD79b.

Alternative or additional FISH analyses, such as INFORMTM(developed by Ventana Co., Arizona) or PATHVISIONTM(Vysis, Illinois) may be carried out on the formalin-fixed and embedded in paraffin tumor tissue to determine the level of sverkhekspressiya CD79b (if present) in the tumor.

Overexpression or amplification CD79b can be estimated using detection analysis in vivo, for example, by administering a molecule (such as an antibody) which binds to apparently detected molecule; tagging apparently detected a label (e.g. a radioactive isotope or a fluorescent tag) and external scan of the patient to determine localiza�AI markers.

As described above, the anti-CD79b antibody according to the invention, in addition to therapeutic applications, can be used in various other purposes. Anti-CD79b antibody according to the invention can be used to determine the stage of development of cancerous tumors expressing CD79b polypeptide (for example, when a radioactive imaging). These antibodies can also be used for purification or immunoprecipitation of CD79b polypeptide from cells, for detection and quantification of CD79b polypeptide in vitro, e.g. in an ELISA-analysis or Western blot analysis, for destruction and elimination of CD79b-expressing cells from a population of mixed cells as in the purification of other cells.

Currently, depending on the stage of cancer, cancer treatment involves performing the following therapy alone or in combination with each other, namely a surgical operation to remove the cancerous tissue, radiation therapy and chemotherapy. Treatment is anti-CD79b antibody may be especially desirable in elderly patients who can not tolerate the toxic side effects of chemotherapy and metastases, when radiation therapy has limited application. Anti-tumor anti-CD79b antibody according to the invention can be used to suppress the growth of CD79b-expressing cancer after PE�an initial diagnosis of the disease or during relapse. For therapeutic purposes, anti-CD79b antibody may be used alone or in combination therapy, for example, together with hormonal therapy, antiangiogenic therapy or therapy with radioactive labelled compounds, or with surgery, cryotherapy, and/or radiation therapy. Treatment is anti-CD79b antibody may be implemented in combination with other forms of standard therapy, or concurrently with standard therapy, either before or after the holding of such therapy. In the treatment of cancer, and in particular, in patients with a high risk of developing the disease, are used chemotherapeutic agents such as TAXOTERE® (docetaxel), TAXOL® (paclitaxel), estramustine and mitoxantrone. In the method according to the invention used to treat or ameliorate the symptoms of cancer, the patient with cancer can be administered an anti-CD79b antibody in combination with the introduction of one or more of the above-mentioned chemotherapeutic agents. In particular, we also consider combination therapy implemented together with the introduction of paclitaxel and its modified derivatives (see, for example, EP0600517). Anti-CD79b antibody is administered together with a therapeutically effective dose of chemotherapeutic agents. In another embodiment of the invention, the anti-CD79b antibody is administered in combination with chemotherap�th to increase the activity and effectiveness of chemotherapeutic agents, for example, paclitaxel. The reference guide for physicians (PDR) indicated dose of means that have been used to treat various cancers. Regimens and doses of the aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the degree of development of this disease, and other factors known to the person skilled in the art and may be determined by the attending physician.

In one of the specific variants of the invention, the patient is administered with a conjugate containing anti-CD79b antibody, anywhereman with a cytotoxic agent. Preferably immunoconjugate associated with protein CD79b, internalized in the cell, resulting in increased therapeutic efficacy immunoconjugate in regard to the destruction of cancer cells to which they bind. In a preferred embodiment of the invention, the cytotoxic agent targeted to a nucleic acid of cancer cells or inhibits its action. Examples of such cytotoxic funds described above, and such means are maytansinoid, calicheamicin, ribonucleases and DNA endonuclease.

Anti-CD79b antibodies or their conjugates with toxins administered to a person in accordance with known methods, such as vnutrepenialnye, for example, in the form of a loading dose or by continuous infusion over a certain period of time, but also intramuscularly, intraperitoneally, inside the cerebrospinal fluid, subcutaneous, intra-articular, synovial fluid inside, intrathecal, oral, topical, or by inhalation. Preferred is intravenous or subcutaneous administration of the antibody.

Other courses of therapy may be combined with the introduction of anti-CD79b antibodies. The combined introduction includes co-administration of separate formulations or a single pharmaceutical preparation and sequential introduction in any order, preferably during the period of time over which both (or all) of the active agent simultaneously exert their biological activity. In this case it is preferable that such combination therapy gave a synergistic therapeutic effect.

It may also be desirable to conduct the combined administration of anti-CD79b antibodies or antibody together with the introduction of antibodies against another tumor antigen associated with the particular cancer.

In another embodiment of the invention, the methods of therapeutic treatment according to the invention include the combined introduction of anti-CD79b antibody (or antibodies) and one or more chemotherapeutic agents or religiuous funds including co-administration of a mixture of various chemotherapeutic agents or other chemotherapy(their) funds (funds) or other therapeutic(their) funds (funds) that also inhibit tumor growth. Chemotherapeutic agents are phosphate estramustine, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyacetone taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Methods of obtaining and schemes such chemotherapeutic agents can be carried out in accordance with the manufacturers instructions, or they can be empirically selected by the specialist. Methods of obtaining and regimen of administration of the chemotherapeutic agents are also described in the publication "Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md (1992). The antibody may be combined with protivovandalnymi connection, for example, anti-estrogen compound such as tamoxifen; antiprogesterone connection, such as onapristone (see, EP 616812) or antiandrogens connection, such as flutamide, in dosages commonly used for the introduction of such molecules. If the cancer being treated is androgen-dependent cancer, the patient may be previously subjected to anti-androgen therapy, and then, after the cancer becomes an�Rogen-independent, the patient can be administered anti-CD79b antibody (and optionally other tools described here).

Sometimes it may also be desirable for co-administration to the patient a means to prevent cardiovascular disease (to prevent or reduce the dysfunction of the myocardium associated with therapy) or one or more cytokines. In addition to the above therapeutic treatment regimens, the patient may undergo surgery to remove the cancer cells, and/or it may be subjected to radiation therapy (e.g. external irradiation or therapy with radioactive labeled agent, such as an antibody) that were conducted before, during or after therapy with antibody. Suitable dosages for any of the above jointly administered funds are used here doses, and these doses can be lowered due to the combined (synergistic) action of the specified means and anti-CD79b antibody.

Antibody-containing composition according to the invention is prepared, divided into doses and administered in accordance with well known medical practice. The factors considered for the preparation of such compositions, are the specific disorder being treated, the particular mammal being treated, the clinical condition of konkretno� patient etiological factor causing this disorder, the site, the tool, method of administration, the scheme of administration and other factors known to practitioners. The specified antibody should be, but not necessarily, made in the form of a composition with one or more modern means used for the prevention or treatment of the disorder. An effective amount of such other funds depends on the amount of the antibody according to the invention present in the composition, the type of disorder or the method of its treatment and other factors discussed above. These other means usually are introduced in the same doses and the same methods that are usually used earlier, either these doses comprise about 1-99% of the previously used doses.

For the prevention or treatment of diseases the appropriate dose and regimen of administration of the drug may be selected by the physician according to known criteria. The appropriate dosage of antibody will depend on the type of disease being treated, as defined above, the severity and course of treatment of the disease, regardless of whether administered the indicated antibody for preventive or therapeutic purposes, from previously conducted treatment, the medical history of the patient and his susceptibility to a given antibody�, and from doctor's appointment. Such an antibody may be administered to the patient once or several times during the course of treatment. Preferably, the antibody is administered by intravenous injection or subcutaneous injection. Depending on the type and severity of the disease, the initial preset dose of the antibody administered to the patient is from about 1 μg/kg to 50 mg/kg of body weight (e.g., about 0.1-15 mg/kg/dose), regardless of whether performed one time or repeated administration or continuous infusion of the antibody. Scheme of dose may include the introduction of an initial loading dose of the anti-CD79b antibody of approximately 4 mg/kg, followed the introduction of weekly maintenance dose of approximately 2 mg/kg anti-CD79b antibody. However, there may be other schemes of doses. A typical daily dosage can range from about 1 μg/kg to 100 mg/kg or more, depending on the aforementioned factors. For re-introduction in the course of several days or more, depending on the condition, the treatment may be carried out until, until you reach the desired suppression of disease symptoms. Monitoring the effect of therapy can be easily carried out by standard methods, from the analysis and in accordance with the criteria known to the doctor and�and other specialists in this field.

In this application, in addition to the introduction of protein-antibody to the patient, we also consider the introduction of antibodies by the method of gene therapy. Such administration of nucleic acid encoding the antibody, is included in the scope of the term "introduction of a therapeutically effective amount of an antibody". See, for example, an application WO96/07321 published March 14, 1996, relating to the use of gene therapy to intracellular production of antibodies.

There are two main ways of introducing the nucleic acid (optionally contained in a vector) into the patient's own cells, in vivo and ex vivo. For in vivo delivery the nucleic acid is directly administered to the patient, and usually on the site, which required the introduction of antibodies. For ex vivo treatment, make a fence of a patient's cells, and then these selected cells are administered nucleic acid, and the modified cells are administered to the patient either directly or, for example, in the form of capsules in porous membranes that are implanted to the patient (see, e.g., U.S. patent 4892538 and 5283187). There are several methods of introducing nucleic acids into viable cells. Such methods may vary depending on, whether administered nucleic acid into cells, cultured in vitro, or administered in vivo into cells of interest of the owner. Techniques suitable for the transfer of nucleic acid into mammalian cells in itro, are the methods with the use of liposomes, electroporation, microinjection, merge cells, a method using DEAE-dextran, the method of precipitation of calcium phosphate, etc. the Most commonly used vector for gene delivery ex vivo is a retroviral vector.

Currently preferred methods of transferring nucleic acids in vivo are transfection with viral vectors (such as adenovirus, herpes simplex virus I or adeno-associated virus) and lipid systems (suitable lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE and DC-Chol). Description currently used protocols for tagging genes and gene therapy can be found in publications Anderson et al.,Science256:808-813 (1992). Cm. also the application WO 93/25673 and cited in her work.

The scope used herein the term "antibody" can include various forms of anti-CD79b antibodies according to the invention. Thus, these antibodies are full-length or intact antibody, antibody fragments, antibody with a native sequence or amino acid variants, humanized, chimeric or hybrid antibodies, immunoconjugates and their functional fragments. In a hybrid antibody sequence of the antibody is attached to a heterologous polypeptide sequence. Antibodies can be modification�is decorated in the Fc-region for the message to the desired effector functions. As discussed in more detail in these sections on the appropriate Fc regions, the "naked" antibody associated with the cell surface can induce cytotoxicity, e.g., through antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in the case of complement-dependent cytotoxicity, or some other mechanisms. Alternatively, if it is desirable to eliminate or reduce effector function, in order to minimize side effects or complications of therapy, can be used some other Fc-region.

In one embodiment of the invention, the antibody competes for binding or essentially binds to the same epitope, bound to the antibodies according to the invention. Also discusses antibodies having biological properties of anti-CD79b antibodies according to the invention, and in particular, including delivery to tumors in vivo, and any inhibition of cell proliferation or cytotoxic properties.

Methods of producing the above antibody is described in detail in the present application.

Anti-CD79b antibody according to the invention can be used for the treatment of CD79b-expressing cancer or ameliorate one or more symptoms of cancer in the mammal. Such cancers are, n� are not limited to, cancer of the hematopoietic system or blood cancer, such as lymphoma, leukemia, myeloma or lymphoid malignancies, but also cancer, cancer of the spleen and lymph nodes. More specific examples of such b-cell-associated cancers include, for example, vysokokachestvennaya, srednestaticheskaya and nizkozameshhennoj lymphoma (including b-cell lymphomas, such as, for example, b-cell lymphoma of lymphoid tissue mucosa, and non-Hodgkin's lymphoma, lymphoma cells of the cortex of the brain, Burkitt's lymphoma, small cell lymphocytic lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, and T cell lymphomas) and leukemia (including secondary leukemia, chronic lymphocytic leukemia, such as b cell leukemia (CD5+-B-lymphocytes), myeloid leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia and myelodysplasia), and other hematological and/or b-cell or T-cell cancers. The term "cancer" includes metastases of any of the above diseases. The specified antibody has the ability to contact at least a part of cancer cells expressing CD79b polypeptide in the mammal. In a preferred embodiment of the invention �specified antibody is effective to destroy or destruction of CD79b-expressing tumor cells, or inhibiting the growth of such tumor cells in vitro or in vivo after binding, a specified antibody to CD79b polypeptide in the cells. This antibody is "naked" anti-CD79b antibody (not anywhereman with any agent). The "naked" antibodies that have cytotoxic properties or properties aimed at the inhibition of cell growth can also be attached to the cytotoxic agent, making them even more effective in the destruction of tumor cells. Cytotoxic properties can be reported to the anti-CD79b antibody, for example, specified by conjugating the antibody with a cytotoxic agent, with the formation described here immunoconjugate. Such cytotoxic agent or growth inhibitory agent are preferably low molecular weight. Preferred are also toxins, such as calicheamicin or maytansinoid and its analogs or derivatives.

The present invention relates to compositions comprising anti-CD79b antibody according to the invention and a carrier. For the treatment of cancer compositions can be administered to a patient in need of such treatment, where said composition may contain one or more anti-CD79b antibodies present in the form of immunoconjugate or in the form of "naked" antibodies. In another embodiment of the invention specified�s compositions can contain these antibodies in combination with other therapeutic agents, such as cytotoxic funds or growth-inhibiting means, including chemotherapeutic agents. The present invention also relates to preparations containing anti-CD79b antibody according to the invention and a carrier. In one embodiment, the antibodies of the specified drug is a therapeutic preparation containing pharmaceutically acceptable carrier.

In another aspect, the present invention relates to isolated nucleic acids encoding anti-CD79b antibody. The term also encompasses nucleic acids encoding the H chain and L, and in particular the remains of the hypervariable region; chain of nucleic acids that encode an antibody with a native sequence, as well as their variants, modifications and humanized versions of the antibody.

The present invention also relates to methods of treating cancerous tumors expressing CD79b polypeptide, or to a weakening of one or more symptoms of cancer in a mammal, where the specified method comprises administering a therapeutically effective amount of an anti-CD79b antibody to the mammal. Antibody-containing therapeutic compositions can be administered for short periods of time (a single injection) or over a long period of time, or periodically, depending on the prescribing physician. Present the image�communication also relates to methods for inhibiting cell growth, expressing CD79b polypeptide, and cytolysis of these cells.

The present invention also relates to kits and industrial products containing at least one anti-CD79b antibody. Kits containing anti-CD79b antibody may be used, for example, for analysis of cytolysis CD79b cells, as well as for purification or immunoprecipitation of CD79b polypeptide from cells. For example, for isolation and purification CD79b specified set may contain anti-CD79b antibody-related fields (e.g., sepharose spheres). Can be obtained in kits which contain the antibodies for detection and quantification of CD79b in vitro, for example using ELISA or Western blot analysis. The antibody used for detection, can be associated with a label such as a fluorescent or radioactive label.

I. Treatment with conjugate “antibody-drug”

It is assumed that the conjugate “antibody-drug” (ADC) according to the invention can be used to treat various diseases or disorders characterized by, for example, sverkhekspressiya tumor antigen. Representative States or hyperproliferative disorders are benign or malignant tumors; leukemias and lymphoid malignant disease. Other diseases are disorders associated with dysfunction of nerve cells, glial cells, hypothalamus, gland cells, macrophages, epithelial cells, stroma and blastocele, and inflammatory, angiogenic and immunologic disorders, including autoimmune disorders.

ADC-compounds that have been identified in animal models and in cell assays can be further tested in higher primates that have tumors, and clinical trials involving human subjects. Clinical trials involving human subjects can be developed to determine the effectiveness of the monoclonal anti-CD79b antibody or immunoconjugate according to the invention in patients suffering from b-cell-proliferative disorder, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. Clinical trials can be designed to assess the effectiveness of the ADC in combination with known therapeutic regimens, such as radiation therapy and/or chemotherapy with the use of known chemotherapeutic� and/or cytotoxic funds.

In General, the disease or disorder being treated is a hyperproliferative disease, such b-cell-proliferative disorder and/or b-cell cancer. Examples of cancer being treated, include, but are not limited to, b-cell-proliferative disorder is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

The cancer may include CD79b-expressing cells, which can contact the ADC according to the invention. To determine the expression levels of CD79b in a cancerous tumor can be carried out various diagnostic/prognostic assays. In one embodiment of the invention, the level of sverkhekspressiya CD79b can be analyzed using IHC. Drenched in paraffin sections of tissues taken by biopsy of the tumor can be subjected to IHC analysis and evaluated on the degree of staining and the number of tumor cells in accordance with the following criteria for the assessment of staining intensity of protein D79b.

For the prevention or treatment of disease the choice of the appropriate dose of the ADC depends on the type of the disease being treated, the above, the severity and course of the disease, the type of molecule, administered for preventive or therapeutic purposes, from previously conducted therapy from the medical history of the patient and his susceptibility to the antibody, and also from a doctor's appointment. The indicated molecule is suitably administered to the patient for one course or for multiple courses of treatment. Depending on the type and severity of the disease, the initial dose of the molecule is assigned for administration to the patient is about 1-15 mg/kg (e.g., 0.1 to 20 mg/kg), and this dose can be administered in a single dose or divided doses, or it can be administered by continuous infusion. A typical daily dosage can range from about 1 μg/kg to 100 mg/kg or more, depending on the aforementioned factors. Representative ADC dose, administered to the patient is from about 0.1 to 10 mg/kg of body weight of the patient.

For re-introduction in the course of several days or more, depending on the patient's condition, treatment may be continued until, until you reach the desired weakening of the symptoms of the disease. Representative scheme of doses comprises administering an initial loading dose prima�but 4 mg/kg, then the weekly introduction of a maintenance dose of anti-ErbB2 antibody of approximately 2 mg/kg. may be applied to other schemes of doses. The effect of this therapy can be easily traced with the use of standard methods and analyses.

J. Combination therapy

Conjugate "antibody-drug" (ADC) according to the invention can be combined with a second compound having anti-cancer properties, to obtain a combined pharmaceutical composition, or it can be applied together with other courses of treatment in the form of combination therapy. The specified second connection such pharmaceutical combination composition or combination course of treatment provides the additional activity of the combination of the ADC, provided that the components of such combination combination do not negatively impact each other.

The latter compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, agent, inhibiting the growth of cells, protivogelmintnoe means and/or means for the prevention of cardiovascular diseases. These molecules are usually present in combination in amounts that are effective to achieve this goal. Pharmaceutical composition containing ADC according �the turbine zobretenie, may also contain a therapeutically effective amount of chemotherapeutic agents, such as formation inhibitor tubulin, topoisomerase inhibitor, or a DNA-binding agent.

In one aspect of the invention, the first compound is an anti-CD79b ADC according to the invention and the second compound is an anti-CD20 antibody (or "naked" antibody or ADC). In one embodiment of the invention, the second compound is an anti-CD20 antibody (rituximab, Rituxan®) or 2H7 (Genentech, Inc., South San Francisco, CA). Other antibodies used in combination immunotherapy with anti-CD79b ADC according to the invention, include, but are not limited to, anti-VEGF antibody (e.g., Avastin®).

Other courses of therapy may be combined with the use of another anti-cancer therapy according to the invention, including, but not limited to, radiation therapy and/or bone marrow transplantation and peripheral blood cells, and/or the introduction of cytotoxic funds, chemotherapeutic agents or growth inhibitory agent. In one of these options chemotherapeutic agent is a means or combination of means, such as, for example, cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapeutic agents such as anti-CD20 antibody (�reamer, Rituxan®) or anti-VEGF antibody (e.g., Avastin®).

Combination therapy can be carried out by simultaneous or sequential carrying out of separate courses of treatment. The sequential mode of conducting such courses, the specified combination of drugs can be introduced in two stages or multiple stages. Such combination therapy includes co-administration of individual drugs or their administration in the form of a single pharmaceutical preparation, and their sequential introduction in any order, preferably during the period of time over which both (or all) of the active agent at the same time show their biological activity.

In one embodiment of the invention, the treatment using the ADC includes the combined introduction identified here an anticancer agent and one or more chemotherapeutic agents or growth inhibitors, including co-administration of a mixture of different chemotherapeutic agents. Chemotherapeutic agents are taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Methods of obtaining and schemes such chemotherapeutic agents can be carried out in accordance with the manufacturer's instructions, or they can empirically chosen specialist. Methods of obtaining and�status doses of chemotherapeutic agents are also described in the publication "Chemotherapy Service", (1992) Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.

Suitable dosages for any of the above jointly administered funds are used here doses, and these doses can be lowered due to the combined (synergistic) action just identified funds and other chemotherapeutic agents or treatments.

Combination therapy may provide “synergy” and “synergistic effect” of active ingredients, i.e., when the effect in the case of joint use of active ingredients, exceeds the sum of effects achievable with the individual use of these compounds. The synergistic effect can be achieved in that case, if the active ingredients (1) prepared in a mixture with each other and introduced or delivered simultaneously in a combined, unified dosage form; (2) delivered by serial or parallel introduction in the form of individual drugs; or (3) delivered in accordance with some other schemes. When administered through alternative therapies synergistic effect can be achieved in the case where the compound is administered or delivered sequentially, e.g., by different injections in separate syringes. In General, during an alternate therapy effective dose of each �active ingredient is administered sequentially, that is, one after another, and during combination therapy effective dose of two or more active ingredients are administered together.

K.Industrial products and kits

In another embodiment, the present invention relates to the commercial product containing the materials used for the treatment, prevention and/or diagnosis of CD79b-expressing cancer. This industrial product is the packaging and the label affixed to the packaging, or an insert that is attached to such packaging. Suitable packings are, for example, bottles, vials, syringes, etc. Such packing can be manufactured from various materials, such as glass or plastic. This package contains a composition which is effective for the treatment, prevention and/or diagnosis of cancer, and may have a sterile inlet (for example, such a package may be a bag for intravenous solution or vessel having a tube, protegem needle for subcutaneous injection). At least one active agent in said composition is an anti-CD79b antibody according to the invention. On the label or on the insert embedded in the package must be stated that such a composition is used for the treatment of cancer. The label or the liner in the package may also contain instrukciya the introduction of the antibody composition to a patient suffering cancer. Alternative specified industrial product may also include a second package containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, ringer's solution and dextrose solution. In addition, such a product may also include other materials necessary from the point of view of industrial production and the consumer, including other buffers, diluents, filters, needles and syringes.

The present invention also relates to kits that can be used for various purposes, e.g., for analyses on cytolysis CD79b-expressing cells, for purification or immunoprecipitation of CD79b polypeptide from cells. For isolation and purification of CD79b polypeptide, the kit can contain an anti-CD79b antibody-related fields (e.g., sepharose spheres). Can be obtained in kits which contain the antibodies for detection and quantification of CD79b polypeptide in vitro, e.g. in an ELISA-analysis or Western blot analysis. The specified set, and article of manufacture that contains packaging and the label affixed to the packaging, or an insert that is attached to such packaging. This package contains a composition comprising at least one anti-CD79b antibody according to the invention. Can also be included�s and other packaging, which contain, e.g., diluents, buffers, control antibodies. The label or the liner in the package can contain a description of the composition, and instructions for use in vitro or detection.

L. the Use of CD79b polypeptides

The present invention encompasses methods of screening compounds to identify compounds that mimic the CD79b polypeptide (agonists) or prevent the action of CD79b polypeptide (antagonists). The screening assays to identify candidate antagonists that are used as medicines, have been developed to identify compounds that bind or form a complex with CD79b polypeptides encoded by the identified genes here, or, conversely, prevent the interaction of the encoded polypeptides with other cellular proteins, including, for example, inhibition of expression of CD79b polypeptide from cells. Such screen-tests include tests that are suitable for large-scale screening of chemical libraries, and therefore suitable for the identification of small molecules drug candidates.

Tests can be designed in a variety of formats, including assays for the binding of protein-protein, biochemical screening assays, immunoassays and cell-based assays, are well known to specialists.

All assays for antagonists are profession� standard, that is, they require contacting the drug candidate with a CD79b polypeptide encoded by a nucleic acid identified here under the conditions and within the period of time that is sufficient for the interaction of these two components.

Analyses on the binding of the specified interaction is binding and the complex formed can be isolated from the reaction mixture or detected in this mixture. In a particular embodiment of the invention, the CD79b polypeptide encoded by the identified genome here, or the drug candidate is immobilized on a solid phase, e.g., on a microtiter tablet, by covalent or non-covalent binding. Non-covalent binding is usually carried out by coating the solid surface of a solution of a CD79b polypeptide and drying. Alternative immobilized antibody, e.g. a monoclonal antibody, specific for the immobilized CD79b polypeptide, may be used to anchor on a solid surface. This analysis is carried out by adding neimmunizirovannah component that can be marked apparently detected by a label, to the immobilized component, e.g., the coated surface containing the anchored component. If the reaction is carried out completely, then unreacted to�mponent removed for example, by washing, and then detecting complexes anchored on the solid surface. If the original neemalirovannym component is detectivea label, the detection of label immobilized on the surface, indicating the formation of the complex. If the original neemalirovannym component shall not be detectivea label, the formation of the complex can be detected, e.g., using a labeled antibody specifically binding the immobilized complex.

If a connection-candidate interacts, but is not associated with a specific CD79b polypeptide encoded by the identified genome here, its interaction with the polypeptide can be analyzed by methods well known methods for the detection of interactions, "protein-protein".

Such analyses involve the use of traditional methods, such as for example, cross-linking, co-immunoprecipitate and the joint cleaned in gradient or chromatographic columns. In addition, monitoring of interaction "protein-protein" can be carried out using a yeast genetic system described by Fields and co-workers (Fields and Song,Nature (London), 340:245-246 (1989); Chien et al.,Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) and Chevray and Nathans,Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many activators of transcription, still� as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain and the other acts as an activation domain of transcription. Expression of the yeast system described in the foregoing publications (generally called "two-component hybrid system"), has the advantage that it uses a hybrid of two proteins, one of which is a protein target that is associated with the DNA-binding domain of GAL4, and the other is an activating protein-candidate associated with an activating domain. Gene expression reporter, GAL1-lacZ under the control of a GAL4-activated promoter depends on the restoration of GAL4 activity by interacting "protein-protein". Colonies containing interacting polypeptides are detected using a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKERTMto identify "protein-protein"interactions between two specific proteins, carried out by methods with application of two-component hybrids, is commercially available and supplied by the company Clontech. This system can also be used to map protein domains involved in specific protein interactions, and the precise amino acid residues that play an important role in such interactive display�tions.

Compounds that inhibit the interaction of a gene encoding identified here CD79b polypeptide, with other intra - or extracellular components can be tested as follows: usually receive a reaction mixture containing the product of the gene and the intra - and extracellular component under certain conditions and within a certain period of time, sufficient for the interaction and binding of the two products. To analyze the ability of the compounds of the candidate to inhibit binding, the reaction is conducted in the absence and in the presence of the tested compounds. In addition, the third reaction mixture can be added placebo, which serves as a positive control. Monitoring the binding (complex formation) of the tested compounds with intra - or extracellular component present in the mixture, were performed as described above. The formation of the complex in the control(s) reaction(s), but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of test compounds with its reaction partner.

For the analysis of antagonists on the CD79b polypeptide may be added to the cells together with the connection, scrinium on a particular activity and the ability of compounds to inhibit interest asset�spine in the presence of CD79b polypeptide means, that the specified connection is an antagonist of a CD79b polypeptide. Alternative antagonists may be detected by combining the CD79b polypeptide and a potential antagonist with membrane-bound receptors CD79b polypeptide or recombinant receptors under conditions that are suitable for analysis on competitive inhibition. The CD79b polypeptide can be labeled, e.g., radioactive label, resulting in different molecules of the CD79b polypeptide associated with the receptor, can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by various methods known in the art, for example, by the methods of panning ligand and FACS sorting. Coligan et al.,Current Protocols in Immun., 1(2): Chapter 5 (1991). If polyadenylated RNA is prepared from a cell responsive to the CD79b polypeptide, preferably carried out gene expression cloning and cDNA library created from this RNA is divided into pools and used for transfection of COS cells or other cells that are not susceptible to the CD79b polypeptide. Transfetsirovannyh cells grown on glass slides, treated with tagged CD79b polypeptide. The CD79b polypeptide can be labeled in various ways, including iodination or inclusion of a site of recognition for the site-specific�certification of protein kinase. After fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified, and then get subpoly that re transferout iterative method of obtaining subpool and re-screening, resulting in a single clone that encodes the intended receptor.

In an alternative method of identification of a receptor tagged CD79b polypeptide can be photouplink linked with cell membrane or can be obtained preparations that Express the receptor molecule. Cross-linked material is separated by electrophoresis in page and exposed to x-ray film. The labeled complex containing the receptor can be excised, divided into peptide fragments, and proteins subjected to microsequencing. Amino acid sequence obtained after microeconomia, can be used to design a set of degenerate oligonucleotide probes for screening the cDNA library, performed for the purpose of identification of the gene encoding the presumed receptor.

In another analysis of antagonists on mammalian cells or a membrane preparation expressing the receptor, can be incubated with labelled CD79b polypeptide in the presence of the connection candidate. Then can be determined the ability of dannulation to enhance or block this interaction.

More specific examples of potential antagonists is an oligonucleotide, which binds to the hybrid immunoglobulin and CD79b polypeptide, and in particular, antibodies including, but not limited to, polyclonal and monoclonal antibodies and their fragments, single-chain antibodies, antiidiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments thereof, and human antibodies and fragments thereof. Alternative a potential antagonist may be a closely related protein, for example, a mutated form of CD79b polypeptide that recognizes the receptor but does not exert any action on it, and thereby inhibits competitive effect of CD79b polypeptide.

Antibodies specifically binding to an identified here CD79b polypeptide, as well as other molecules identified in the above screening assays, can be administered in the form of pharmaceutical compositions for the treatment of various diseases, including cancer.

If the CD79b polypeptide is intracellular, and as inhibitors using whole antibodies, are preferred internalize antibodies. However, to deliver the antibody or antibody fragment in the cell, can also be used lipofectin or liposomes. If you use fragments of antibodies, it is preferred�is relatively the shortest inhibitory fragment, which specifically binds with the binding domain of the target protein. For example, on the basis of sequences of variable region of the antibody can be designed peptide molecules that retain the ability to bind to the sequence of the target protein. Such peptides can be synthesized by chemical method and/or can be produced by methods of recombinant DNA. See, for example, Marasco et al.,Proc. Natl. Acad. Sci. USA,90:7889-7893 (1993).

The drug described here may also contain more than one active connection is needed to treat a particular disease, and preferably, the compounds with complementary activities that do not negatively influence each other. Alternative or additionally, said composition may contain an agent that enhance its function, such as, for example, cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are usually present in combination in amounts effective to achieve the desired objective.

M.Derivatives of antibodies

Antibodies according to the invention can be further modified to include other nonprotein molecules that are known in the art and are readily available. Preferred molecules suitable �La derivatization of antibodies, are water-soluble polymers. Non-limiting examples of water-soluble polymers are polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, copolymers of polypropyleneoxide/ethylene oxide, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. In industrial production, preference should be given to Propionaldehyde of polyethylene glycol due to its stability in water. This polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if you attach more than one polymer, these polymers can have the same or different molecules. In General, the number and/or type of polymers used for derivatization may be selected based on several factors including, but not limited to, the particular properties or functions of the antibody, which needs to be improved, regardless of the specific modalities of therapy in which �Udet to use a specified derivative of the antibody, etc.

N.Method of screening

In another embodiment, the present invention relates to a method for determining the presence of CD79b polypeptide in a sample suspected to contain a CD79b polypeptide, where the specified method comprises treating the sample with a conjugate "antibody-drug" that binds to a CD79b polypeptide, and determining the level of binding of the specified conjugate "antibody-drug" with the CD79b polypeptide in the sample, where the presence of such binding indicates the presence of CD79b polypeptide in the sample. Such a pattern may, but need not, contain cells (which may be cancer cells), presumably expressing CD79b polypeptide. Conjugate "antibody-drug" used in this way, maybe, but not necessary-detectable labeled, attached to a solid carrier, or etc.

In another embodiment, the present invention relates to a method for diagnosing the presence of tumor in the mammal, where the specified method comprises (a) contacting the test sample containing tissue cells isolated from a mammal with the conjugate "antibody-drug" that binds to a CD79b polypeptide, and (b) detection of complex formation between the conjugate "antibody-drug" and the CD79b polypeptide in testirovanie, where the formation of the complex indicates the presence of tumor in the mammal. Conjugate "antibody-drug" used in this way, maybe, but not necessary-detectable labeled, attached to a solid carrier or the like, and/or the test sample of tissue cells can be obtained from the individual with suspected cancer.

IV.Other methods of using anti-CD79b antibodies and immunoconjugates

A.Diagnostic methods and methods of detection

In one aspect of the invention, the anti-CD79b antibodies and immunoconjugates according to the invention can be used to detect the presence of CD79b in a biological sample. Used here, the term "detection" includes the quantitative and qualitative detection. In some embodiments of the invention, the biological sample contains cells or tissues. In some embodiments of the invention such tissues are normal or cancer tissue, which, compared with other tissues, Express CD79b at a higher level, for example, b cell and/or tissue associated with the b-cells.

In one of its aspects the present invention relates to a method of detecting the presence of CD79b in a biological sample. In some embodiments of the invention the method comprises contacting the biological sample with an anti-CD79b antibody� in conditions conducive to the binding of anti-CD79b antibody to CD79b, and detection of complex formation between the anti-CD79b antibody and CD79b.

In one of its aspects the present invention relates to a method for diagnosing disorders associated with increased levels of expression of CD79b. In some embodiments of the invention the method comprises contacting the test cell with an anti-CD79b antibody; determining the level of expression (either quantitative or qualitative) CD79b in the tested cells by detection of the binding of anti-CD79b antibody to CD79b; and comparing levels of CD79b expression in the tested cells CD79b expression level in control cells (e.g., normal cells derived from the same tissue from which the tested cells, or in cells compared to normal cells Express CD79b), where higher levels of CD79b expression in the tested cells, compared to the control cells indicates the presence of a disease associated with elevated levels of expression of CD79b. In some embodiments of the invention, the test cells are obtained from the individual with a suspected disorder associated with elevated levels of expression of CD79b. In some embodiments of the invention such disorder is a cellular proliferative disease such as cancer or a tumor.

Representative cell-proliferative disorders that can be diagnosed using antibodies according to the invention are b-cell disorder and/or cell-proliferative disorders, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In some embodiments of the invention a method of diagnosis or detection, such as the method described above, enables the detection of the binding of anti-CD79b antibody to CD79b, expressed on the cell surface or in a membrane preparation isolated from cells expressing on their surface CD79b. In some embodiments of the invention said method comprises contacting cells with an anti-CD79b antibody under conditions conducive to binding of anti-CD79b antibody to CD79b, and detection of complex formation between the anti-CD79b antibody and CD79b on the cell surface. A representative analysis for the detection of the binding of anti-CD79b antibody to CD79b, expressed on the surface of cells, I�is "FACS"analysis.

To detect binding of anti-CD79b antibody to CD79b can be applied and some other methods. Such methods include, but are not limited to, assays for binding to the antigen are well known in the art, such as Western blot analyses, radioimmunoassays, ELISA (ELISA), "sandwich"immunoassays, analysis using immunoprecipitation, fluorescent immunoassays and immunoassays using protein A, and immunohistochemical analyses (IHC).

In some embodiments of the invention anti-CD79b antibodies are labeled. Labels include, but are not limited to, markers or molecules that can be detected directly (such as fluorescent, chromophoric, electronmobility, chemiluminescent and radioactive labels), as well as molecules such as enzymes or ligands, that are detected by indirect, e.g., through an enzymatic reaction or molecular interaction. Representative labels include, but are not limited to, radioisotopes32P,14C,125I,3H and131I, fluorophores such as chelate complexes of rare earth metals or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., Firefly luciferase and bacterial luciferase (patent school� No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, for example, glucose oxidase, galactosidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidase, such as uricase and xanthine-oxidase attached to the enzyme that uses hydrogen peroxide for the oxidation dye precursor such as HRP; lactoperoxidase or microbiocides; Biotin/avidin, spin labels, bacteriophobia labels, stable free radicals, etc.

In some embodiments of the invention, the anti-CD79b antibody is immobilized on an insoluble matrix. Immobilization leads to the separation of the anti-CD79b antibody of any CD79b, remaining in solution in the free state. This procedure is usually carried out by insolubilization anti-CD79b antibodies of the procedure of analysis, as is the case for adsorption of the water-insoluble matrix or surface (Bennich et al., U.S. patent 3720760), or by covalent bonding (e.g., cross-linking with glutaraldehyde) or by insolubilization anti-CD79b antibody after formation of a complex between the anti-CD79b antibody and CD79b, for example, by immunoprecipitation.

Any of the above variants of diagnosis or detection may be carried out with the use�m immunoconjugate according to the invention is an anti-CD79b antibody or along with this antibody.

B.Therapeutic methods

The antibody or immunoconjugate according to the invention can be used, for example, in therapeutic methods in vitro, ex vivo and in vivo. In one of its aspects the present invention relates to methods of inhibiting the growth or proliferation of cells, either in vivo or in vitro, where the specified method comprises treating cells with anti-CD79b antibody or immunoconjugate under conditions conducive to binding immunoconjugate with CD79b. The term "inhibition of growth or proliferation of cells means lower growth or proliferation of cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and includes the induction of cell death. In some embodiments of the invention the specified cell is a tumor cell. In some embodiments of the invention the specified cell is a b cell. In some embodiments of the invention the specified cell is a xenograft, for example, illustrated in the description of the present application.

In one aspect of the invention, the antibody or immunoconjugate according to the invention is used for treating or preventing b-cell-proliferative disorder. In some embodiments of the invention specified cell-proliferative disorder is associated with elevated expression and/or activity CD79b. For example, in some embodiments from�of bretania b-cell-proliferative disorder is associated with an increased level of expression of CD79b on the surface of b-cells. In some embodiments of the invention In a cell-proliferative disorder is a tumor or cancer. Examples of b-cell-proliferative disorders susceptible to treatment with antibodies or immunoconjugate according to the invention are, but are not limited to, lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to methods of treating b-cell-proliferative disorder, comprising administering to the individual an effective amount of an anti-CD79b antibody or its immunoconjugate. In some embodiments of the invention a method of treating b-cell-proliferative disorder comprises administering to the individual an effective amount of a pharmaceutical preparation containing an anti-CD79b antibody or anti-CD79b immunoconjugate, and optionally at least one additional therapeutic agent, such as the means described below. In some embodiments of the invention a method of treating b-cell-proliferative are terribly up�STV comprises administering to the individual an effective amount of a pharmaceutical preparation, contains (1) immunoconjugates comprising anti-CD79b antibody and cytotoxic agent, and optionally (2) at least one additional therapeutic agent, such as the means described below.

In one aspect of the invention, at least some of the antibody or immunoconjugate according to the invention can contact CD79b grown from the species, not human. Accordingly, antibodies or immunoconjugate according to the invention can be used to bind to CD79b, for example, in a cell culture containing CD79b, in humans or other mammals (e.g., chimpanzees, baboons, marmosets, dog-like apes and macaques, the rhesus monkey and pigs or mice) having a CD79b, with which the antibody cross-reacts or immunoconjugate according to the invention. In one embodiment of the invention, the anti-CD79b antibody or immunoconjugate can be used to deliver CD79b in b cells by contacting the antibody or immunoconjugate with CD79b with the formation of the complex of the "antibody-antigen" or "immunoconjugate-antigen, and thus to ensure penetration of conjugated cytotoxin immunoconjugate inside cells. In one embodiment of the invention specified is human CD79b CD79b.

In one embodiment of the invention, the anti-CD79b antibody or immuno�nyugat can be used in the method of CD79b binding of the individual, suffering from a disorder associated with increased expression and/or activity CD79b, where the specified method comprises administering to the individual the antibody or immunoconjugate to implement CD79b binding in an individual. In one embodiment of the invention bound antibody or bound immunoconjugate internalized in b-cells expressing CD79b. In one embodiment of the invention specified is human CD79b CD79b, and the specified individual is. Alternative individual can be a mammal expressing CD79b, which binds anti-CD79b antibody. In addition, the specified individual may be a mammal, which is introduced CD79b (e.g., by delivery CD79b or expression of the transgene encoding CD79b).

Anti-CD79b antibody or immunoconjugate can be administered to man for therapeutic purposes. In addition, the anti-CD79b antibody or immunoconjugate can be administered to a mammal, not a person who has expressed CD79b, which cross-connects the antibody (e.g., a Primate, pig, rats or mice), where the specified introduction is carried out for treatment of this animal or in order to use this animal as a model of human disease. In the latter case, such animal models can be used dlawrence therapeutic efficacy of antibodies or immunoconjugates (for example, determine the dose and time of treatment).

During therapy antibody or immunoconjugate according to the invention can be used alone or in combination with other therapeutic compositions. So, for example, antibodies or immunoconjugate according to the invention can be administered together with at least one additional therapeutic agent and/or adjuvant. In some embodiments of the invention, the other therapeutic agent are cytotoxic agent, chemotherapeutic agent, or growth-inhibitory agent. In one of these options chemotherapeutic agent is a means or combination of means, such as, for example, cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapeutic agents such as anti-CD20 antibody (e.g., Rituxan®) or anti-VEGF antibody (e.g., Avastin®), where the specified combination therapy can be used for treating cancer and/or b-cell disorders such as b-cell-proliferative disorders, including lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic Le�goats (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

So the above combination therapy involves the combined administration (where two or more therapeutic agents are included in the same preparation or in separate preparations) and a separate introduction, in which the introduction of antibodies or immunoconjugate according to the invention can be carried out before, during and/or after administration of the additional therapeutic agent and/or adjuvant. Antibodies or immunoconjugate according to the invention can also be used in combination with radiation therapy.

The antibody or immunoconjugate according to the invention (and any additional therapeutic agent or adjuvant) can be administered by any appropriate means, including parenteral administration, subcutaneous administration, intraperitoneal administration, intrapulmonary administration and intranasal introduction, and if it is necessary for local treatment, introduction to the affected area. Parenteral injections are intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate can be introduced by periodic infusion, particularly with declining doses of the antibody or immuno�conjugate. The dose may be administered by any suitable method, for example, by injection, such as intravenous or subcutaneous injection, partly depending on whether such administration immediately or over a long period of time.

Antibodies or immunoconjugate according to the invention is prepared, divided into doses and administered in accordance with well known medical practice. The factors considered for the preparation of such compositions, are the specific disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiological factor causing this disorder, the site, the tool, method of administration, the scheme of administration and other factors known to practitioners. The specified antibody or immunoconjugate should be, but not necessarily, made in the form of a composition with one or more modern means used for the prevention or treatment of the disorder. An effective amount of such other funds depends on the amount of the antibody according to the invention present in the composition, the type of disorder or the method of its treatment and other factors discussed above. These other means usually injected in the same dose and those�and the same techniques, which usually previously used, or these doses comprise about 1-99% of the previously used doses, or such funds may be administered at any dose and by any means that can be, if necessary, determined empirically or by the clinical method.

For the prevention or treatment of disease appropriate dose of the antibody or immunoconjugate according to the invention (when used alone or in combination with one or more other therapeutic agents such as chemotherapeutic agents) depends on the type of disease being treated, the type of antibody or immunoconjugate, the severity and course of treatment of disease, administered regardless of whether the specified antibody or immunoconjugate for preventive or therapeutic purposes, from previously conducted treatment, the medical history of the patient and his susceptibility to the antibody or immunoconjugate and from doctor's appointment. Such an antibody or immunoconjugate can be administered to the patient once or several times during the course of treatment. Depending on the type and severity of the disease early, pre-set dose of the antibody or immunoconjugate entered the patient is about 1-100 mg/kg (e.g., 0.1 to 20 mg/kg/dose), regardless of whether single or multiple �maintenance or continuous infusion of the antibody. One typical daily dosage can range from about 1 μg/kg to 100 mg/kg or more, depending on the aforementioned factors. For re-introduction in the course of several days or more, depending on the condition, the treatment may be carried out until, until you reach the desired suppression of disease symptoms. One representative dose of the indicated antibodies or immunoconjugate can range from about 0.05 mg/kg to 10 mg/kg. Thus, the patient can be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg 4,0 mg/kg or 10 mg/kg of antibody or immunoconjugate (or any combination). Such doses may be administered intermittently, e.g. every week or every three weeks (e.g., so that the patient received approximately from 2 to 20, or for example, about 6 doses of antibodies or immunoconjugate). At first it may be introduced a higher loading dose, and then can be entered one or more lower doses. Representative scheme of doses comprises administering an initial loading dose of approximately 4 mg/kg, then the weekly introduction of a maintenance dose of about 2 mg/kg of the antibody. However can be applied to other schemes of doses. Monitoring the effect of therapy can be easily performed using standard methods of analisou.

C.Tests for activity

Anti-CD79b antibodies and immunoconjugates according to the invention can be characterized for their physical/chemical and/or biological activity using various assays known in the art.

1.Tests for activity

In one of its aspects the present invention relates to assays to identify anti-CD79b antibodies or their immunoconjugates having biological activity. Biological activity may include, for example, the ability to inhibit the growth or proliferation of cells (for example, the activity aimed at the destruction of cells") or the ability to induce cell death, including programmed cell death (apoptosis). The present invention also relates to antibodies or immunoconjugates having such biological activity in vivo and/or in vitro.

In some embodiments of the invention, the anti-CD79b antibody or its immunoconjugate tested for the ability to inhibit the growth or proliferation of cells in vitro. Tests for the inhibition of growth or cell proliferation are well known in the art. Some of the tests on cell proliferation are called tests for "cell killing", to determine cell viability. One of these tests is luminescent analysis on isresponsible�ü cells CellTiter-Glo TMwhich is commercially available and was developed by Promega (Madison, WI). This analysis allows to determine the number of viable cells in culture based on a quantitative assessment of the presence of ATP, which is an indicator of the presence of metabolically active cells. Cm. Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. patent No. 6602677. The analysis can be performed in 96 - or 384-well format, making it possible to conduct highly automated scrinia (HTS). Cm. Cree et al. (1995) AntiCancer Drugs 6:398-404. The test procedure involves adding the single reagent (reagent CellTiter-Glo®) directly in cultured cells. This leads to lysis of cells and the production of luminescent signal produced by luciferase reaction. The fluorescent signal is proportional to the number present the Asia-Pacific region, which is directly proportional to the number of viable cells present in culture. Data can be recorded on a luminometer or imaging device, a camera, charge-coupled. The fluorescent signal obtained at the output of such a device, expressed in relative light units (RLU).

Another analysis on the proliferation of cells represents the analysis of "MTT", namely colorimetric analysis, which measure the level of oxidation of bromide 3-(4,5-dimethyl�eazol-2-yl)-2,5-diphenyltetrazolium to formazan under the action of mitochondrial reductase. Similar to the analysis of CellTiter-GloTMthis analysis allows us to determine the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005) Cancer Res. 65:3877-3882.

In one aspect of the invention, the anti-CD79b antibody test for the ability to induce cell death in vitro. Tests for the induction of cell death are well known in the art. In some embodiments of the invention, such analyses allow us to determine, for example, loss of membrane integrity as indicated by the uptake of propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD. In a representative analysis of PI uptake, the cells are cultured in modified according to the method of Dulbecco environment Needle (DMEM):environment of Hams F-12 (50:50), to which were added 10% thermoinactivation FBS (Hyclone) and 2 mm L-glutamine. Thus, this analysis is carried out in the absence of complement and immune effector cells. Cells plated in 100×20-mm Cup at a density of 3×106cells in a Cup and leave overnight for adhesion. Then the medium is removed and replaced with fresh medium or medium containing various concentrations of antibody or immunoconjugate. Then the cells were incubated for 3 days. After treatment, the monolayers washed with PBS and separated by trypsinization. After that, the cells are centrifuged at 1200 R/mi� for 5 minutes at 4°C, then the precipitate was resuspended in 3 ml cold Ca2+-binding buffer (10 mm Hepes, pH of 7.4, 140 mm NaCl, 2.5 mm CaCl2) and divided into aliquots in test tubes (35 mm, 12×75) with a tight fitting lid (1 ml per tube, 3 tubes per treatment group) for removal of cell clusters. Then in a test-tube add PI (10 μg/ml). The samples are analyzed on a flow cytometer FACSCAN® and using a computer program FACSCONVERT® CellQuest (Becton Dickinson). Thus can be identified antibodies or immunoconjugate that induce statistically significant levels of cell death as determined by PI uptake.

In one aspect of the invention, the anti-CD79b antibody or immunoconjugate tested for the ability to induce apoptosis (programmed cell death) in vitro. Representative antibody test or immunoconjugate, inducing apoptosis, is the analysis on the binding of annexin. In a representative analysis on the binding of annexin cells are cultivated and sown in cups as described in the previous paragraph. The medium is removed and replaced with fresh medium or medium containing 0.001-10 μg/ml antibody or immunoconjugate. After incubation for three days, the monolayers washed with PBS and separated by trypsinization. After that, the cells are centrifuged, was resuspended in Ca2+binding �where and divided into aliquots in test tubes, as described in the previous paragraph. In test tubes add labeled annexin (e.g. annexin V-FITZ) (1 μg/ml). The samples are analyzed on a flow cytometer FACSCAN™ and with the help of a computer program FACSCONVERT™ CellQuest (BD Biosciences). Thus identify antibodies or immunoconjugate which induce statistically significant levels of binding to annexin compared with the control. Other representative antibody test or immunoconjugate that induce apoptosis, is a colorimetric ELISA-analysis using DNA from the histone, which allows the detection minucioso degradation of genomic DNA. Such analysis can be accomplished using, for example, an ELISA-kit for the detection of cell death (Roche, Palo Alto, CA).

The cells used in any of the above in vitro tests, are cells or cell lines that normally Express CD79b or which were designed so that they expressively CD79b. Such cells are tumor cells expressing CD79b compared to normal cells derived from the same tissue. Such cells are also cell lines (including tumor cell lines) that Express CD79b, and cell lines that do not normally Express CD79b, but were transfected by a nucleic acid that encodes CD79b.

The water aspect of the invention, the anti-CD79b antibody or its immunoconjugate tested for the ability to inhibit the growth or proliferation of cells in vivo. In some embodiments of the invention, the anti-CD79b antibody or its immunoconjugate tested for the ability to inhibit tumor growth in vivo. For this kind of testing can be used a model system in vivo. In a representative system of xenotransplanted human tumor cells is administered to an animal with a weakened immune system, not a person, for example, the SCID mice. This animal is administered an antibody or immunoconjugate according to the invention. Then assess the ability of the antibody or immunoconjugate to inhibit or reduce tumor growth. In some embodiments, the above system of xenotransplantation, human tumor cells are tumor cells, derived from human. Such cages suitable for the preparation of models of xenotransplantation, are cells of the human leukemia cell line of human lymphoma, which include, but are not limited to, cells of BJAB-luc (for example, EBV-negative cell line of Burkitt lymphoma, transfetsirovannyh luciferase reporter gene), Ramos cells (ATCC, Manassas, VA, CRL-1923), cells SuDHL-4 (DSMZ, Braunschweig, Germany, AAC 495), DoHH2 cells (see Kluin-Neilemans, H. C. et al., Leukemia 5:221-224 (1991), and Kluin-Neilemans, H. C. et al., Leukemia 8:1385-1391 (1994)), the cells Granta-519 (see Jadayel, D. M. et al, Leukemia 11(1):64-72 (1997)). In some embodiments of the invention, human tumor cells are administered to an animal with a weakened IMM�nicecom, not human, by subcutaneous injection or transplantation in appropriate area, for example, in the fat body of the mammary gland.

2.Assays for binding and other tests

In one aspect of the invention, the anti-CD79b antibody test for antigen-binding activity. For example, in some embodiments of the invention, the anti-CD79b antibody test for the ability to bind to CD79b, expressed on the cell surface. Such testing may be performed using FACS analysis.

In one aspect of the invention to identify a monoclonal antibody that competes with murine MA79b antibody, humanized MA79b antibody.v17 and/or humanized MA79b antibody.v28 and/or humanized MA79b antibody.v32 for binding to CD79b, can be analyzed in a competitive binding. In some embodiments of the invention, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound murine MA79b antibody, humanized antibody MA79bv.17 and/or humanized MA79b antibody.v18 and/or humanized MA79b antibody.v28 and/or humanized MA79b antibody.v32. Representative assays for competitive binding include, but are not limited to, routine tests, such as tests, described in the publication�ation Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed description of representative methods for the mapping of the epitope to which the antibody binds can be found in the publication of Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). It is believed that the two antibodies bind to the same epitope if each blocks the binding of the other by 50% or more.

In a representative analysis on competitive binding of immobilized CD79b incubated in a solution comprising the first labeled antibody that binds to CD79b (e.g., murine MA79b antibody, humanized antibody MA79bv.17 and/or humanized MA79b antibody.v18 and/or humanized MA79b antibody.v28 and/or humanized MA79b antibody.v32) and a second unlabeled antibody that is being tested on its ability to compete with the first antibody for binding to CD79b. The second antibody may be present in the supernatant of hybridomas. As a control, immobilized CD79b incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions conducive to binding of the first antibody to CD79b, excess unbound antibody is removed, and determine the amount of label associated with immobilized CD79b. If the amount of label associated with immobilized CD79b in the sample is significantly lower than the number m�weave in a control sample, this means that the second antibody is competing with the first antibody for binding to CD79b. In some embodiments of the invention immobilized CD79b is present on the cell surface or in a membrane preparation from cells expressing on their surface CD79b.

In one aspect of the invention, the purified anti-CD79b antibodies can be further characterized by conducting a series of tests, including, but not limited to, N-terminal sequencing, amino acid analysis, size exclusion high performance liquid chromatography (HPLC) carried out in adenocarinoma conditions, mass spectrometry, ion exchange chromatography and papain hydrolysis.

In one of its variants the present invention relates to a modified antibody that possesses some but not all effector functions, which makes it a desirable candidate for use in many purposes, namely in the case where the important factor is the half-life of antibodies in vivo, and some effector functions (such as complement and ADCC) are either unnecessary, or undesirable. In some embodiments of the invention determine the activity of antibodies to Fc guarantee that will be retained the desired properties. To confirm the reduction/depletion of CDC and/or ADCC activity can be conducted�s tests for cytotoxicity in vitro and/or in vivo. For example, in order to guarantee that the antibody will not communicate with FcγR (and therefore probably would not have ADCC activity), but will retain the ability to bind to FcRn can be analyzed for binding to Fc-receptor (FcR). Primary cells mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is systematized in table 3 on page 464 of publication of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). Example in vitro analysis to assess ADCC activity of interest molecules described in U.S. patents №№ 5500362 or 5821337. Suitable effector cells for such assays are mononuclear cells of peripheral blood (PBMC) and natural killer cells (PC). Alternative or additionally, ADCC activity of interest molecules can be assessed in vivo, e.g., in animal models, such as the model described in the publication Clynes et al. PNAS (USA) 95:652-656 (1998). To confirm that the antibody is unable to communicate with C1q, and therefore it does not have CDC activity can also be analyzed for binding to C1q. For the analysis of complement activation may be conducted by the CDC analysis, for example, the analysis described in the publication Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Evaluation of binding to FcRn, and so�e clearance/time-life in vivo can also be implemented by methods, known in the art.

The following examples are only for illustrative purposes and are not considered as limiting the scope of the invention.

All patents and publications cited in the description of the present application, is fully introduced into the present description by reference.

Examples

Commercially available reagents referred to in the examples were used according to manufacturer's instructions, unless otherwise agreed. The antibodies used in the examples are commercially available antibodies, and such antibodies include, but are not limited to, anti-CD79b antibody (antibody purchased by the company Biomeda (Foster City, CA) or BDbioscience (San Diego, CA) or Ancell (Bayport, MN), anti-CD79b antibody (isolated from hybridomas deposited with the ATCC as HB11413 July 20, 1993), and chimeric anti-CD79b antibody (containing the variable domains of antibodies produced from hybridomas deposited with the ATCC as HB11413 20 July 1993). The source of those cells identified in the following examples and throughout the description of the invention under registration numbers of ADS, is the American type culture collection, Manassas, VA.

Example 1: Obtaining a humanized anti-CD79b antibody

Numbers of residues are given by Kabata (Kabat et al., Sequences of proteins of iunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). In the description of every�retenu used one-letter notation of amino acids. The degeneracy of the DNA represented by IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T, M=A/C, R=A/G, S=G/C, W= A/T, Y=C/T).

A. Hybrid humanized anti-CD79b antibody

Received various humanized anti-CD79b antibody. The sequence of domains VL and VH of murine MA79b antibody (MA79b) (Roswell Park Cancer Institute; Okazaki et al., Blood, 81:84-94 (1993)) was aligned with sequences of domains of the human consensus VL Kappa I (huKI) and human consensus VH subgroup III (huIII). To obtain HVR-hybrid used frame acceptor VH region, which differed from the domain of the human consensus VH subgroup III in 3 positions: R71A, N73T and L78A (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992)). Hypervariable region of murine MA79b (MA79b) was attached to the frame region acceptor human consensus sequences and received a direct HVR-hybrid MA79b (here called "hybrid MA79b" or "MA79b-hybrid" or "MA79b-bound "humanized antibody" or "huMA79b-in hybrid"). In the VL domain the following areas were annexed to the human consensus acceptor sequence at positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) (figure 7A-B). In the VH domain was annexed region at positions 26-35 (H1), 49-65 (H2) and 93-102 (H3)(figure 8A-B). The publication also available in et al. (Also available in et al., J. Mol. Biol., 262:732-745 (1996)) analyzed the crystal structure of the complex antigen-antibody, and was detected, Thu� position 49, 93 and 94 of the heavy chain are part of the contact area, and, in the case of the humanized antibodies, they are included in the definition HVR-H2 and HVR-H3.

Variant obtained by direct connection (huMA79b-hybrid), was produced by the method of Kunkel mutagenesis as Fab presented on the phage, and as IgG, using a separate oligonucleotide for each hypervariable region. Appropriate clones were evaluated by DNA sequencing.

B. Options hybrids of humanized anti-CD79b antibodies

Options hybrid anti-CD79b antibodies that include a variety of mutants in the hypervariable regions MA79b-associated "humanized" antibodies, obtained using phage libraries. Options hybrid anti-CD79b antibodies comprise mutations at one position in the HVR (figure 9) or mutations in a number of positions in HVR (figure 10).

C. Selection of phage

For selection of phage extracellular domain of CD79b (huCD79becd) (2 µg/ml) were immobilizovana in PBS in microtiter tablets MaxiSorp (Nunc) overnight at 4°C. the plates were blocked for 1 hour using casein blocker (Pierce). Phage were harvested from the culture supernatant and suspended in PBS containing 0.5% BSA and 0.05% Tween 20 (PBSBT). After adding ragovoy library and selection of phage for 2 hours, the wells of a microtiter plate was extensively washed with PBS containing 0.05% Tween 20 (PBST)to remove unbound phage, and the bound phage were suirable by incubation of the wells with 100 mm HCl for 30 minutes. The stiffness of selection can be increased in the process of conducting successive rounds of selection by increasing the number of washes with PBST or by incubation with soluble huCD79becdto increase the period of time prior to elution.

- Eluted phage was neutralized with 1 M Treece, pH 8, and were amplified using cells XL1-Blue and phage-assistant M13/KO7, and then cultured overnight at 37°C in 2YT, 50 μg/ml of carbenicillin. The titers of phage, - eluted with the target-containing wells were compared with the titers of phage isolated from the wells containing the target, to assess the level of enrichment.

D. Obtaining Fab and receiving IgG

In order Fab protein expression for measuring the affinity, between the heavy chain and g3 in the phage vector representation by embedding the stop codon. Clones were transferred into cells of E. coli 34B8 and were cultured in complete medium C. R. A. P. at 30°C (Presta et al. Cancer Res. 57:4593-4599 (1997)). The cells were collected by centrifugation, suspended in PBS, 100 µm PMSF, 100μm of benzamidine, 2.4 mm EDTA, and destroyed in the clear with the use of microfluidizer. Fab was purified using affinity chromatography on G-proteins.

For screening variants of IgG was first produced in 293 cells. Vectors encoding the VL and VH (25 g) was transferred into 293 cells using uGene system. 500 µl of FuGene was mixed with 4.5 ml of DMEM containing no FBS and incubated at room temperature for 5 minutes. To this mixture was added each circuit (25 µg) and incubated at room temperature for 20 minutes and then transferred to flasks for transfection overnight at 37°C in 5% CO2. The next day, the medium containing the mixture for transfection, was removed and replaced with 23 ml of medium PS04 containing 0.1 ml/l trace elements (A0934) and 10 mg/l insulin (A0940). Cells were incubated for another 5 days and then the medium was collected at 1000 Rev./min for 5 minutes and subjected to sterile filtration through a 0.22-μm filter, binding to the protein present in small quantities. After adding 2.5 ml of 0.1% PMSF for every 125 ml of medium, samples can be stored at 4°C.

E. Determination of affinity (Biacore analysis)

To determine the affinity of MA79b-related options "humanized" antibody, extracellular domain of human CD79b (huCD79becd) expressively in CHO cells alone or in the form Fc-hybrid (huCD79becd-Fc) and purified by standard methods. In addition, the peptide of 16 amino acids (ARSEDRYRNPKGSACK) (SEQ ID NO: 16) containing the epitope for MA79b, synthesized by standard methods.

Characterization of the epitope for MA79b antibody (labeled on figure 19 as a "test peptide") described in the application for U.S. patent No. 11/462336, filed August 3, 2006, the Epitope for MA79 was localized in extracellular peptide region, located at a considerable distance from the transmembrane domain, and was present in full-length and truncated forms of human CD79b (see Cragg, Blood, 100(9):3068-76 (2002)), related to normal and malignant B-cells (Hashimoto, S. et al., Mol. Iunol., 32(9):651-9 (1995); Alfarano et al., Blood, 93(7):2327-35 (1999)). The truncated form of CD79b did not contain full-sized extracellular Ig-like domain (extracellular Ig-like domain that is not present in playerwindow truncated form of CD79b presented in the box on figure 19).

The level of binding of Fab and IgG variants of MA79b, MA79b-associated "humanized" antibodies or MA79b-related options "humanized" antibodies with immobilized huCD79becdor huCD79b-Fc or with the peptide of 16 amino acids containing the epitope for MA79b, measured by surface plasmon resonance. The affinity measurement was performed by surface plasmon resonance using a BIAcoreTM-2000. Antigen, huCD79becdor huCD79b-Fc was immobilizovana (approximately 50-200 EO) in 10 mm sodium acetate, pH of 4.8, at the touch chip CM5. The magnitude of the affinity depends on the number of immobilized huCD79becdthat is largely due to the avidity effect. For this reason, the affinity values obtained for samples in different days were normalized MA79b, which was also used�IAOD as standard. In those experiments, which evaluated the level of binding of the peptide of 16 amino acids containing the epitope for MA79b (ARSEDRYRNPKGSACK) (SEQ ID NO: 16), biotinylating peptide was immobilizovana (approximately 20 EO) on a sensor chip coated with streptavidin. Cleaned MA79b-attached version of "humanized" antibody (Fab or IgG) (2-fold serial dilution of 0.5 to 1000 nm in PBST) was injected at a flow rate of 30 μl/min. Each sample was analyzed in 4-minutes of the Association and a 10-minute dissociation. After each injection, the chip was regenerated using 10 mm glycine, pH of 1.7.

The magnitude of the response of the binding reactions was corrected by subtracting the values obtained for the control flow cells, from the values obtained for the flow-through cuvette containing MA79b-attached version of "humanized" antibody (Fab or IgG). For kinetic analysis used langourously model 1:1 curve according to konand koff.

F. Analysis on binding (FACS-analysis)

To further determine the level of binding of the Fab-fragment of MA79b-associated "humanized" antibody or its variants evaluated the level of binding of the Fab variants and/or IgG to the cells of the DoHH-2 by FACS analysis. Furthermore, using FACS analysis evaluated the level of binding of the MA79b-related options "humanitarian�about" antibodies with cells of BJAB, containing luciferase.

For conducting FACS analysis of Fab variants of MA79b-related options "humanized" antibody (MA79b-associated "humanized" antibodies (variant IgG used as a control)) cells, DoHH-2 (1×106in a volume of 100 µl) was first incubated in the presence or in the absence of 1 μg of the original murine monoclonal anti-CD79b antibody (MA79b) for 30 minutes and then was added 1 μg of individual Fab option (or control antibodies). As a "second" detection antibodies used Feh-konjugierten murine antibody against human light chain Ig Kappa (clone G20-193, BD Biosciences, San Diego, CA), as all versions are Fab light chain Kappa, and the cells DoHH-2 do not Express on their surface light chain Kappa.

For additional FACS analysis of IgG variants MA79b-related options "humanized" antibodies (variant IgG chMA79b used as a control), and 1.0 μg, 0.1 μg or 0.01 μg of antibody was titrated into a million BJAB cells containing luciferase. As a "second" detection antibodies used Feh-konjugierten murine antibody against human Ig.

G. Determination of affinity (analysis of Scatchard)

To further determine the level of binding of IgG variants having modifications in HVR-L2 and HVR-H3 (huMA79b L2/H3), we analyzed the binding sodirova�tion of IgG variants with BJAB cells, expressing human and CD79b CD79b dog-like apes, and then analyzed Scatchard.

For analysis of Scatchard 0.5 nm of the I125-labeled MA79b or huMA79b L2/H3 subjected to competitive binding with unlabeled MA79b or huMA79b L2/H3, respectively, in concentrations ranging from 50 to 0.02 nm (12-stage serial dilution 1:2) in the presence transfetsirovannyh cell line BJAB stably expressing CD79b copacobana monkeys and endogenous human CD79b. After 4-hour incubation at 4°C the cells were washed, and the cell precipitate was read on a gamma counter (automatic gamma counter 1470 WIZARD Automatic Damme Counter; Perkin Elmer, Walthem, MA). All calculations were performed with three replications, and the counting was performed for 10 minutes. To calculate the Kd used the average number of counts per minute (CPM), and such a calculation was carried out using a New computer program Ligand (Genentech, South San Francisco, CA).

Results and discussion

A. the Results of producing a humanized anti-CD79b antibodies

Human acceptor skeleton of the region used to produce a humanized anti-CD79b antibody contains a VL domain consensus sequence the human Kappa I and variant of the VH domain of the human consensus sequence of subgroup III. Variant of the VH domain has 3 substitutions in the human�Oh consensus sequences in the provisions R71A, N73T and L78A. The sequence of domains VL and VH MA79b aligned with human sequences Kappa I and subgroup III, where each HVR was identified and then incorporated into the human acceptor frame area with obtaining HVR-hybrid, which can be presented on the phage as Fab (figures 7 and 8).

Phage is MA79b-hybrid in the form of Fab, associated with immobilized huCD79becd(data not shown). However, if the sequence huMA79b-hybrid expressives as IgG, FACS-analysis on the affinity of its binding with huCD79becdpointed to the fact that the binding affinity was decreased more than 100-fold (data not shown), and Biacore analysis indicated a more than 50-fold reduction (figure 11).

1. Repair CDR

MA79b-related options "humanized" antibodies that possess the ability to bind to immobilized huCD79becdidentified using the following substitutions in the sequences.

Changes in HVR in VL were only available in libraries containing substitutions at one position, and such modifications is shown in figure 9 (for the mutations L1:Q27K (SEQ ID NO: 17; mutations SPL-2), (for mutations L2:L54R (SEQ ID NO: 18), E55K (SEQ ID NO: 19)), and (for mutations L3: E93S (SEQ ID NO: 20; mutation SPL-5), E93K (SEQ ID NO: 21)).

Changes in HVR in the L2, L3, H1 and H3 in the HVR were only available in libraries containing substitutions at several positions, and still� changes is shown in figure 10 ( for mutations L2:S52R, N53K, E55G and S56R (SEQ ID NO: 22; mutation L2-2); N53R (SEQ ID NO: 23); S52R, N53K, E55G and S56N (SEQ ID NO: 24); S52R, N53K, E55K and S56R (SEQ ID NO: 25); S52R, N53Y, E55K and S56R (SEQ ID NO: 26; mutation L2-29); S52R, N53K and E55K (SEQ ID NO: 27); S52R, N53K and E55A (SEQ ID NO: 28); S52G, N53I, E55A and S56R (SEQ ID NO: 29); S52R, N53K, E55R (SEQ ID NO: 30); S52R, N53K and E55G (SEQ ID NO: 31; mutation L2-38); S52R, N53H, E55K and S56R (SEQ ID NO: 32); A51S, S52R, N53Y, E55S and S56R (SEQ ID NO: 33); A51G, N53K, E55L and S56R (SEQ ID NO: 34); L54R and E55K (SEQ ID NO: 35); N53K and E55G (SEQ ID NO: 36); S52R, N53Y, E55R and S56R (SEQ ID NO: 37); S52R, N53R, E55R and S56T (SEQ ID NO: 38); S52R, N53R, E55G and S56R (SEQ ID NO: 39); S52R, N53Q, L54R, E55K and S56R (SEQ ID NO: 40); S52R, N53K, E55L and S56R (SEQ ID NO: 41); S52R, N53K, E55K and S56N (SEQ ID NO: 42); S52R, N53K, E55G and S56T (SEQ ID NO: 43); S52R, N53K, E55G and S56G (SEQ ID NO: 44); S52R, N53K, E55A and S56R (SEQ ID NO: 45)), (mutationL3:E93A (SEQ ID NO: 46); E93Q (SEQ ID NO: 47); no mutation (SEQ ID NO: 48); E93D (SEQ ID NO: 49); E93L (SEQ ID NO: 50); Q89N, Q90N, E93G and T97N (SEQ ID NO: 51); Q90P, S91D, D94A and L96R (SEQ ID NO: 52); Q89D, S91R and E93A (SEQ ID NO: 53)), (for mutation, H1:T28P, S30T, S31R and E35S (SEQ ID NO: 54); T28P, S30R and E35Q (SEQ ID NO: 55); T28P, S30T and E35N (SEQ ID NO: 56); T28P, S30T, S31R and E36N (SEQ ID NO: 57; mutation H1-6)); S30N, S31R and E35N (SEQ ID NO: 58); T28S and S30K (SEQ ID NO: 59); G26P, T28S, F29L, S30C, S31T, W33F and E35D (SEQ ID NO: 60); T28Y and S30T (SEQ ID NO: 61); T28P, S30G, S31R, I34V and E35N (SEQ ID NO: 62); S30K and S31K (SEQ ID NO: 63); T28P, S30T and E35Q (SEQ ID NO: 64); T28P, S30R and S31R (SEQ ID NO: 65); T28P, F29V, S30G, S31R and E35S (SEQ ID NO: 66); T28P, S30N, S31R and E35N (SEQ ID NO: 67; mutation H1-1); T28G, S30T and E35S (SEQ ID NO: 68); S30T, I34L and E35S (SEQ ID NO: 69); S30T (SEQ ID NO: 70); S31G and E35N (SEQ ID NO: 71); S30R, S31R and E35N (SEQ ID NO: 72); T28S, S30R and E35N (SEQ ID NO: 73); T28S, S30R, S31R and E35N (SEQ ID NO: 74); T28S, S30R and S31R (SEQ ID NO: 75); T28S, S30P, I34L and E35Q (SEQ ID NO: 76); T28P, S30T and S31R (SEQ ID NO: 77); T28P and S31G (SEQ ID NO: 78); T28P, S30R and E35S (SEQ ID NO: 79); T28P S30R and E35N (SEQ ID NO: 80); T28P, S30R and S31G (SEQ ID NO: 81); T28P, S30N and S31R (SEQ ID NO: 82); T28P, S30N, S31G and E35N (SEQ ID NO: 83); T28N, F29V, I34L and E35S (SEQ ID NO: 84); Y27F, T28P, S30T and E35S (SEQ ID NO: 85); and Y27F, T28P, S30N, S31R and E35N (SEQ ID NO: 86)) and (for H3 mutations:V98I and F100L (SEQ ID NO: 87; mutation of H3-12); the mutation is absent (SEQ ID NO: 88); Y99K and F100L (SEQ ID NO: 89); F100L (SEQ ID NO: 90); V98I (SEQ ID NO: 91); V98F, Y99C and F100L (SEQ ID NO: 92); F100L (SEQ ID NO: 93); V98I, Y99R and F100L (SEQ ID NO: 94; mutation of H3-10); V98I, Y99K and F100L (SEQ ID NO: 95); V98I and Y99R (SEQ ID NO: 96); V98I (SEQ ID NO: 97); D101S (SEQ ID NO: 98); Y99V and F100L (SEQ ID NO: 99); Y99R and F100L (SEQ ID NO: 100); Y99R (SEQ ID NO: 101); Y99F and F100L (SEQ ID NO: 102); V98I and F100L (SEQ ID NO: 103); V98I (SEQ ID NO: 104); V96R, Y99C and F100L (SEQ ID NO: 105); and V96I (SEQ ID NO: 106)).

Selection of clones was carried out in a different format in the form of Fab for FACS analysis and in the form of IgG for further analysis Biacore and Scatchard.

a. The definition of affinity (Biacore analysis)

As shown in figure 11, which illustrates the Biacore analysis, this method CDR-repair allows you to identify many changes in a separate sequence, which increase the affinity MA79b-associated "humanized" antibodies. Analysis conducted using surface plasmon resonance showed that although none of the tested variants with one HVR-replacement had no affinity similar to the affinity MA79b, however, the combination of substitutions identified in HVR-L2 and HVR-H3 (MA79b-associated variant of "humanized" antibodies L2/H3; also referred to here huMA79b L2/H3), resulted in the formation of variants (figure 11), about�latausha affinity, the similar affinity of the MA79b antibody when binding to immobilized huCD79becdor huCD79becd-Fc or a peptide of 16 amino acids containing the epitope for MA79b as defined in the Biacore analysis.

Analysis of the binding of the monomer (Fab) and dimer (IgG) MA79b with antigen (huCD79becd-Fc) (figure 11, row 1, columns for Ms. Fab and IgG) gave reason to assume that the component with 100-fold greater avidity and present in MA79b, may be absent in variants with improved affinity. In particular, MA79b-bound version of "humanized" antibody L2-2 (also referred to here huMA79b L2-2) that detects a 5-fold increase in the level of Monomeric binding compared to MA79b (figure 11, rows 1 and 3, columns for Ms. Fab), after changing the format huMA79b L2-2 IgG (figure 11, row 4, columns for Ms. Fab to IgG) of any apparent increase in affinity was observed. In addition, the original HVR-bound "humanized" MA79b antibody (huMA79b-hybrid) showed loss of binding of a component with avidity (figure 11, row 2, columns for Ms. Fab to IgG). The ability of increasing the level of binding through avidity may be desirable when binding to antigens on the cell surface.

b. The definition of affinity (analysis of Scatchard)

As was identified in the analysis of Scatchard, this method of CDR repair allows you to quickl� to identify the set of substitutions in a separate sequence, which increase the affinity MA79b-associated "humanized" antibodies. In particular, the analysis of cell binding showed that the affinity of binding of the MA79b and MA79b-associated case of "humanized" antibodies L2/H3 (huMA79b L2/H3) (obtained in another format such as IgG) BJAB cells stably expressing CD79b dog-like apes and endogenous human CD79b, had a Kd value of 0.63 nm (MA79b; Kd=0,63±0,14 nm and 0.52 nm (huMA79b L2/H3; Kd=0,52±0.1 nm), respectively (data not shown), as was identified in the analysis of Scatchard.

c. Determining the level of binding (FACS-analysis)

As was assessed by FACS-analysis, this method of CDR repair allows to identify the set of substitutions in a separate sequence, resulting in increased level of binding of the MA79b-associated "humanized" antibody (huMA79b-hybrid) cells, DoHH-2 (data not shown). In particular, FACS analysis of Fab variants (mutations L2-2, H3-10 and H1-1) isolated from libraries of SP and SR 6 libraries, cells, DoHH-2, indicated the binding of Fab variants and huMA79b-hybrid (presented in a new format such as IgG) with cells DoHH-2 (data not shown). In addition, FACS analysis of Fab variants showed that the binding of the Fab variants with cells DoHH-2 prevented by pre-incubation with murine anti-CD79b monoclonal antibody (MA79b) (data not shown).

p> 2. Repair of frame

Modification of the sequence HVR entered in HVR-L2 huMA79b L2/H3 variant, was radically different from the modifications observed in any of the human germ line. It was discovered that a variant of the huMA79b L2/H3, with its conjugation with DM1, effectively inhibits tumor growth in mice with model xenotransplanted in vivo (table 9). Since the analysis for Monomeric binding (Fab) and dimeric binding (IgG) huMA79b L2/H3 variant with the antigen indicated a loss of avidity (figure 11), the frame repair performed, as described below.

To determine the role of the provisions of the frame of the residues in the dimer binding to the antigen, designed variant with the provisions "of all remnants of the frame region in which potentially important provisions murine residues frame region were included in MA79b HVR-bound "humanized" antibody (huMA79b-hybrid). This option (called the figure 12 embodiment, having "all the remnants of the frame region") that does not contain any modifications in HVR, had the dimeric binding affinity similar to the affinity of binding to the chimeric MA79b antibody (chMA79b) (figure 12), as determined using Biacore analysis and analysis of Scatchard.

IgG variants, including murine skeleton residues in positions 4 and/or 47 (VL) and/or in the provisions 47, 48, 67, 69, 71, 73, 74, 78 and/or 80 (VH), Paul�Chali in order to identify a minimum set of provisions of a frame region, required to maintain high affinity binding of the dimer (figure 12). The remains of a mouse framed region shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14). It was found that the position of the frame region 47 in VL and 75 and 80 in VH not play an important role, as shown by the analysis of the MA79b-associated variant "humanized antibody" 17 (huMA79b.v17) (figure 12, row labeled 17).

MA79b-associated variant "humanized antibody" 18 (huMA79b.v18; figure 12, row labeled 18), which includes murine residues frame region in positions 4 in VL, and 48, 67, 69, 71, 73 and 78 in VH, as well as replacement in HVR-H3 (marked on figure 12 as “H3-10” and described above as a mutation H3-10), including V98I, Y99R and F100L, discover an additional 2-fold increase (figure 12, row labeled 28) the level of binding of the dimer compared to option 17 (figure 12, row labeled 17).

In order to avoid possible difficulties in obtaining these antibodies, the potential of the site, forming seasparrow acid (Asp-Gly) in HVR-L1 MA79b-related options "humanized antibody", was removed by transformation D28 in Glu (glutamic acid) (D28E; see variant 28; also designated herein as "huMA79b.v28”; figure 12, row labeled 28). Also acceptable are other substitutions that can be introduced to provide stability VL MA79b-associated variants �humanized antibody", including replacement D28 at Ser (serine) (D28E; see variant 32; also denoted here “huMA79b.v32”; figure 12, row labeled 32).

MA79b-associated variant "humanized antibody" 28 (huMA79b.v28; figure 12, row labeled 28), which includes: (1) murine residues frame region in positions 4 in VL, and 48, 67, 69, 71, 73 and 78 in VH, as well as includes (2) replacement in HVR-H3 (marked on figure 12 as “H3-10” and described above as a mutation H3-10), including V98I, Y99R and F100L, and, in addition, includes: (3) replacement in HVR-L1 (D28E described above) were characterized via Biacore analysis.

MA79b-associated variant "humanized antibody" 32 (huMA79b.v32; figure 12, row labeled 32), which includes: (1) murine residues frame region in positions 4 in VL, and 48, 67, 69, 71, 73 and 78 in VH, as well as includes (2) replacement in HVR-H3 (marked on figure 12 as “H3-10” and described above as a mutation H3-10), including V98I, Y99R and F100L, and, in addition, includes: (3) replacement in HVR-L1 (D28S described above) were characterized via Biacore analysis.

a. The definition of affinity (Biacore analysis)

As shown in figure 12, illustrating the Biacore analysis, this method of reparation frame circuit allows to identify the set of substitutions in a separate sequence that improve the binding affinity of the hybrid "MA79b-humanized antibody" with huCD79becd. Analyses performed by surface plasmon RES�nance, showed that MA79b-associated variant "humanized antibody" 28 (huMA79b.v28; with murine residues frame region in positions 4 in VL, 48, 67, 69, 71, 73 and 78 in VH, as well as mutation of H3-10 in HVR-H3 (V98I, Y99R and F100L (also described above) and D28E mutation in HVR-L1 (introduced for stability reasons, see above); figure 12, row labeled 28) and MA79b-associated variant "humanized antibody" 32 (huMA79b.v32; with murine residues frame region in positions 4 in VL, 47, 48, 67, 69, 71, 73 and 78 in VH, as well as mutation of H3-10 in HVR-H3 (V98I, Y99R and F100L (also described above) and with a mutation D28S in HVR-L1 (introduced for stability reasons, see above); figure 12, row labeled 32) have a great affinity binding to immobilized huCD79becdequivalent to the binding affinity of the chimeric MA79b antibody (chMA79b) with the specified antigen, as determined using Biacore analysis.

b. The definition of affinity (analysis of Scatchard)

As was assessed by analysis of Scatchard similar to Biacore analysis, this method of reparation frame circuit allows to identify the set of substitutions in a separate sequence that improve the binding affinity of the hybrid "MA79b-the humanized antibody (huMA79b-hybrid). Tests for binding to cells showed that the binding affinity MA79b, MA79b-associated case of "humanized" antibodies 28 (huMA79b.v28; see figure 12, several, oboznachenie�28 th) (obtained in the new format as IgG) and MA79b-associated case of "humanized" antibody 32 (huMA79b.v32; see figure 12, row labeled 32) BJAB cells stably expressing CD79b dog-like apes and endogenous human CD79b, Kd has a value equal to 0.63 nm (MA79b; Kd=0,63±0,14 nm) of 0.44 nm (huMA79b.v28; Kd=0,44±0,04 nm and 0.24 nm (huMA79b.v32; Kd=0,24±0,02 nm), respectively (data not shown), as was determined by analysis of Scatchard.

c. Determining the level of binding (FACS-analysis)

As was determined by FACS-analysis, this method of reparation frame circuit allows to identify the set of substitutions in a separate sequence that improve the binding of the hybrid "MA79b-the humanized antibody (MA79b-hybrid) BJAB cells containing the luciferase (data not shown). In particular, FACS-analysis of IgG variants of MA79b-related options "humanized" antibodies (variants of huMA79b.v28 and huMA79b.v32) BJAB cells containing luciferase, showed binding with the specified BJAB cells containing the luciferase (data not shown).

B. Discussion of the production of humanized anti-CD79b antibodies

To identify substitutions in the HVR 1-6, which increase the affinity of binding was carried out in the repair CDR by attaching 6 mice HVR MA79b (defined as positions 24-34 (L1), 50-56 (L2), 89-97 (L3), 26-35 (H1), 49-65 (H2) and 93-102 (H3)) into the human consensus sequence VL Kappa I and VH subgroup III (containing A71, T73 and A78). Replacements� in the sequence HVR, identified in the figure 10 and 11, or a combination of these substitutions lead to the formation of the humanized variants of MA79b with affinity similar to the affinity MA79b.

Alternative reparation frame region was applied to re-capture the avidity of binding to the dimer by adding residues frame region 4 in VL, and 48, 67 and 69 in VH to huMA79b-hybrid (which includes murine residues frame region at positions 71, 73 and 78 in VH) (figure 12; MA79b-associated variant of "humanized" antibody 17 (huMA79b.v17)). The affinity of binding of the variants with substitutions in the framework region of the antibody against the antigen huCD79becdthere was also further increased by the introduction of 3 substitutions in the HVR-H3: V98I, Y99R and F100L (figure 12; MA79b-associated variant of "humanized" antibodies 18 (huMA79b.v18)). The potential of education website izospinovoi acid in HVR-L1 was removed by introducing D28E mutation (figure 12; MA79b-associated variant of "humanized" antibodies 28 (huMA79b.v28)).

Example 2: Obtaining conjugates anti-CD79b antibody-drug" (ADC)

To analyze the efficacy of IgG variants of MA79b-related options "humanized" antibodies listed MA79b-related options "humanized" antibodies conjugated to drugs, such as DM1. Options, conjugated with DM1 are variants having substitutions in the HVR-L2 � HVR-H3 (huMA79b L2/H3), huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32.

Medicines used to obtain conjugates anti-CD79b antibody-drug" (ADC), are maytansinoid DM1 and derivatives dolastatin 10, namely monomethylaniline E (MMAE) and monomethylaniline F (MMAF). (application USA 2005/0276812; 2005/0238649; Doronina et al., Bioconjug. Chem., 17:114-123 (2006); Doronina et al., Nat. Biotechnol., 21:778-784 (2003); Erickson et al., Cancer Res., 66:4426-4433 (2006), who fully entered into the present description by reference). The linkers used to produce the ADC are BMPEO, SPP or SMCC (also referred to here as the "MCC") for DM1 or MC or MC-vc-PAB for MMAE and MMAF. In the case of DM1, the antibodies were attached to a thio group DM1 through the ε-amino group of lysine using the linker reagent SMCC. Alternatively, in the case of DM1, the antibodies were attached to DM1 through e-amino group of lysine using the linker SPP. SPP (N-Succinimidyl-4-(2'-pyridyldithio)pentanoate) reacts with the Epsilon-amino group of lysine with the formation of a reactive 2-pyridyldithio the linker to the protein. Under the action of SPP linkers, after reaction with the free sulfhydryl (e.g., DM1), Peregrina group is removed, resulting in DM1, attached recoverable through disulfide bonds. DM1, attached through a linker SPP is released in reducing conditions (i.e., voltage�emer, in the cells), and DM1, attached via the SMCC linker is resistant to cleavage under the conditions of restoration. In addition, ADC SMCC-DM1 induces cell toxicity if the ADC is internalized and delivered to the lysosome, releasing lysine-Nε-DM1, which is an effective antimitotic agent, present in the cells, and when it emerges from the cell, lysine-Nε-DM1 becomes non-toxic (Erickson et al., Cancer Res., 66:4426-4433 (2006)). In the case of MMAE and MMAF antibodies were attached to MMAE or MMAF through the cysteine via maleimidomethyl-valine-citrulline(vc)-p-aminobenzeneboronic (MC-vc-PAB). In the case of MMAF these antibodies were attached to alternative MMAF through the cysteine via multimedialny (MC) linker. MC-vc-PAB linker is cleaved extracellular proteases, such as cathepsin In, and after cleavage of the released free drug (Doronina et al., Nat. Biotechnol., 21:778-784 (2003)), and the linker MC is resistant to cleavage by intracellular proteases.

Conjugates of the antibody-drug" (ADC) for anti-CD79b antibodies were obtained using SMCC DM1 and in accordance with the procedure described in the application US 2005/0276812. After cleaning anti-CD79b antibodies underwent buffer exchange with the introduction of a solution containing 50 mm potassium phosphate and 2 mm EDTA, pH 7.0. SMCC (Pierce Biotechnology, Rockord, IL) was dissolved in dimethylacetamide (DMA) and added to the antibody solution to obtain the final molar ratio of SMCC/Ab=10:1. The reaction was performed for three hours at room temperature with stirring. SMCC-modified antibody was then purified on demineralization column (GE Healthcare HiTrap (G-25), equilibrated in 35 mm sodium citrate with 150 mm NaCl and 2 mm EDTA, pH 6.0. DM1, dissolved in DMA, was added to SMCC-drug antibodies with obtaining molar ratio of DM1 to antibody of 10:1. The reaction was performed for 4-20 hours at room temperature with stirring. A solution of DM1-modified antibodies were subjected to diafiltration 20 volumes of PBS to remove unreacted DM1, and then sterile filtered and stored at 4°C. Typically, when performing this procedure, the yield of antibody was 40-60%. This drug is usually more than 95% was Monomeric, as it was estimated using gel filtration and light scattering method with a laser. Because the maximum absorption of DM1 was observed at 252 nm, the amount of drug that is associated with the antibody can be determined by measuring the differential absorption at 252 and 280 nm. Usually the ratio of drug to antibody ratio was 3:4.

Conjugates described herein "antibody-drug" (ADC) for anti-CD79b antibodies can be obtained used�eat linkers SPP-DM1 in accordance with the procedure described in the application US 2005/0276812. After cleaning anti-CD79b antibodies underwent buffer exchange with the introduction of a solution containing 50 mm of potassium and phosphorus 2mm EDTA, pH 7.0. SPP (Immunogen) was dissolved in DMA and added to the antibody solution to obtain the final molar ratio SPP/Ab=10:1, with the exact ratio depends on the desired loading of the drug to the antibody. Based on the relationship of 10:1, we can obtain the ratio of the drug to the antibody of approximately 3-4. SPP was left to react for 3-4 hours at room temperature with stirring. SPP-modified antibody was then purified on demineralization column (GE Healthcare HiTrap (G-25), equilibrated in 35 mm sodium citrate with 150 mm NaCl and 2 mm EDTA, pH 6.0, or phosphate-buffered saline, pH 7.4. DM1 was dissolved in DMA and added to SPP-drug antibodies with obtaining molar ratio of DM1 to antibody of 10:1, which gives 3-4-fold molar excess compared to SPP-linkers present in the antibody. Reaction with DM1 was performed for 4-20 hours at room temperature c stirring. A solution of DM1-modified antibodies were subjected to diafiltration 20 volumes of PBS to remove unreacted DM1, and then sterile filtered and stored at 4°C. Typically, when performing this procedure, the yield of antibody was 40-60%. This conjugate "antibody-drug �the case" is usually more than 95% was Monomeric, as it was estimated using gel filtration and light scattering method with a laser. The amount of bound drug was determined by measuring the differential absorption at 252 and 280 nm, as described for obtaining conjugates SMCC-DM1 (as described above).

Conjugates described herein "antibody-drug" (ADC) for anti-CD79b antibodies can also be obtained using a connection "drug-linker", namely MC-MMAF, MC-MMAE, MC-val-cit(vc)-PAB-MMAE or MC-val-cit(vc)-PAB-MMAF, in accordance with the procedure described in the application U.S. 2005/0238649. Purified anti-CD79b antibody was dissolved in 500 mm sodium borate and 500 mm sodium chloride at pH 8.0, and then treated with an excess of 100 mm of dithiothreitol (DTT). After incubation at 37°C for about 30 minutes, the buffer was replaced by elution of the resin Sephadex G25, and the mixture was suirable PBS with 1 mm DTPA. The amount of the thiol/Ab were assessed by determining the concentration of the recovered antibodies, based on the optical density of the solution and 280 nm, and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of absorbance at 412 nm. The recovered antibody was dissolved in PBS and cooled on ice. Conjugate "drug-linker", for example, MC-val-cit(vc)-PAB-MMAE, in DMSO, dissolved in acetonitrile and water and then added to the cooled copied�nnomo the antibody in PBS. After incubation for 1 hour was added an excess of maleimide to quench the reaction, and any unreacted thiol group of the antibody was kopirovali. The reaction mixture was concentrated by centrifuge ultrafiltration, and then conjugate "antibody-drug" purified and desalted by elution through G25 resin in PBS, then filtered through a 0.2-μm filters under sterile conditions, and frozen for storage.

Conjugates of the antibody-drug" (obtained using the described here anti-CD79b antibody) was diluted with 2×10 μg/ml in the analytical environment. Conjugates were attached by cross-linking the linker SMCC (for joining SPP to maytansinoids to DM1 toxin can be used alternative disulfide linker)(see US application 2005/0276812 and US 2005/0238649). In addition, the conjugates may be connected by MC-valine-citrulline(vc)-PAB or MC with derivatives of dolastatin 10, the toxin monomethylaniline E (MMAE) or the toxin monomethylaniline F (MMAF) (see application for U.S. patent No. 11/141344, filed may 31, 2005, and patent application U.S. No. 10/983340, filed November 5, 2004). Negative control includes conjugates on the basis of HERCEPTIN® (trastuzumab) (anti-HER2 antibody) (SMCC-DM1 or SPP-DM1 or MC-vc-MMAE or MC-vc-MMAF). Positive controls can include free form L-DM1, an equivalent load�full dose of the conjugate. Samples before dilution, intensively stirred to ensure a homogeneous mixture.

Anti-CD79b antibodies for conjugation with drugs are chimeric MA79b antibody (chMA79b) and variant L2/H3 antibody, huMA79b, and describes scnes variants of huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32 (see example 1). Other antibodies for conjugation can be any antibody described herein (see example 1).

Example 3: in vivo Analysis on the destruction of tumor cells

A. Xenograft

To analyze the efficacy of IgG variants of MA79b-related options "humanized" antibodies having replacements in HVR-L2 and HVR-H3 (huMA79b L2/H3), a variant of the huMA79b L2/H3 conjugated to DM1, and analyzed the influence of the conjugated variant on tumors in mice.

In particular, can be analyzed the ability of antibodies to ensure the regression of tumors in many xenograft models, including cells RAMOS, BJAB cells (cell line of Burkitt lymphoma, which contains a translocation t(2;8)(p112;q24) (IGK-MYC), mutated P53 gene, which is negative for Epstein-Barr (EBV)) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), cells Granta 519 (cell line lymphoma cells of the cerebral cortex, which contains: the translocation t(11;14)(q13;q32) (BCL1-IGH), leading to sverkhekspressiya detected D1 (BCL1); deletions P16INK4B and P16INK4A and is EBV) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), the cells U698M (b-cell line lymphoblastic lymphosarcoma; (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001) and DoHH2 cells (follicular cell line lymphoma containing the translocation responsible for the development of follicular lymphoma, namely t(14;18)(q32;q21), which leads to sverkhekspressiya Bcl-2-regulated Ig heavy chain; the P16INK4A deletion, and translocation t(8;14)(q24;q32) (IGH-MYC), and is EBV negative) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)).

For analysis on the effectiveness of the MA79b-related options "humanized antibody", the female mice CB17 ICR SCID (aged 6-8 weeks, supplied by the laboratory of Charles Rivers Laboratories; Hollister, CA) were subcutaneously inoculable 2×107BJAB cells containing the luciferase cells or Granta-519 by injection into the flanks of mice CB17 ICR SCID, and these mice were left up until the average size of tumor transplants are not reached 200 mm2. Day 0 means the day on which the tumor size averaged 200 mm2and in which was entered the first or only one dose, administered after this treatment, if it is not specifically covered below. The tumor volume was expressed in mm3, was measured by caliper in two dimensions and calculated by the formula: V=0.5 a×b2whereaandbmean large and small diameters of the tumor, respectively. Yes�nye, collected for each experimental group were expressed as mean ± RMS from. Groups of 10 mice were injected with a single intravenous (i.v.) dose 50-210 mcg is associated with an antibody drugs/m2mouse (corresponding to 1-4 mg/kg mouse), namely, conjugates were introduced "version of the MA79b-associated humanized antibody or control antibody-drug". During the experiment the tumors were measured once or twice a week. Body weight of mice was measured once or twice a week during the whole experiment. When tumor volume reached 3000 mm3or when the tumor was discovered life-threatening signs of ulceration, the mice were euthanized. All protocols for animal experiments were approved by the Institutional Committee on animal care and use (IACUC).

Used by linkers linking the antibody and the toxin are thioether cross-linking the linker SMCC, communicating with DM1. Other linkers can be disulfide linker SPP or thioether cross-linking the linker SMCC to DM1 or MC or MC-valine-citrulline(vc)-PAB or a dipeptide linker reagent (valine-citrulline(vc)) with maleimide component and pair-aminobenzeneboronic (PAB) carolinensis component for monomethylaniline E (MMAE) or monomethylaniline F (MMAF). Used �okinami are DM1. Additional toxins can be MMAE or MMAF.

Anti-CD79b antibodies used in this experiment are chimeric MA79b antibody (chMA79b) described in the application for U.S. patent No. 11/462336, filed August 3, 2006, and described here MA79b-related options "humanized" antibodies (see example 1A). Other antibodies may be commercially available antibodies, including anti-CD79b antibody, and monoclonal antibody MA79b obtained from hybridomas deposited with the ATCC as HB11413 July 20, 1993

Negative control includes conjugates based on anti-HER2 antibody (HERCEPTIN® (trastuzumab))(SMCC-DM1).

B. Results

1. BJAB xenograft containing luciferase

After the 35-day course of treatment with a conjugate of the drugs at the doses indicated in table 9, namely, MA79b-associated variant humanized antibody L2/H3 (variants of huMA79b L2/H3) (obtained in the new format, IgG) and chimeric anti-CD79b antibody (chMA79b), conjugated with DM1 (huMA79b L2/H3-SMCC-DM1 and chMA79b-SMCC-DM1, respectively), it was revealed that the inhibition of growth of BJAB tumors containing luciferase SCID mice compared with the negative control, namely, the antibody is HERCEPTIN® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1). For all ADC and control was administered one dose of ADC (as shown in table 9) on day 0. In particular, L2/H3-SMCC-DM1 antibodies huMA79b (obtained in the new format as IgG) and chMA79b-MCC-DM1 significantly inhibited tumor growth (figure 20). In addition, table 9 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3).

Table 9
The injected antibody (processing)PRCRDose "drug - DM1
(µg/m2)
Dose Ab
(mg/kg)
The ratio (Drug/
Ab)
Control anti-HER2-SMCC-DM10/100/1010023,3
chMA79b-SMCC-DM13/103/101002,42,9
chMA79b-SMCC-DM11/100/1050 1,22,9
huMA79b L2/H3-SMCC-DM12/100/101002,92,4
huMA79b L2/H3-SMCC-DM10/100/10501,42,4

2. Xenograft Granta-519

After the 14-day course of treatment with a conjugate of the drugs at the doses indicated in table 10), MA79b-associated variant humanized antibody, namely version 17, version 18, version 28 and variant 32 (huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32, respectively) (obtained in the new format as IgG) and chimeric anti-CD79b antibody (chMA79b), conjugated with DM1 (huMA79b.v17-SMCC-DM1, huMA79b.v18-SMCC-DM1, huMA79b.v28-SMCC-DM1, huMA79b.v32-SMCC-DM1 and chMA79b-SMCC-DM1, respectively), revealed inhibition of tumor growth Granta-519 SCID mice compared with the negative control, namely the antibody HERCEPTIN® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1). For all ADC and control was administered one dose of ADC (as shown in table 9) on day 0. In particular, the antibody, huMA79b.v28-SMCC-DM1, huMA79b.v32-SMCC-DM1, huMA79b.v17-SMCC-DM1 and huMA79b.v18-SMCC-DM1 (obtained in the new format as IgG) and chMA79b-SMCC-DM1 significantly inhibited tumor growth (figure 21A).

�besides treatment with antibodies huMA79b.v28-SMCC-DM1, huMA79b.v32-SMCC-DM1, huMA79b.v17-SMCC-DM1, huMA79b.v18-SMCC-DM1 and chMA79b-SMCC-DM1, and a control antibody HERCEPTIN® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1) did not lead to a decrease in the percentage of body weight of mice (figure 21B). Moreover, table 10 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3).

Table 10
The injected antibody (processing)PRCRDose "drug - DM1
(µg/m2)
Dose Ab
(mg/kg)
The ratio (Drug/Ab)
Control anti-HER2-SMCC-DM10/100/1020843,4
chMA79b-SMCC-DM10/100/10107 23,6
chMA79b-SMCC-DM11/100/1021343,6
huMA79b.v17-SMCC-DM10/100/1020243,4
huMA79b.v18-SMCC-DM14/100/1019643,3
huMA79b.v28-SMCC-DM10/100/1010123,4
huMA79b.v28-SMCC-DM12/102/1020243,4
huMA79b.v32-SMCC-DM10/100/1017242,9

Based on the ability of ADC "MA79b-associated humanized antibody" significantly inhibit the progression of�whole in xenotransplantation, we can say that the CD79b molecule can be an excellent target for the treatment of tumors in mammals, including b-cell associated cancers, such as lymphomas (i.e. non-Hodgkin's lymphoma), leukemias (i.e. chronic lymphocytic leukemia) and other cancers of hematopoietic cells. In addition, MA79b-associated humanized ADC can be used to reduce tumor growth in vivo, including b-cell associated cancers, such as lymphomas (i.e. non-Hodgkin's lymphoma), leukemias (i.e. chronic lymphocytic leukemia) and other cancers of hematopoietic cells.

Example 4: Colocalization anti-CD79b antibodies

To determine the site of delivery MA79b-related humanized antibodies and their variants after internalization into the cell, can be carried out researches on colocalization anti-CD79b antibody that is internalized into b-cell lines, namely cell line Ramos. LAMP-1 is a marker for late endosomes and lysosomes (Kleijmeer et al., Journal of Cell Biology, 139(3):639-649 (1997); Hunziker et al., Bioessays, 18:379-389 (1996); Mellman et al., Annu. Rev. Dev. Biology, 12:575-625 (1996)), including compartments MHC class II (MIIC), which represent a compartment that is similar to the late endosome/the lysosome. HLA-DM is a marker for MIIC.

The Ramos cells were incubated for 3 hours at 37°C with 1 µg/ml MA79b humanized antibodies and their variants with FcR block (Miltenyi) and 25 μg/ml Alexa647-transferrin (Molecular Probes) containing carbonate medium (Gibco) in the presence of 10 μg/ml leupeptin (Roche) and 5 μm of pepstatin (Roche) for inhibiting decomposition of lysosomes. Then the cells twice washed, fixed with 3% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature, quenched with 50 mm NH4Cl (Sigma), and made permeable using a 0.4% saponin/2% FBS/1% BSA for 20 minutes, then incubated with 1 μg/ml antibody against mouse Cy3 (Jackson Iunoresearch) for 20 minutes. Then the reaction was blocked for 20 minutes with mouse IgG (Molecular Probes), and the mixture is then incubated for 30 minutes with signal amplifier Image-iT FX (Molecular Probes). Finally, the cells were incubated with Zenon Alexa488-labeled mouse antibody against LAMP1 (BD Pharmingen), a marker for lysosomes and MIIC (lysosome-like compartment that is part of the path MHC class II), for 20 minutes, and then fixed with 3% PFA. The cells were resuspended in 20 µl tapaninaho buffer and left for adhesion on glass slides coated with polylysine (Sigma), and then placed on a coverslip using a DAPI-containing vector VectaShield (Vector Laboratories). For immunofluorescence MIIC or lysosomes, the cells were fixed, made permeable, and the signal was amplified, as described below, and then subjected to co-staining labeled with Zenon Alexa555-HLA-DM (BD Pharmingen) and Alexa488-Lamp1 in the presence of excess mouse IgG, in accordance with the manufacturers instructions (Molecular Probes).

Accordingly, colocalization MA79b-associated humane�specialized antibodies or their variants with MIIC or lysosomes In cell lines, as was assessed using immunofluorescence, may indicate that these molecules are essential tools for treating tumors in mammals, including b-cell associated cancers, such as lymphomas (i.e. non-Hodgkin's lymphoma), leukemias (i.e. chronic lymphocytic leukemia) and other cancers of hematopoietic cells.

Example 5: Obtain designed on the basis of cysteine anti-CD79b antibodies

Constructed of cysteine-based anti-CD79b antibodies were obtained as described in the present application.

DNA encoding the MA79b antibody (light chain, SEQ ID NO: 4, figure 4; and a heavy chain, SEQ ID NO: 5, figure 5), was subjected to mutagenesis methods described herein to modify the light and heavy chains. The DNA that encodes the MA79b antibody (heavy chain, SEQ ID NO: 5; figure 5), can also be subjected to mutagenesis methods described herein for modifying the Fc region of the heavy chain.

DNA encoding the antibody, huMA79b.v17 (heavy chain, SEQ ID NO: 304, figure 15), was subjected to mutagenesis methods described herein for modifying the heavy chain. DNA encoding the antibody, huMA79b.v17 (light chain, SEQ ID NO: 303; figure 15; and a heavy chain SEQ ID NO: 304; figure 15), may also be subjected to mutagenesis methods described herein to modify the light chain or the Fc-region of the heavy chain.

DNA encoding the antibody, huMA79b.v18 (cord�sexless chain, SEQ ID NO: 306, figure 16), was subjected to mutagenesis methods described herein for modifying the heavy chain. DNA encoding the antibody, huMA79b.v18 (light chain, SEQ ID NO: 305; figure 16; and a heavy chain SEQ ID NO: 306; figure 16), may also be subjected to mutagenesis methods described herein to modify the light chain or the Fc-region of the heavy chain.

DNA encoding the antibody, huMA79b.v28 (heavy chain, SEQ ID NO: 308, figure 17), was subjected to mutagenesis methods described herein for modifying the heavy chain. DNA encoding the antibody, huMA79b.v28 (light chain, SEQ ID NO: 307; figure 17; and heavy chain SEQ ID NO: 308; figure 17), may also be subjected to mutagenesis methods described herein to modify the light chain or the Fc-region of the heavy chain.

DNA encoding the antibody, huMA79b.v32 (light chain, SEQ ID NO: 310; figure 18; and a heavy chain SEQ ID NO: 309; figure 18), may also be subjected to mutagenesis methods described herein to modify the light chain and heavy chain.

DNA encoding the antibody against CD79b dog-like apes (light chain, SEQ ID NO: 241, figure 45; and heavy chain, SEQ ID NO: 243, figure 47), was subjected to mutagenesis methods described herein to modify the light and heavy chains. The DNA that encodes the antibody against CD79b dog-like apes (heavy chain, SEQ ID NO: 243; figure 47), may also be subjected to mutagenesis methods described herein for modifying the Fc region of the heavy C�PI.

To obtain designed on the basis of cysteine anti-CD79b antibodies DNA encoding light chain, was subjected to mutagenesis by replacing valine with cysteine at position 205 in Cabatu in light chain (position 209 in accordance with a sequential numbering), as shown in figure 27 (light chain SEQ ID NO: 235 thio-MAB MA79b) and figure 49 (light chain SEQ ID NO: 300 thio-MAb against CD79b dog-like apes (ch10D10)). DNA encoding a heavy chain, was subjected to mutagenesis by replacing alanine cysteine in the heavy chain at position 118 in accordance with the European numbering (position 118 in accordance with a consecutive numbering system; room 114 on Kabuto), as shown in figure 48 (heavy chain SEQ ID NO: 244 thio-Mab antibodies against CD79b dog-like apes (ch10D10)), figure 28 (heavy chain SEQ ID NO: 236 thio-Mab MA79b), figure 24 (heavy chain SEQ ID NO: 228 thio-MAb huMA79b.v17), figure 25 (heavy chain SEQ ID NO: 230 thio-MAb huMA79b.v18) and figure 26 (heavy chain SEQ ID NO: 232 thio-MAb huMA79b.v28). Fc-region of an anti-CD79b antibody may be subjected to mutagenesis by replacing serine with cysteine at position 400 in the Fc-region of the heavy chain in accordance with the European numbering system (position 400 in accordance with a sequential numbering; number by Kabata-396), as shown in table 2-4.

A. Getting designed on the basis of cysteine anti-CD79b antibodies for conjugation by restore�Oia and re-oxidation

Full-size, constructed on the basis of cysteine anti-CD79b monoclonal antibody (Mab) expressively in CHO cells and purified by carrying out affinity chromatography on protein A, and then size exclusion chromatography. Purified antibodies were diluted in 500 mm sodium borate and 500 mm sodium chloride at about pH 8.0, and then restored approximately 50-100-fold molar excess of 1 mm TCEP (hydrochloride Tris(2-carboxyethyl)phosphine; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA) for approximately 1-2 hours at 37°C. the Recovered thio-Mab was diluted and loaded on a HiTrap column S in 10 mm sodium acetate, pH 5, and suirable PBS containing 0.3 M sodium chloride. - Eluted restored thio-Mab was treated with 2 mm dehydroascorbic acid (dhAA) at pH 7 for 3 hours or 2 mm aqueous copper sulfate (CuSO4) at room temperature over night. It may also be effective oxidation in air. The buffer was replaced by elution of the resin Sephadex G25, and the mixture was suirable PBS with 1 mm DTPA. The amount of the thiol/Ab was calculated by determining the concentration of the recovered antibodies, based on the optical density of the solution at 280 nm and the concentration of the thiol by conducting the reaction with DTNB (Aldrich, Milwaukee, WI) and determination of absorbance at 412 nm.

Example 6: Obtaining conjugates "designed�tion of cysteine-based anti-CD79b antibody-drug" by the reaction of conjugation constructed on the basis of cysteine anti-CD79b antibodies with intermediate connection "drug-linker"

After the procedure the recovery and re-oxidation as described in example 5, is constructed on the basis of cysteine anti-CD79b antibody diluted in PBS buffer (phosphate buffered saline) and cooled on ice. About 1.5 molar equivalents of the intermediate "auristatin drug-linker", such as MC-MMAE (maleimidomethyl-monomethylaniline E), MC-MMAF, MC-val-cit-PAB-MMAE or MC-val-cit-PAB-MMAF containing thiol-reactive functional group, such as maleimido, in relation to cysteines introduced into the antibody was dissolved in DMSO, is diluted in acetonitrile and water was added to the cooled restored and re-oxidized antibody in PBS. After about one hour there was added an excess of maleimide to quench the reaction, and any unreacted thiol group of the antibody was kopirovali. The reaction mixture was concentrated by centrifugal ultrafiltration, and then conjugate "constructed on the basis of cysteine anti-CD79b antibody-drug" purified and desalted by elution through G25 resin in PBS, then filtered through a 0.2-μm filters under sterile conditions, and frozen for storage.

Getting MAb-BMPEO-DM1 huMA79b.v18-HC(A118C) is performed as follows. The free cysteine on huMA79b.v18-HC(A118C) thio-MAb modified bis-maleimido - reagent�m BM(PEO) 3(Pierce Chemical), whereby the unreacted maleimido group remained on the surface of the antibody. This is carried out by dissolving BM(PEO)350% mixture of ethanol/water to achieve a concentration of 10 mm and adding a 10-fold molar excess of BM(PEO)3in a solution containing thio-MAb huMA79b.v18-HC(A118C) in phosphate-buffered saline at a concentration of approximately 1.6 mg/ml (10 micromol), and then left for 1 hour for the reaction. Excess BM(PEO)3were removed by gel filtration (HiTrap column, Pharmacia) in 30 mm citrate, pH 6, in the presence of 150 mm NaCl-buffer. Approximately 10-fold excess DM1 dissolved in dimethylacetamide (DMA) was added to intermediate compound thio-MAb-BMPEO huMA79b.v18-HC(A118C). For dissolution of the reagent, namely molecule drugs, may be used dimethylformamide (DMF). The reaction mixture was left overnight to complete the reaction, and then subjected to gel filtration or dialysis in PBS to remove unreacted drug. Gel filtration on S200 columns in PBS was performed to remove high molecular weight aggregates and downloaded purified thio-MAb-BMPEO-DM1 huMA79b.v18-HC(A118C).

In accordance with the same protocols received control thio-antibody hu-anti-HER2-HC(A118C)-BMPEO-DM1, the control thio-antibody hu-anti-HER2-HC(A118C)-MC-MMAF, control tio�ntitle hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE and control thio-antibody anti-CD22-HC(A118C)-MC-MMAF.

In accordance with the above procedures have been received and tested the following conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" (TDC):

1. thio-huMA79b.v18-HC(A118C)-MC-MMAF, obtained by conjugation of A118C-thio-huMA79b.v18-HC(A118C) and MC-MMAF;

2. thio-huMA79b.v18-HC(A118C)-BMPEO-DM1, obtained by conjugation of A118C-thio-huMA79b.v18-HC(A118C) and BMPEO-DM1;

3. thio-huMA79b.v18-HC(A118C)-MCvcPAB-MMAE obtained by conjugation of A118C-thio-huMA79b.v18-HC(A118C) and MC-val-cit-PAB-MMAE;

4. thio-huMA79b.v28-HC(A118C)-MC-MMAF, obtained by conjugation of A118C-thio-huMA79b.v28-HC(A118C) and MC-MMAF;

5. thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, obtained by conjugation of thio-huMA79b.v28-HC(A118C) and BMPEO-DM1;

6. thio-huMA79b.v28-HC(A118C)-MC-val-cit-PAB-MMAE, obtained by conjugation of thio-huMA79b.v28-HC(A118C) and MC-val-cit-PAB-MMAE;

7. thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MC-MMAF, obtained by conjugation of A118C-thio-anti-cynoCD79b(ch10D10)-HC(A118C) and MC-MMAF;

8. thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM1, obtained by conjugation of A118C-thio-anti-cynoCD79b(ch10D10)-HC(A118C) and BMPEO-DM1;

9. thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MCvcPAB-MMAE obtained by conjugation of A118C-thio-anti-cynoCD79b(ch10D10)-HC(A118C) and MC-val-cit-PAB-MMAE;

10. thio-MA79b-HC(A118C)-MC-MMAF, obtained by conjugation of thio-MA79b-HC(A118C) and MC-MMAF; and

11. thio-MA79b-LC(V205C)-MC-MMAF, obtained by conjugation of thio-MA79b-LC(V205C) and MC-MMAF.

Example 7: Characterization of the binding affinity of the conjugates is "constructed on the basis of cysteine thio Mab-a drug with the cell surface antigen

The affinity of binding of the conjugates "thio-huMA79b.v18, thio-huMA79b.v28-drug and conjugates "thio-MA79b-drug" with CD79b, expressed on BJAB cells containing luciferase was assessed using FACS analysis. In addition, the affinity of binding of the conjugates "thio-anti-cynoCD79b(ch10D10) antibody-drug" with CD79b, expressed on BJAB cells expressing CD79b dog-like apes, were determined using FACS analysis.

Briefly, approximately 1×106cells in 100 µl were subjected contacted with various amounts of 1.0 μg, 0.1 μg or 0.01 μg Ab per million cells of BJAB containing luciferase, or BJAB cells expressing CD79b dog-like apes (for anti-cynoCD79b thio-Mab)) one of the following conjugates anti-CD79b thio-MAb-drug" or "naked" antibodies (unconjugated Ab, used as control): (1) thio-MA79b-LC(V205C)-MC-MMAF or (2) thio-MA79b-HC(A118C)-MC-MMAF (figures 29A-B, respectively; (3) thio-huMA79b.v18-HC(A118C)-MC-MMAF, (4) thio-huMA79b.v18-HC(A118C)-MC-vcPAB-MMAE or (5) thio-huMA79b.v18-HC(A118C)-BMPEO-DM1 (figure 30B-D, respectively); (6) thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, (7) thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 or (8) thio-huMA79b.v28-HC(A118C)-MC-MMAF (see figure 31B-31D, respectively); or (9) thio-anti-cynoCDb79(ch10D10)-HC(A118C)-MCvcPAB-MMAE, (10) thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM1 or (11) thio-anti-cynoCD79b(ch10D10)-HC(A11C)-MC-MMAF (see figures 32B-32D, respectively). Feh-konjugierten murine antibody against human Ig was used as a "second" detection antibody (BD Cat#555787).

Anti-CD79b antibody associated with the cell surface were detected using PE-conjugated mouse antibodies against human Ig. In the graphs shown in figures 29-32, the binding to the antigen is approximately the same for all tested conjugates "thio-MAb-drug".

Example 8: in vitro Analysis to reduce the proliferation of cells under the action of the conjugates anti-CD79b thio-Mab-drug"

The efficiency of the conjugates in vitro "anti-CD79b thio-Mab-drug" (including thio-huMA79b.v18-HC(A118C)-MCMMAF, thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE and thio-huMA79b.v18-HC(A118C)-BMPEO-DM1) was determined by analysis on cell proliferation (figure 41A, BJAB cells containing luciferase; figure 41B, Granta-519; figure 41C, WSU-DLCL2). Fluorescent analysis of cell viability CellTiter-Glo® is a commercially available (Promega Corp., Madison, WI) and is a specially developed analysis carried out by the method of recombinant expression of Coleoptera luciferase (US 5583024; US 5674713; US 5700670). In this analysis on the proliferation of cells determine the number of viable cells in culture based on the presence of ATP, which is showing�Telem metabolic activity of cells (Crouch et al., J. Immunol. Metho., 160:81-88 (1993); US 6602677). Analysis of CellTiter-Glo® was performed in 96-well format that allowed for automated high-performance screening (HTS) (Cree et al., AntiCancer Drugs 6:398-404 (1995)). The procedure is specially designed analysis involves adding the single reagent (reagent CellTiter-Glo®), which is directly added to the cells, cultured in medium containing serum.

A procedure performed in the format of "add-mix-measure" allows the lysis of the cells with the production of a luminescent signal proportional to the presence of ATP. The substrate, luciferin, beetles, subjected to oxidative decarboxylation under the action of recombinant Firefly luciferase, followed by conversion of ATP to AMP and generation of photons. The number of viable cells relative luminescence units (RLU). Data can be recorded on a luminometer or imaging device has a camera with a CCD. Output fluorescent signal expressed as RLU measured depending on time. % RLU is the percent of relative luminescence units normalized data for control, that is, the "antibody that has not been anywhereman with medicine." Alternative photons generated by the emitted fluorescent�eat can be counted in a scintillation counter in the presence of scintillation fluid. Light units can be further represented as CPS (counts per second).

The efficiency of the conjugates "thio-Mab-drug" were determined using analysis of cell proliferation, conducted in accordance with the Protocol adapted for fluorescent analysis of cell viability (CellTiter Glo Luminescent Cell Viability Assay, Promega Corp. Technical bulletin TB288; Mendoza et al., Cancer Res., 62:5485-5488 (2002)):

1. Aliquot 40 ml of cell culture containing about 3000 cells BJAB, Granta-519 or WSU-DLCL2 in the medium was placed in each well of 384-well plates with opaque walls.

2. TDC (conjugate "thio-Mab-drug") (10 µl) was added to experimental wells with four replications to final concentration 10000, 3333, 1111, 370, 123, 41, 13,7, 4,6 or 1.5 ng/ml, and in control wells was added only the environment, not containing conjugate with a drug, and incubated for 3 days.

3. Plates were equilibrated to room temperature for approximately 30 minutes.

4. Added reagent CellTiter-Glo (50 µl).

5. The contents were stirred for 2 minutes on an orbital shaker to induce cell lysis.

6. The plate was incubated at room temperature for 10 minutes to stabilize luminescent si�Nala.

7. Luminescence was recorded on the chart and expressed in % RLU (relative luminescence units). According to the data obtained for cells in 0,51 ng/ml, incubated with the medium containing no conjugate with drug, building schedule.

Wednesday: cells BJAB, Granta-519 and WSU-DLCL2 were cultured in RPMI1640 medium/10% FBS/2 mm glutamine.

Example 9: assay for inhibition of tumor growth in vivo using conjugates anti-CD79b thio-Mab-drug"

A. Granta-519 (lymphoma cells of the cerebral cortex of man)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft Granta-519 (lymphoma cells of the cortex of the human brain) in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are shown below in table 11.

Control Ab was a hu-anti-HER2-MC-MMAF or MA79b-MC-MMAF. The control thio-MAb HC(A118C) was a thio-MAb thio-hu-anti-HER2-HC(A118C)-MMAF. The results are presented in table 11 and figure 33.

Figure 33A shows a graph of the average tumor volume in time for CNS�of transplantat Granta-519 in mice CB17 SCID, which were injected with the conjugate TDC "heavy chain A118C or a light chain with V205C anti-CD79b antibody" in the doses presented in table 11. In particular, the introduction of thio-chMA79b-HC(A118C)-MC-MMAF and thio-chMA79b-LC(V205C)-MC-MMAF resulted in inhibition of tumor growth compared with the negative control (anti-hu-HER2-MC-MMAF and thio-hu-anti-HER2-HC(A118C)-MC-MMAF). As another control used MA79b-MC-MMAF.

In addition, in the same study, for each group, which injected dose, was determined by the percentage change of body weight during the first 14 days. The results (figure 33B) showed that the introduction of these conjugates "thio-Mab-drug" does not lead to a significant decrease in the percentage of body weight or loss of weight during this time period.

Moreover, table 11 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 11
Reduction of tumor volume in vivo by introducing conjugate tio chMA79b-HC(A118C) or thio-chMA79b-LC(V205CMMAF CB17 mice with SCID xenograft Granta-519
The injected antibodyPRCRDose MMAF
(µg/m2)
Dose Ab
(mg/kg)
DAR (Drug/
Ab)
Control hu-anti-HER2-MC-MMAF0/80/84136,84,0
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/90/91916,81,85
Control chMA79b-MC-MMAF1/80/81002,33,0
Control chMA79b-MC-MMAF8/91/93006,83,0
Tio chMA79b-HC(A118C)-MC-MMAF0/80/8632,31,9
Ti�-chMA79b-HC(A118C)-MC-MMAF 4/90/91906,81,9
Tio chMA79b-LC(V205C)-MC-MMAF0/80/8602,31,8
Tio chMA79b-LC(V205C)-MC-MMAF5/94/91806,81,8

B. BJAB Xenograft containing luciferase (Burkitt's)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of other conjugates of drugs in BJAB xenograft containing luciferase (Burkitt's) in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are shown below in table 12.

Control antibody was a huMA79b.v28 (anywhereman with SMCC-DM1). The control thio-MAb HC(A118C) was an antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), T.�-Mab thio-huMA79b.v28-HC(A118C) or thio-Mab thio-hu-anti-CD22(10F4v3)-HC(A118C) (anywhereman with MC-MMAF). The results are presented below in table 12 and figure 34.

Figure 34A shows a graph of the average tumor volume in time for BJAB xenografts containing luciferase in mice CB17 SCID treated with conjugates "thio-MAb huMA79b.v28-HC(A118C)-medicament", as shown in table 12. In particular, the introduction of conjugate thio-Mab, "thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MC-MMAF and thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE-drug" led to the inhibition of tumor growth compared with the growth of the tumor after treatment with conjugate "antibody-drug" (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF and thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE) used as a negative control. Another control consisted of a thio-huMA79b.v28-HC(A118C), huMA79b.v28-SMCC-DM1 and thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF.

In addition, in the same study, for each group, which injected dose, was determined by the percentage change of body weight for the first 7 days. The results (figure 34B) showed that the introduction of these conjugates "thio-Mab-drug" does not lead to a significant decrease in the percentage of body weight or loss of weight during this time period.

Moreover, table 12 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume using any� period of time after administration dropped to less than 50% of the volume of the tumor, measured on day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 12
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with BJAB xenograft containing luciferase
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µg/m2)
Dose Ab
(mg/kg)
DAR (Drug/
Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/100/10572To 1.86
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF1/100/105821,9
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/100/1046 21,55
Control huMA79b.v28-SMCC-DM12/103/1010123,4
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM13/102/105521,85
Thio-huMA79b.v28-HC(A118C)-MC-MMAF0/1010/105721,95
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/1010/105421,87
Thio-control huMA79b.v28-HC(A118C)0/100/10NA2NA
Thio control hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF1/104/105921,96

C. Xenograft WSU-DLCL2 (diff�heat large cell lymphoma)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft follicular lymphoma (diffuse large cell lymphoma) WSU-DLCL2 in mice CB17 SCID. The drug conjugates and doses are presented in table 13 below.

Control antibody was a huMA79b.v28 (anywhereman with SMCC-DM1). The control thio-MAb HC(A118C) was an antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), thio-Mab thio-huMA79b.v28-HC(A118C) or thio-Mab thio-hu-anti-CD22(10F4v3)-HC(A118C) (anywhereman with MC-MMAF). The results are presented in table 13 below.

Figure 35A shows a graph of the average tumor volume depending on time of the xenograft WSU-DLCL2 (diffuse large cell lymphoma) in mice CB17 SCID treated with conjugates TDC "heavy chain with AS anti-CD79b antibody" in the doses given in table 13. In particular, the introduction of conjugate thio-Mab, "thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MC-MMAF and thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE" resulted in inhibition of tumor growth compared with tumor growth after treatment with the negative controls (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, �IO-hu-anti-HER2-HC(A118C)-MC-MMAF and thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE), thio-huMA79b.v28-HC(A118C). Another control consisted of a thio-huMA79b.v28-HC(A118C), huMA79b.v28-SMCC-DM1 and thio-hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF.

TDC thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, obviously, is the most effective of all tested agents in this study.

In addition, in the same study, for each group, which injected dose, was determined by the percentage change of body weight for the first 7 days. The results (figure 35B) showed that the introduction of these conjugates "thio-Mab-drug" does not lead to a significant decrease in the percentage of body weight or loss of weight during this time period.

Moreover, table 13 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 13
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenograft WSU-DLCL2
The injected antibody PRCRDose MMAF, MMAE or DM1
(µg/m2)
Dose Ab
(mg/
kg)
DAR (Drug/Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/100/101144To 1.86
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/100/1011541,9
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/100/109241,55
Control huMA79b.v28-SMCC-DM11/100/1020243,4
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM10/100/1011041,5
Thio-huMA79b.v28-HC(A118C)-MC-MMAF3/101/1011541,95
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE4/103/1010841,87
Thio-control huMA79b.v28-HC(A118C)0/100/10NA4NA
Thio-control 10F4v3-HC(A118C)-MC-MMAF1/100/1011841,96
Thio-control huMA79b.v28-HC(A118C)0/100/10NA4NA

D. Xenograft DOHH2 (follicular lymphoma)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different�bubbled conjugates of drugs and various doses, evaluated the ability of the conjugates "thio-Mab-drug" to reduce the amount of b-cell tumors in mice CB17 SCID model DOHH2 xenografts. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are shown below in table 14.

Control antibody was a huMA79b.v28 (anywhereman with SMCC-DM1). The control thio-MAb HC(A118C) was an antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), thio-Mab thio-huMA79b.v28-HC(A118C) or thio-Mab thio-hu-anti-CD22-HC(A118C) (anywhereman with MC-MMAF). The results are presented below in table 12 and figure 36.

Figure 36A shows a graph of the average tumor volume depending on time of the xenograft DOHH2 cells in mice CB17 SCID treated with conjugates TDC "heavy chain A118C" in the doses given in table 14. In particular, the introduction of conjugate thio-Mab, "thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MC-MMAF and thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE-a drug in the doses shown in table 14, resulted in inhibition of tumor growth compared with the growth of the tumor after treatment with conjugate "antibody-drug" (thio-control thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-control thio-hu-anti-HER2-HC(A118C)-MC-MMAF and thio-control thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE) used as a negative control. Another control PR�dstable a thio-control huMA79b.v28-HC(A118C), thio control anti-CD22-HC(A118C)-MC-MMAF, thio-control huMA79b.v28-HC(A118C) and control huMA79b.v28-SMCC-DM1.

Moreover, table 14 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 14
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)DM1, MMAF and MMAE mice CB17 SCID with DOHH2 xenograft
The injected antibodyPRCRDose MMAF or DM1
(µg/m2)
Dose Ab
(mg/
kg)
DAR (Drug/
Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/90/91144To 1.86
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF 0/90/911541,9
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/90/99241,55
Control huMA79b.v28-SMCC-DM11/81/820243,4
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM11/91/911041,85
Thio-huMA79b.v28-HC(A118C)-MC-MMAF5/94/911541,95
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/99/910841,87
Thio - control huMA79b.v28-HC(A118C)1/90/9NA 4NA
Thio control anti-CD22-HC(A118C)-MC-MMAF0/90/911841,96

E. Xenograft BJAB cells containing the luciferase (Burkitt's)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugate "antibody-drug" and various doses, evaluated the effectiveness of other conjugates of drugs in BJAB xenograft containing luciferase (Burkitt's) in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are presented below in table 15.

Control antibody was a carrier (only buffer (ADC)). The control thio-MAb HC(A118C) was an antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1, MCvcPAB-MMAE or MC-MMAF), thio-Mab thio-huMA79b.v28-HC(A118C) or thio-Mab thio-hu-anti-CD22(10F4v3)-HC(A118C) (anywhereman with MC-MMAF). The results are presented below in table 15.

Figure 37A shows a graph of the average tumor volume in time for BJAB xenografts containing luciferase, m�Shea CB17 SCID, processed TDC "heavy chain A118C anti-CD79b antibody" in the doses given in table 15. In particular, the introduction of the "thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE and thio-huMA79b.v28-HC(A118C)-MC-MMAF" resulted in inhibition of tumor growth compared with tumor growth after treatment with the negative controls (thio-anti-HER2-HC(A118C)-BMPEO-DM1, thio-anti-HER2-HC(A118C)-MCvcPAB-MMAE, thio-anti-HER2-HC(A118C)-MC-MMAF). Another control consisted of a thio-huMA79b.v28-HC(A118C) and thio-10F4v3-HC(A118C)-MC-MMAF.

Moreover, table 15 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 15
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with BJAB xenograft containing luciferase
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µg/m2)
Dose Ab
(mg/
kg)
DAR (Drug/Ab)
control media0/100/10NANANA
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/101/10572To 1.86
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/100/102311,55
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/100/102911,9
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM12/100/102711,85
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM14/100/105521,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE4/101/102711,9
Thio-huMA79b.v28-HC(A118C)-MC-MMAF3/81/82811,9
Thio-control huMA79b.v28-HC(A118C)0/100/10NA1NA
Thio-control 10F4v3-HC(A118C)-MC-MMAF0/101/103011,96

F. Xenograft Granta-519 (lymphoma cells of the cerebral cortex of man)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft Granta-519 (lymphoma cells of the cortex of the human brain) in mice CB17 SCID. Conjugates of drugs� and dose (administered on day 0 for all ADC and control) are presented below in table 16.

The control thio-MAb HC(A118C) was a thio-MAb thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1 or MC-MMAF). The results are presented in table 16 and figure 38.

Figure 38A shows a graph of the average volume of the tumor in time to xenotransplantation Granta-519 in mice CB17 SCID, which were injected with the conjugate TDC "heavy chain A118C anti-CD79b antibody" in the doses presented in table 16. In particular, the introduction of conjugates thio-MAb "thio-huMA79b.v28-HC(A118C)-UMRAO-DM1 and thio-huMA79b.v28-HC(AS)-MC-MMAF-a drug in a dosage shown in table 16, resulted in inhibition of tumor growth compared with the drug conjugates used as a control.

In addition, in the same study, for each group, which injected dose, was determined by the percentage change of body weight during the first 14 days. The results (figure 38B) showed that the introduction of these conjugates "thio-Mab-drug" did not lead to a decrease in the percentage of body weight or loss of weight during this time period.

Table 16 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume using �the any period of time after administration dropped to 0 mm 3). (DAR = the ratio of drug to antibody).

Table 16
Reduction of tumor volume in vivo by introducing conjugate thio-huMA79b.v28-HC(A118C)-DM1 and MMAF CB17 mice with SCID xenograft Granta-519
The injected antibodyPRCRDose MMAF or DM1
(ág/
m2)
Dose Ab
(mg/
kg)
DAR (Drug /Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/80/834212To 1.86
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/80/8346121,9
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM10/60/65521,85
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM10/80/8 11041,85
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM14/84/821981,85
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM13/85/8329121,85
Thio-huMA79b.v28-HC(A118C)-MC-MMAF1/81/85721,95
Thio-huMA79b.v28-HC(A118C)-MC-MMAF2/81/811541,95
Thio-huMA79b.v28-HC(A118C)-MC-MMAF6/82/822981,95
Thio-huMA79b.v28-HC(A118C)-MC-MMAF4/84/8344121,95

G. Xenograft WSU-DLCL2 (d�Fresnoy large cell lymphoma)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft WSU-DLCL2 (diffuse large cell lymphoma) in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are presented below in table 17.

Control antibody was a carrier (only buffer (ADC)). The control thio-MAb was an antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1, MCvcPAB-MMAE or MC-MMAF). The results are presented in table 17 and figure 39.

Figure 39 shows a graph of the average tumor volume depending on time of the xenograft WSU-DLCL2 in mice CB17 SCID treated with conjugates TDC "heavy chain with AS anti-CD79b antibody" in the doses given in table 17. In particular, the introduction of conjugate thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MC-MMAF and thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE (at the dose of Ab 0.5 mg/kg, 1.0 mg/kg 2.0 mg/kg and 4.0 mg/kg) resulted in inhibition of tumor growth compared with tumor growth after treatment with the negative controls (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE, thio-hu-anti-HER2-HC(A118C)-MC-MMAF and A-novtel�).

Moreover, table 17 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 17
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenograft WSU-DLCL2
The injected antibodyPRCRDose MMAF, MMAE
or DM1
(µg/m2)
Dose
Ab
(mg/
kg)
DAR (Drug/Ab)
control media0/90/9NANANA
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/90/9114 4To 1.86
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/90/99241,55
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/90/911541,9
Thio-huMA79b.v28-HC(A118C)-
MC-MMAF
5/92/911241,9
Thio-huMA79b.v28-HC(A118C)-
BMPEO-DM1
4/90/911041,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE1/90/9140,51,9
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/90/9271,01,9
Tio huM79b.v28-HC(A118C)-MCvcPAB-MMAE 2/91/9552,01,9
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE1/97/91104,01,9

N. Xenograft Granta-519 (lymphoma cells of the cerebral cortex of man)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft Granta-519 (lymphoma cells of the cortex of the human brain) in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are presented in table 18 below.

The control thio-MAb was a thio-hu-anti-HER2-HC(A118C) (anywhereman with BMPEO-DM1 and thio antibody-MAb thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE. The results are presented in table 18 below.

Figure 40A shows a graph of the average volume of the tumor in time to xenotransplantation Granta-519 in mice CB17 SCID, which were injected with the conjugate TDC "heavy chain A118C EN�and-CD79b antibody in a dosage presented in table 18. In particular, the introduction of thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 and thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE (at a dose of Ab 1.0 mg/kg 2.0 mg/kg and 4.0 mg/kg) resulted in inhibition of tumor growth compared with the negative controls (thio-anti-HER2-HC(A118C)-BMPEO-DM1 and thio-anti-HER2-HC(A118C)-MCvcPAB-MMAE.

Moreover, in table 18 indicates the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 18
Reduction of tumor volume in vivo by introducing conjugate thio-HuMA79b.v28-HC(A118C)DM1 and MMAE CB17 mice with SCID xenograft Granta-519
The injected antibodyPRCRDose
MMAF,
ME
or DM1
(µg/m2)
Dose
Ab
(mg/
kg)
DAR (Drug/Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/10 0/101144To 1.86
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE2/101/109241,55
Thio-huMA79b.v28-HC(A118C)-
BMPEO-DM1
3/100/1011041,85
Thio-huMA79b.v28-HC(A118C)-
MCvcPAB-MMAE
0/101/10130,51,87
Thio-huMA79b.v28-HC(A118C)-
MCvcPAB-MMAE
1/100/10271,01,87
Thio-huMA79b.v28-HC(A118C)-
MCvcPAB-MMAE
1/107/10542,01,87
Thio-huMA79b.v28-HC(A118C)-
MCvcPAB-MMAE
0/1010/10108 4,01,87

I. Xenotransplant BJAB containing CD79b dog-like apes (BJAB-cynoCD79b)

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft cells BJAB (Burkitt's), expressing CD79b dog-like apes (BJAB-cynoCD79b), in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are presented in table 18 below.

Control Ab was a carrier (only buffer). The control thio-MAb was an antibody thio-Mab, namely, thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF and thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE. The results are presented below in table 19 and in figure 50.

Figure 50 shows a graph of the inhibition of tumor growth depending on time of the xenograft (BJAB-cynoCD79b) in mice CB17 SCID treated TDC "heavy chain A118C anti-CD79b antibody" in the doses given in table 19. In particular, the introduction of thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE and thio-huMA79b.v28-HC(A118C)-MC-MMAF and thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM1, thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MCvcPAB-MMAE and thio-anti-cynoCD7b(ch10D10)-HC(A118C)-MC-MMAF resulted in inhibition of tumor growth compared with tumor growth after treatment with the negative controls (thio-anti-HER2-HC(A118C)-BMPEO-DM1, thio-anti-HER2-HC(A118C)-MCvcPAB-MMAE, thio-anti-HER2-HC(A118C)-MC-MMAF and A-media).

Moreover, in table 19 indicates the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 19
Reduction of tumor volume in vivo by introducing conjugate thio anti-cyno CD79b(ch10D10)-HC(A118C) DM1, MMAF or MMAE or thio-HuMA79b.v28 DM1, MMAF or MMAE CB17 mice with SCID xenograft BJAB-cynoCD79b
The injected antibodyPRCRDose MMAF, MMAE
or
DM1
(µg/m2)
Dose
Ab
(mg/kg)
DAR (Drug/
Ab)
control media0/90/9NANANA
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM1 0/90/9572To 1.86
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE0/90/92311,55
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/90/92911,9
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM13/81/85321,8
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MCvcPAB-MMAE1/92/9271To 1.86
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MC-MMAF0/91/92811,9
Thio-huMA79b.v28-HC(A118C)-
BMPEO-DM1
3/90/9 5521,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE2/92/92711,9
Thio-huMA79b.v28-HC(A118C)-
MC-MMAF
7/91/92811,9

J. Xenograft BJAB-cynoCD79b

In a similar study, conducted in accordance with the same Protocol analysis xenotransplanted described in example 3 (see above), but with different conjugates of drugs and various doses, evaluated the effectiveness of the conjugates "thio-Mab-drug" in the xenograft cells BJAB (Burkitt's), expressing CD79b dog-like apes (BJAB-cynoCD79b), in mice CB17 SCID. Conjugates of the drug and dose (administered on day 0 for all ADC and control) are presented below in table 19.

The control thio-MAb was an antibody thio-MAb, namely, thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-huMA79b.v28-HC(A118C) and thio-anti-cynoCD79b(ch10D10)-HC(A118C). The results are presented below in table 20 and in figure 51.

Figure 51 shows a graph of the inhibition of tumor growth in dependence�completely from time to xenografts BJAB-cynoCD79b in mice CB17 SCID, processed TDC "heavy chain A118C anti-CD79b antibody" in the doses given in table 20. In particular, the introduction of thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 and thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM1 resulted in inhibition of tumor growth compared with tumor growth after treatment with the negative controls (thio-anti-HER2-HC(A118C)-BMPEO-DM1). Another control consisted of a thio-hu-MA79b.v28-HC(A118C) and thio-anti-cynoCD79b(ch10D10)-HC(A118C).

The results are presented below in table 20. Table 20 shows the number of mice out of the total number of tested mice that detect PR (PR = partial regression) (where the tumor volume over any period of time after administration dropped to less than 50% of the tumor volume measured at day 0) or CR (CR = complete remission (where the tumor volume over any period of time after administration dropped to 0 mm3), NA = not determined. (DAR = the ratio of drug to antibody).

Table 20
Reduction of tumor volume in vivo by introducing conjugate thio anti-cyno CD79b(ch10D10)-HC(A118C) DM1 or thio-HuMA79b.v28-HC(A118C)DM1 mice CB17 SCID with xenograft BJAB-cynoCD79b
The injected antibodyPRCRDose
MMAF,
ME
or DM1
(µg/m2)
Dose
Ab
(m�/
kg)
DAR (Drug/Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/100/10572To 1.86
Thio-control huMA79b.v28-HC
(A118C)
0/100/10NA2NA
Thio control anti-cynoCD79b(ch10D10)-HC(A118C)0/100/10NA2NA
Thio-huMA79b.v28-HC(A118C)-
BMPEO-DM1
1/100/102711,85
Thio-huMA79b.v28-HC(A118C)-
BMPEO-DM1
0/102/105521,85
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM10/100/10271 1,8
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM10/101/105321,8

The above description is sufficient for the practical implementation of the present invention by a person skilled in the art. Scope of the present invention is not limited to the stated structure, since the stated option was presented only to illustrate certain aspects of the invention, and the scope of the present invention may include any structures that are the functional equivalent of the claimed designs. The claimed material should not be construed as indicating that the material was inadequate for the practical implementation of any aspect of the invention, including the best variant of its implementation, and those specifically illustrated versions should not be construed as limiting the scope of the claims. Indeed, based on the above description, a person skilled in the art can be made various modifications, which, in addition to the illustrated and described in this application, will also be included in the scope of the attached claims.

1. A humanized BCC�struisbaai anti-CD79b antibodies with cysteine substitutions, contains
(a) light chain and heavy chain and
(b) at least one free cysteine amino acid residue in a position selected from: V205C on Kabuto and AS in accordance with the European numbering system, where AS in accordance with the European numbering system is located in the heavy chain and where V205C on Cabatu is located in the light chain, and optionally, where the specified anti-CD79b antibody contains CDR selected from:
(i) HVR-L1 sequence KASQSVDYEGDSFLN (SEQ ID NO:194);
(ii) HVR-L2 sequence AASNLES (SEQ ID NO:195);
(iii) HVR-L3 sequence QQSNEDPLT (SEQ ID NO:196);
(iv) HVR-H1 sequence GYTFSSYWIE (SEQ ID NO:202);
(v) HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO:203); and
(vi) HVR-H3 sequence TRRVPIRLDY (SEQ ID NO:204);
(i) HVR-L1 sequence KASQSVDYDGDSFLN (SEQ ID NO:156);
(ii) HVR-L2 sequence AASNLES (SEQ ID NO:157);
(iii) HVR-L3 sequence QQSNEDPLT (SEQ ID NO:158);
(iv) HVR-H1 sequence GYTFSSYWIE (SEQ ID NO:164);
(v) HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO:165); and
(vi) HVR-H3 sequence TRRVPVYFDY (SEQ ID NO:166);
(i) HVR-L1 sequence KASQSVDYDGDSFLN (SEQ ID NO:175);
(ii) HVR-L2 sequence AASNLES (SEQ ID NO:176);
(iii) HVR-L3 sequence QQSNEDPLT (SEQ ID NO:177);
(iv) HVR-H1 sequence GYTFSSYWIE (SEQ ID NO:183);
(iv) HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO:184); and
(vi) HVR-H3 sequence TRRVPIRLDY (SEQ ID NO:185); and
(i) HVR-L1 consisten with�eTelestia KASQSVDYSGDSFLN (SEQ ID NO:213);
(ii) HVR-L2 sequence AASNLES (SEQ ID NO:176);
(iii) HVR-L3 sequence QQSNEDPLT (SEQ ID NO:177);
(iv) HVR-H1 sequence GYTFSSYWIE (SEQ ID NO:221);
(iv) HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO:222); and
(vi) HVR-H3 sequence TRRVPIRLDY (SEQ ID NO:223).

2. A humanized engineered anti-CD79b antibodies with cysteine substitutions according to claim 1, containing:
(i) HVR-L1 sequence KASQSVDYEGDSFLN (SEQ ID NO:194);
(ii) HVR-L2 sequence AASNLES (SEQ ID NO:195);
(iii) HVR-L3 sequence QQSNEDPLT (SEQ ID NO:196);
(iv) HVR-H1 sequence GYTFSSYWIE (SEQ ID NO:202);
(v) HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO:203); and
(vi) HVR-H3 sequence TRRVPIRLDY (SEQ ID NO:204).

3. A humanized engineered anti-CD79b antibodies with cysteine substitutions according to claim 2, in which the variable domain light chain contains the sequence of SEQ ID NO:207, and the variable domain of the heavy chain contains the sequence of SEQ ID NO:208.

4. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 1 comprising the sequence of SEQ ID NO:255.

5. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 1 comprising the sequence of SEQ ID NO:284.

6. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 1, wherein the light chain contains the sequence of SEQ ID NO:233, and the heavy chain contains the sequence of SEQ ID NO:232.

7. Designed�tion anti-CD79b antibodies with cysteine substitutions according to claim 1, where at least one free amino acid cysteine residue is located in the heavy chain.

8. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 7, where the heavy chain antibody further comprises an additional amino acid residue cysteine at position selected from: V5C, AS, AS, S112C on Kabuto and TS, V282C, S375C, S400C, in accordance with the European numbering system where each position is located in the heavy chain.

9. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 1, wherein the at least one free amino acid cysteine residue is located in a light chain.

10. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 9, where the light chain of the antibody contains additional free amino acid in a position selected from: V15C, V110C, S114C, S121C, S127C, S168C on Kabuto, where each position is located in the light chain.

11. Engineered anti-CD79b antibodies with cysteine substitutions according to claim 1, wherein the antibody obtained by the method comprising replacing one or more amino acid residues in the native sequence of the parent anti-CD79b antibody at a cysteine, and where the parent antibody contains a sequence of the heavy chain selected from: SEQ ID NO:308, 304, 306 and 310, and/or the light chain sequence selected from: SEQ ID NO:307, 303, 305 and 309.

12. Connection-conjugate the antibodies�about (Ab)-a drug (D)", directed against CD79b, where the antibody is a humanized engineered anti-CD79b antibodies with cysteine substitutions according to any one of claims.1-11, and the drug is a molecule of the drug, where the antibody and the drug is covalently bound through at least one free cysteine amino acid residue with the formation of compound-conjugate.

13. The compound-conjugate of claim 12, wherein the humanized engineered anti-CD79b antibodies with cysteine substitutions attached to a molecule of the drug (D) using a linker molecule (L) by at least one free amino acid cysteine residue, where the compound has formula I
Ab-(L-D)p(I)
where p is 1, 3, 4, or preferably 2.

14. The compound-conjugate of claim 13, wherein L is a
-Aa-Ww-Yy-,
where a represents the extension component, covalently linked to the thiol of the cysteine engineered antibodies with cysteine substitutions (Ab);
and is 0 or 1;
each W independently represents an amino acid unit;
w is an integer from 0 to 12;
Y is a GS spacer component covalently linked to a molecule of the drug and
y is 0, 1 or 2.

15. Connection-conjuga� according to claim 13, where D is selected from the group consisting of maytansinoid, auristatin and dolastatin.

16. The compound-conjugate of claim 15, where D represents auristatin or dolastatin and where D is a molecule of the drug of formula (DEor DF:

where each R2and R6is methyl; each R3and R4represents isopropyl; R5represents H; R7represents sec-butyl; each of R8independently selected from-och3HE and H; R9represents H; R10represents aryl; Z represents-O - or-NH; R11represents H, C1-C8alkyl or -(R13O)m-R14where R13represents C2-alkyl, R14represents C1-alkyl and m is 1; R18represents-C(R8)2-C(R8)2-aryl.

17. The compound-conjugate of claim 13, wherein the conjugate "antibody-drug" has the formula
(a)

where RAV is a couple aminobenzoylamino, a R17is a divalent radical selected from (CH2)rWith3-C8carbocycle, -O-(CH2)rarylene, (CH2)rarylene, -aralen-(CH2)r-, (CH2)r-(C3-C8 -carbocycle), (C3-C8-carbocyclic)-(CH2)r-, C3-C8-heterocyclyl, (CH2)r-(C3-C8-heterocyclyl), -(C3-C8-heterocyclyl)-(CH2)r-, -(CH2)rC(O)NRb(CH2)r, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-, -(CH2)C(O)NRb(CH2CH2O)r-, -(CH2)rC(O)NRb(CH2CH2O)r-CH2-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-CH2- and -(CH2CH2O)rC(O)NRb(CH2)r-, where Rbrepresents H, C1-C6alkyl, phenyl or benzyl, and r independently is an integer from 1 to 10.

18. The compound-conjugate of claim 14, where Wwis a valine-citrulline.

19. The compound-conjugate of claim 14, having the formula

where R17is a divalent radical selected from (CH2)rWith3-C8carbocycle, -O-(CH2)rarylene, (CH2)rarylene, Allen-(CH2)r, (CH2)r-(C3-C8-carbocycle), -(C3-C8-carbocyclic)-(CH2)r-, C3-C8-heterocyclyl, (CH2)r-(C3-� 8-heterocyclyl), (C3-C8-heterocyclyl)-(CH2)r-, -(CH2)rC(O)NRb(CH2)r, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-, (CH2)C(O)NRb(CH2CH2O)r, (CH2)rC(O)NRb(CH2CH2O)r-CH2, - (CH2CH2O)rC(O)NRb(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-CH2- and -(CH2CH2O)rC(O)NRb(CH2)rwhere Rbrepresents H, C1-C6alkyl, phenyl or benzyl and r independently is an integer of 1-10.

20. The compound-conjugate of claim 19, where R17represents (CH2)5or (CH2)2.

21. The compound-conjugate of claim 13, having the formula

22. The compound-conjugate of claim 13, wherein L represents SMCC, SPP, SPDB or UMRAO.

23. The compound-conjugate of claim 13, wherein D represents either MMAE, preferably having the structure

where the wavy line indicates the site of binding to the linker L; or MMAF, preferably having the structure

where the wavy line indicates the binding site to the linker L.

24. Connection-conjugate "antibody (Ab)-a drug�, directed against CD79b, selected from the structures:

where Val is a valine; Cit is a citrulline; p is 1, 2, 3 or 4;

where Val is a valine; Cit is a citrulline; p is 1, 2, 3 or 4;

where p is 1, 2, 3 or 4;
or

where p is 1, 2, 3 or 4, wherein the antibody is a humanized anti-CD79b antibody according to any one of claims.1-11, where the antibody konjugierten by at least one free cysteine amino acid residues.

25. Connection-conjugate "antibody-drug" according to claim 24 having the structure

where Val is a valine; Cit is a citrulline; p is 1, 2, 3 or 4, where the antibody is an antibody according to any one of claims.1-11, where the antibody konjugierten by at least one free cysteine amino acid residues.

26. Connection-conjugate "antibody-drug" according to claim 25, where the antibody is an antibody according to claim 4.

27. Connection-conjugate "antibody-drug" according to claim 25, where the antibody is an antibody according to claim 5.

28. Connection-conjugate "antibody-drug" according to claim 24 having the structure
,br/> where Val is a valine; Cit is a citrulline; p is 1, 2, 3 or 4, where the antibody is an antibody according to any one of claims.1-11, where the antibody konjugierten by at least one free cysteine amino acid residues.

29. Connection-conjugate "antibody-drug" according to claim 28, where the antibody is an antibody according to claim 2.

30. Connection-conjugate "antibody-drug" according to claim 28, where the antibody is an antibody according to claim 3.

31. Connection-conjugate "antibody-drug" according to claim 28, where the antibody is an antibody according to claim 4.

32. Connection-conjugate "antibody-drug" according to claim 28, where the antibody is an antibody according to claim 5.

33. Connection-conjugate "antibody-drug" according to claim 28, where the antibody is an antibody according to claim 6.

34. Engineered anti-CD79b antibodies with cysteine substitutions according to any one of claims.1-11, where a parent anti-CD79b antibody is selected from monoclonal antibodies, especificacao antibody, chimeric antibody, human antibody, humanized antibody, and antibody fragment, preferably a Fab fragment.

35. Connection-conjugate "antibody-drug" according to any of claims.13-23, 26, 27 or 29 to 33, where the parent anti-CD79b antibody is selected from monoclonal antibodies, especifismo antibodies, chimeric antibodies, an�of icela person a humanized antibody, and antibody fragment, preferably a Fab fragment.

36. Conjugate "antibody-drug" according to any of claims.24-32, where the antibody obtained by the method comprising replacing one or more amino acid residues of a parent anti-CD79b antibody by cysteine, where the parent antibody contains:
(a) SEQ ID NO:307 and/or SEQ ID NO:308;
(b) SEQ ID NO:303 and/or SEQ ID NO:304;
c) SEQ ID NO:305 and/or SEQ ID NO:306 or
(d) SEQ ID NO:309 and/or SEQ ID NO:310.

37. Pharmaceutical composition for treating cancer containing cells expressing or sverkhekspressiya CD79b, containing an effective amount of the engineered anti-CD79b antibodies with cysteine substitutions according to any one of claims.1-11, or a conjugate "antibody-drug" according to any of claims.12-32 and a pharmaceutically acceptable solvent, carrier or excipient.

38. The pharmaceutical composition according to claim 37, further comprising a therapeutically effective amount of another drug.

39. The pharmaceutical composition according to claim 38, where the drug is selected from the group consisting of bortezomib, tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, involving vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, metotrekstat, vinblastine, ka�Bolotina, paclitaxel, 5-fluorouracil, doxorubicin, melphalan, prednisone and docetaxel.

40. The pharmaceutical composition according to claim 38, where the medicament is an anti-CD20 antibody.

41. The pharmaceutical composition according to claim 38 or 40, further comprising one or more of cyclophosphamide, hydroxydaunorubicin, adriamycin and doxorubicin.

42. Method detection CD79b protein in the sample, presumably containing the specified protein, where the specified method comprises bringing into contact of the specified sample with engineered anti-CD79b antibodies with cysteine substitutions according to claim 1 and determining binding of the indicated antibodies with the specified CD79b protein in a given sample, where the specified antibody is covalently linked to a label and where the binding of an antibody with said protein is indicative of the presence of the indicated protein in a given sample.

43. A method according to claim 42, where the detection is quantitative.

44. A method according to claim 42, where detection is qualitative.

45. A method according to claim 42, where the specified antibody is covalently linked to a label selected from a fluorescent dye, a radioisotope, Biotin or ligand that forms a complex with the metal; or where the specified pattern contains the cell, presumably expressing the indicated protein CD79b, or where the specified cell is a malignant cell hematopoetic�coy tumors.

46. Method of detection of cancer cells expressing or sverkhekspressiya CD79b carried out in a biological sample, comprising:
(a) treatment of cells with compound-conjugate "antibody-drug" according to claim 12 and
(b) detection of binding of the compound-conjugate "antibody-drug" cells,
where cancer cells are cells of hematopoietic tumors.

47. A method according to claim 46, where the detection is quantitative.

48. A method according to claim 46, where detection is qualitative.

49. Method for inhibiting cell proliferation, capable of handling the tumor cells of a mammal in an environment for culturing cells compound-conjugate "antibody-drug" according to claim 12, thereby inhibiting the proliferation of tumor cells, where tumor mammalian cells, preferably cells are hematopoietic tumors expressing or sverkhekspressiya CD79b.

50. The antibody according to any one of claims.1-11 for use in the method of treating hematopoietic cancers.

51. The antibody according to claim 50, where the hematopoietic cancer is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large b-cell lymphoma, aggressive NHL, asymptomatic NHL, follicular lymphoma, relapsed aggressive NHL, relapsing� asymptomatic NHL untreatable the NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

52. The antibody according to claim 50, where the method involves the introduction of an effective amount of another drug to the patient in combination with an antibody.

53. The antibody according to claim 52, where the drug is selected from the group consisting of bortezomib, tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, involving vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, metotrekstat, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, melphalan, prednisone and docetaxel.

54. The antibody according to claim 52, where the medicament is an anti-CD20 antibody.

55. The antibody according to claim 52 or 54, further comprising one or more of cyclophosphamide, hydroxydaunorubicin, adriamycin and doxorubicin.

56. Connection-conjugate "antibody (Ab)-a drug (D) according to any one of claims.12 to 33, 35 and 36 for use in the method of treating hematopoietic cancers.

57. Connection-conjugate "antibody (Ab)-a drug (D) according to claim 56, g�e hematopoietic cancer is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large b-cell lymphoma, aggressive NHL, asymptomatic NHL, follicular lymphoma, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

58. Connection-conjugate "antibody (Ab)-a drug (D) according to claim 56, where the method involves the introduction of an effective amount of another drug to the patient in combination with the compound-conjugate "antibody-drug".

59. Connection-conjugate "antibody (Ab)-a drug (D) according to claim 58, where the drug is selected from the group consisting of bortezomib, tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, involving vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, metotrekstat, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, melphalan, prednisone and docetaxel.

60. Connection-conjugate "antibody (Ab)-a drug (D)" �on p. 58, where the medicament is an anti-CD20 antibody.

61. Connection-conjugate "antibody (Ab)-a drug (D) according to claim 58 or 60, further comprising one or more of cyclophosphamide, hydroxydaunorubicin, adriamycin and doxorubicin.

62. The pharmaceutical composition according to claim 37 for use in the method of treating hematopoietic cancers.

63. The pharmaceutical composition according to claim 62, where the hematopoietic cancer is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large b-cell lymphoma, aggressive NHL, asymptomatic NHL, follicular lymphoma, relapsed aggressive NHL, relapsed asymptomatic NHL, not treatable NHL, not treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia reticuloendotheliosis (re), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

64. The pharmaceutical composition according to claim 62, where the method involves the introduction of an effective amount of another drug to the patient in combination with the pharmaceutical composition.

65. The pharmaceutical composition according to claim 64, where the drug is selected from the group consisting of bortezomib, tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, full�of strata, involving vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, metotrekstat, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, melphalan, prednisone and docetaxel.

66. The pharmaceutical composition according to claim 64, where the medicament is an anti-CD20 antibody.

67. The pharmaceutical composition according to claim 64 or 66, further comprising one or more of cyclophosphamide, hydroxydaunorubicin, adriamycin and doxorubicin.

68. A method of producing compound-conjugate "antibody-drug" containing a humanized engineered anti-CD79b antibodies with cysteine substitutions (Ab) according to claim 1 and a molecule auristatin medicines (D), where the specified engineered antibodies with cysteine substitutions attached to D of the linker molecule (L) by at least one engineered cysteine amino acid residues, where the specified connection has the formula I
Ab-(L-D)p,(I)
where p is 1, 2, 3 or 4; the method comprises the stage of:
(a) interaction of engineered cysteine group engineered antibodies with cysteine substitutions with the linker reagent with obtaining the intermediate "antibody-linker" Ab-L, and
(b) interaction Ab-L with Akti�iravani group drug D, with education, thus, conjugate "antibody-drug",
or provides for stages:
(c) interaction of nucleophilic groups in the molecule of the drug with a linker reagent with the formation of the intermediate connection "drug-linker" D-L and
(d) interaction between the D-L with an engineered antibodies with cysteine substitutions, with education, thus, conjugate "antibody-drug",
where the method optionally provides for the stage of ekspressirovali engineered antibodies with cysteine substitutions in the cells of the Chinese hamster ovary (Cho) cells.

69. A method according to claim 68, further providing the step of processing the expressed engineered antibodies with cysteine substitutions reducing agent, where the reducing agent is preferably selected from TSER and DTT.

70. A method according to claim 69, further providing the step of processing the expressed engineered antibodies with cysteine substitutions oxidizing agent, where the specified oxidizing agent is preferably selected from copper sulfate, dehydroascorbic acid and air.



 

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