Anti-cd79b antibodies and immunoconjugates and methods for using them

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

 

Cross-reference to related applications

In this application, submitted in accordance with article 37, § 1.53(b) of the code of Federal Regulations (CFR), the priority of the provisional application for U.S. patent No. 60/950052, filed July 16, 2007, provisional application for U.S. patent reg. No. 61/025137, filed January 31, 2008, provisional application for U.S. patent reg. No. 61/032790, filed on 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, where the description of each application in its entirety is introduced into the present application by reference.

The scope of the invention

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

The level of technology

In the United States of malignant tumors (cancer), after heart diseases 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 tumor masses and invasion of adjacent tissues such neoplastic tumor cells, with the formation of malignant what's cells, which, ultimately, is spread through the bloodstream or lymphatic system to regional lymph nodes and in the peripheral region according to the mechanism, called metastasis. When cancer cells proliferate in conditions in which normal cells can not grow. The very cancer manifests itself in various forms of a wide range characterized by varying degrees of invasiveness and aggressiveness.

Cancers involving cells generated in the process of hematopoiesis, i.e. the process through which are formed cellular elements of the blood, such as lymphocytes, leukocytes, platelets, red blood cells and natural killer cells, called cancer of the haematopoietic system. Lymphocytes, which can be detected in the blood and in the lymphatic tissues and play a critical role in the immune response, are divided into two main 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 bone marrow, expressive on its surface antigennegative antibody. After first contact "untrained" In-cells with the antigen for which membrane-bound antibody is specific, cells begin to rapidly divide, and their sweat is msto differentiated in the In-memory cells and effector cells, called "plasma cells". In-memory cells have a longer life time and continue to Express the membrane-bound antibody, which has the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody, and instead they produce 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 undergo rearrangeable genes, which leads to the production of T-cell receptor and undergo positive and negative selection, which facilitates the determination of the phenotype on the cell surface of Mature T cells. Characteristic cell surface markers of Mature T cells are CD3 complexes:T-cell receptor and one used, CD4 or CD8.

In an attempt to identify effective cellular targets for cancer therapy studies have been conducted on the identification of transmembrane or other membrane-bound polypeptides that are specifically expressed on the surface of cancer cells of one or more specific types compared to one or n is the number of normal non-cancer cells. In most cases, these membrane-bound polypeptides in a large number of them are expressed on the surface of cancer cells, but not on the surface of cancer cells. The identification of these associated with tumor polypeptide antigens on the cell surface allows specifically destroy tumor target cells by treatment using antibodies. In this regard, it should be noted that therapy on the basis of the antibodies proved very effective for the treatment of certain cancerous tumors. 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 humanitariannet 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 found on the surface of normal and slocate the state of b-lymphocytes. Both of these antibodies recombinante produced in the cells SNO.

In an attempt to identify effective cellular targets for cancer therapy studies have been conducted to identify (1) polypeptides that are not membrane-bound and which, unlike normal non-cancer cells of specific types, specifically responsible for the generation of one or more cancer cells of a particular type, (2) polypeptides that are produced by cancer cells on the expression level significantly exceeding the level of expression of the polypeptides of one or more normal non-cancerous cells, or (3) polypeptide, the expression of which, in particular, is limited to tissues of the same type (or a very limited number of tissues other types), affected and non-affected cancer (e.g., normal tissue of the prostate gland and tumor tissue of the prostate gland). Such polypeptides can be localized inside the cells, or they can secretariats cancer cells. Further, such polypeptides can be expressed not by cancer cells, and the cells that produce and/or secrete the polypeptides, providing a potentiating effect on cancer cells or action, stimulating the growth of cancer cells. In most cases, such sekretiruemyi polypeptides ablauts the proteins, which provide cancer cells but not normal cells, the preferential growth, and such polypeptides are, for example, angiogenic factors, factors, cell adhesion, growth factors, etc. it is assumed that identification of antagonists of these polypeptides, which are not membrane-bound, will identify effective therapeutic agent for the treatment of these cancers. In addition, identification of the nature of the expression of such polypeptides can be used for the diagnosis of specific cancers in mammals.

In spite of the above advances in anticancer therapy mammals, the need for 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, namely, membrane-bound, secreted polypeptides or intracellular polypeptides, the expression of which, in particular, is limited to tissues of only one type (or a very limited number of tissues other types), hematopoietic tissues affected and not affected by cancer; and the use of such polypeptides and nucleic KIS is on, 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 signal component of the b-cell receptor consisting of covalently bound heterodimer containing CD79a (Igα, mb-1) and CD79b (Igβ, B29). Each of CD79a and CD79b contains the extracellular domain of the immunoglobulin (Ig), transmembrane domain, the intracellular signaling domain and the activation domain of immunoreceptor with the tyrosine motif (ITAM). CD79 is expressed on B-cells and in cells of non-Hodgkin 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 (2001)). The average level of surface expression of CD79b 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 antigen CD79b and has minimal or no antigenicity has antigenicity when administered to patients, particularly during prolonged treatment. The present invention meets these and other requirements. the present invention relates to an anti-CD79b antibody, which do not have the disadvantages inherent in contemporary therapeutic compositions, and also have other advantages which will be apparent from the following detailed description.

The use of conjugates of the antibody-drug" (ADC), i.e. immunoconjugates, in order for local delivery of cytotoxic or cytostatic agents, for example, drugs to destroy 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 can lead to the production of toxic levels 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 Antibodies 84:Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). In attempts to improve therapeutic index, that is, to maximize the efficacy and minimize the toxicity of the ADC, all efforts were directed at improving the CE is aktivnosti 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 drug and the release of drugs (Lambert J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drugs used in the conjugates of the antibody-drug"are bacterial protein toxins, such as diphtheria toxin, plant protein toxins, such as ricin, and small molecules, 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), see above). Drugs can influence the cytotoxic and cytostatic mechanisms, including binding to tubulin, binding to DNA or inhibition of topoisomerase. Some cytotoxic drugs in their conjugation with major antibodies or ligands of proteins-receptors tend to loss or reduction of activity.

Drugs, namely auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), i.e., synthetic analogues of dolastatin (WO 02/088172)were conjugial who are: (i) with chimeric monoclonal antibodies cBR96 (specific to the antigen Lewis Y on carcinomas); (ii) with SAS, which is specific to CD30, present on hematological malignancies (Klussman et al. (2004) Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; Francisco et al. (2003) Blood 102(4):1458-1465; publication of 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) anti-EphB2R antibody N used to treat cancer colorectal (Mao et al. (2004) Cancer Research 64(3):781-788); (v) with the antibody against E-selectin (Bhaskar et al. (2003) Cancer Res. 63:6387-6397); (vi) with trastuzumab (HERCEPTIN®, application U.S. 2005/0238649), and (vii) anti-CD30 antibodies (WO 03/043583). Options auristatin is described in U.S. patent 5767237 and 6124431. Monomethylaniline E, conjugated with monoclonal antibodies described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published on 28 March 2004 Auristatin analogues of MMAE and MMAF were conjugated with different antibodies (application USA 2005/0238649).

The standard method of attachment, i.e. covalent binding molecule drugs with the antibody, essentially allows you to obtain a heterogeneous mixture of molecules, in which medicines are attached in different parts of the molecule antibodies. So, for example, cytotoxic drugs are usually kongugiruut with the antibodies is through a large number of lysine residues of the antibody to obtain a heterogeneous mixture of conjugate “antibody-drug”. Depending on the reaction conditions heterogeneous mixture usually contains a certain number of antibodies, which are attached from 0 and up to about 8 or more molecules associated medicines. In addition, in each sub-conjugates, with respect to the molecules of the drug to antibody molecules equal to a specific integer, may be a heterogeneous mixture, in which the molecule drugs attached at different sites of antibodies. Analytical and preparative methods may be unsuitable for the separation and characterization of molecules conjugates of the antibody-drug” in a heterogeneous mixture resulting from the reaction of conjugation. Antibodies are large, complex, and differ in their structure of biomolecules, which in most cases have many 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 co-solvents. Furthermore, the method of multi-conjugation can be playable, due to the difficulties of regulating the reaction conditions and characterization of reagents and intermediates.

In the best of the e most amines, which are protonated and less nucleophilic at pH~7, thiols cysteine are reactive at neutral pH. Because the free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins containing cysteine residues, are often oxidized form and are linked by disulfide oligomers, or they contain an internal disulfide bridge group. Extracellular proteins typically do not contain free thiol groups (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, page 55). Thiol group of cysteine antibodies are usually more reactive, more nucleophilic towards electrophilic reagents conjugation than amino or hydroxyl group antibodies. Cysteine residues were introduced into proteins by means of genetic engineering with the formation of covalent bonds with ligands or forming new intramolecular 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 thiol groups of cysteine by substituting a different amino acid residues of the protein cysteine residues may be linked to certain what problemami, especially if there are unbound (free Cys) residues or residues, which are relatively accessible for the reaction or oxidation. In concentrated protein solutions, regardless of whether they are on periplasmic E. coli, supernatant culture, or are partially or completely purified proteins, unrelated Cys residues on the surface of the protein can contact and be oxidized with the formation of intermolecular disulfides, and therefore dimers or multimers protein. The formation of disulfide dimers according to the new residue Cys inability to form conjugates with drug, ligand, or other label. In addition, if the protein as a result of oxidation forms an intramolecular disulfide bond between the new designed Cys and existing Cys residue, both thiol group of Cys become unavailable for operation in the active centre and to the interaction. In addition, this protein may become inactive or non-specific as a result of improper installation or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311:1-9).

Constructed on the basis of cysteine antibodies were obtained in the form of FAB fragments antibody (thio-Fab) and expressed as full-size monoclonal IgG antibody (thio-Mab) (Junutula, J.R. et al. (2008) J. Immunol Methods 332:41 to 52; application U.S. 2007/0092940, containing the s which is introduced into the present description by reference). Antibodies thio-Fab and thio-Mab was conjugated via linkers in the provisions of the newly introduced cysteine thiols using reactive thiol linker reagents and reagents "drug-linker" obtaining conjugates of the antibody-drug" (thio-ADC).

All work cited here, including patent applications and publications in their entirety are introduced in the present description by reference.

The invention

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

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

In one of its aspects the present invention relates to humanitarianlaw anti-CD79b antibody, where the monovalent affinity of the antibody (e.g. affinity of the antibody used as a Fab fragment against CD79b) or the affinity of the bivalent form antibodies against CD79b (e.g., affinity of the antibody used as an IgG fragment against CD79b) is essentially the same, lower or higher than the monovalent affinity or affinity of divalent, respectively, a murine antibody (e.g. affinity of a murine antibody, used as a Fab fragment or IgG fragment against CD79b)or a chimeric antibody (e.g., the affinity of the chimeric antibody, used as a Fab fragment or IgG fragment against CD79b), containing the sequence of the variable domain of the light and tarlouze or comprising or essentially consisting of a specified 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 humanitarianlaw anti-CD79b antibody, where the specified affinity of the antibody in its bivalent form in relation 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 the 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 Maine is our least one residue of the sequence, presented 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 a sequence HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 15 (SEQ ID NO: 164-166).

In one of its aspects the present invention relates to an antibody comprising the variable domain of the light chain containing a sequence 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 a sequence 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 the variable domain of the light chain containing a sequence 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 a sequence 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 the variable domain of the light chain containing a sequence HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 17 (SEQ ID NO: 194-196).

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

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

In one of its aspects the present invention relates to an 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 an anti-CD79b antibody containing the variable domain of the light chain selected from SEQ ID NO: 169, 188, 207 226.

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

In one of its aspects the present invention is tositsa to designed on the basis of cysteine anti-CD79b antibody, containing one or more free amino acids cysteine, where the specified constructed on the basis of cysteine anti-CD79b antibody binds to CD79b polypeptide, and where the specified antibody was obtained by a process comprising replacing 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 the 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 on the basis of cysteine anti-CD79b antibody may be a monoclonal antibody, antibody fragment, chimeric antibody, humanitariannet antibody, single-chain antibody or antibody that competitively inhibits the binding of antibodies against the CD79b polypeptide with him for the criterion antigenic epitope. Antibodies according to the invention can be, but not necessarily, conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin or maytansinoid. Antibodies according to the invention can be, but not necessarily, produced in the cells of SNO 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, used for diagnostic purposes, can be detektirano labeled, attached to a solid carrier or the like

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

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

In one of its aspects the present invention relates to an industrial product with a container and a composition contained within the 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 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 of its aspects the present invention relates to the use of anti-CD79b antibodies according to the invention in order to obtain a medicinal product 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 in order to obtain a medicinal product 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 in order to obtain a medicinal product for therapeutic and/or prophylactic Les is possible 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 the method includes contacting the specified cell with the antibody according to the invention and thereby inhibiting growth of the specified cells. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of therapeutic treatment of a mammal suffering from a cancer containing cells expressing CD79b, where the method includes the introduction of the specified mammal a therapeutically effective amount of the antibody according to the invention and thus effective treatment specified mammal. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of treating or preventing cell proliferative disorders associated with an increased level of expression of CD79b,where the method includes the introduction of the individual, in need of such treatment, an effective amount of the antibody according to the invention and thus effective treatment or prevention of a specified cell-proliferative disorder. In one embodiment of the invention specified proliferative disorder is cancer. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a 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 upon the growth potentiating action CD79b, where the method includes contacting the specified cell with an effective amount of the antibody according to the invention and thereby inhibit growth of these cells. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a 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, at least partially, depends on the growth-potentiating actions CD79b, where the method includes contacting the specified cells effective to the number of antibodies according to the invention and thus effective treatment of the indicated tumor. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of treatment of cancer, comprising the administration to a patient the pharmaceutical compositions containing the described here immunoconjugate and acceptable diluent, carrier or filler.

In one of its aspects the present invention relates to a method of inhibiting proliferation of b cells, comprising treating the cells with 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 for detecting the presence of CD79b in the sample, which is assumed to contain CD79b, where the method includes processing the specified sample with an antibody according to the invention and determining the level of binding of the indicated antibody to CD79b in the specified sample, where the specified binding of an antibody to CD79b in the 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 for the diagnosis of cell proliferative disorders, assotsiirovannogo increase in the number of cells, such as b-cells expressing CD79b, where the method includes contacting the test cells in the biological sample with any of the above antibodies; determining the level of antibodies bound to the test cells in the sample by detecting binding of the indicated antibody to CD79b; and comparing levels of antibodies bound to cells in a control sample, where the level of bound antibody normalize by the number of CD79b-expressing cells in the test and control samples, and where a higher level of bound antibody in the test sample compared to a control sample indicates the presence of a cell proliferative disorder associated with cells, expressing CD79b.

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

In one of its aspects the present and the finding relates to a method of binding antibodies according to the invention with the cell, expressing CD79b, where the method includes contacting the specified cells with the indicated antibody according to the invention. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

Brief description of figures

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

The figure 2 presents the amino acid sequence (SEQ ID NO: 2)derived from the coding sequence of SEQ ID NO: 1, presented in figure 1.

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

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

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

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

In figures 7A-B shows an alignment of sequences of the variable light chain: consensus sequences of human light chain Kappa I (marked "huKI"; SEQ ID NO: 9) with VL FR1, VL FR2, VL FR3, and VL FR4 (SEQ ID NO: 139-142, respectively), murine anti-CD79b antibody (labeled "MA79b"; SEQ ID NO: 10), MA79b-linked "gumanitarnogo" antibodies (labeled "huMA79b-hybrid; SEQ ID NO: 11), MA79b-associated variants 17 "gumanitarnogo" antibodies (labeled "huMA79b.v17"; SEQ ID NO: 169), MA79b-associated case 18 "gumanitarnogo" antibodies (labeled "huMA79b.v18"; SEQ ID NO: 188), MA79b-associated case 28 "gumanitarnogo" antibodies (Ref the frame "huMA79b.v28"; SEQ ID NO: 207) and MA79b-associated variant 32 "gumanitarnogo" antibodies (labeled "huMA79b.v32"; SEQ ID NO: 226). Positions are numbered according to Kabat, and the hypervariable region (HVR) of MA79b attached to a consensus framework region of the variable region of the light chain Kappa I, specified in the framework.

In figures 8A-B illustrates the alignment of sequences of the variable regions of the heavy chains: consensus sequences of human heavy chain subgroup III (marked "humIII"; SEQ ID NO: 13) with VH FR1, VH FR2, VH FR3, and VH FR4 (SEQ ID NO: 143-146), murine anti-CD79b antibody (labeled "MA79b"; SEQ ID NO: 14), MA79b-linked "gumanitarnogo" antibodies (labeled "huMA79b-hybrid"; SEQ ID NO: 15) (containing 71A, 73T and 78A), MA79b-associated case 17 "gumanitarnogo" antibodies (labeled "huMA79b.v17"; SEQ ID NO: 170) (containing 71A, 73T and 78A), MA79b-associated case 18 "gumanitarnogo" antibodies (labeled "huMA79b.v18"; SEQ ID NO: 189) (containing 71A, 73T and 78A), MA79b-associated case 28 "gumanitarnogo" antibodies (labeled "huMA79b.v28"; SEQ ID NO: 208) (containing 71A, 73T and 78A) and MA79b-associated variant 32 "gumanitarnogo" antibodies (labeled "huMA79b.v32"; SEQ ID NO: 227) (containing 71A, 73T and 78A). Positions are numbered according to Kabat, and the hypervariable region (HVR) of MA79b attached to a consensus framework region of the variable region of the heavy chain p is gruppy III, specified in the framework.

Figure 9 shows various HVR sequence selected options MA79b-linked "gumanitarnogo" antibodies (SEQ ID NOs: 17-21), where each option has a single amino acid substitution in one HVR MA79b-linked "gumanitarnogo" antibodies (HVR-L1 (SEQ ID NO: 131); HVR-L2 (SEQ ID NO: 132); HVR-L3 (SEQ ID NO: 133)). The sequence of the variable region of the light chain and the variable region of the heavy chain outside the specified one amino acid replacement, 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-linked "gumanitarnogo" antibodies was not observed.

The figure 10 shows various HVR sequence selected options MA79b-linked "gumanitarnogo" antibody (SEQ ID NO: 22-106), including huMA79b L2-2 (also designated here "L2") and huMA79b H3-10 (also called here "H3"), where each option has a lot of amino acid substitutions in one area HVR MA79b-linked "gumanitarnogo" antibodies (HVR-L2 (SEQ ID NO: 132); HVR-L3 (SEQ ID NO: 133); HVR-H1 (SEQ ID NO: 134); part HVR-H3 (SEQ ID NO: 136) is shown in figure 10 as SEQ ID NO: 107). The sequence of the variable region of the light chain and the variable region of the heavy chain outside of these amino acid substitutions are identical sequence huMA79b-hybrid and the figure is not shown. Any and what changes in HVR-L1 (SEQ ID NO: 131) or HVR-H2 (SEQ ID NO: 135) MA79b-linked "gumanitarnogo" antibodies was not observed.

The figure 11 illustrates Biacore analysis of selected anti-CD79b antibodies, including murine anti-CD79b antibody (labeled "MA79b"), MA79b-bound "humanitariannet" antibody (labeled "huMA79b-hybrid") and options MA79b-linked "gumanitarnogo" 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 (mutations 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 the Fc (huCD79becd-Fc) and the peptide of 16 amino acids, containing the epitope for MA79b and chMA79b (SEQ ID NO: 16).

Figure 12 illustrates Biacore analysis of selected anti-CD79b antibodies, including MA79b-bound "humanitariannet" antibody (labeled "huMA79b-hybrid") and options MA79b-linked "gumanitarnogo" antibodies (labeled 1-34 in the first column or as a "whole frame" in the first column) against the extracellular domain of human CD79b (antigen huCD79becd). Options MA79b-linked "gumanitarnogo" antibodies include options "all frame fields", in which there are potentially important mouse skeleton remains, and options (marked 1-34) with combinations of mutations in the frame region in the presence or in the absence of mutations HVR variable regions of the heavy chain and variations the nutrient region of the light chain. Option 17 MA79b-linked "gumanitarnogo" antibodies (designated here "huMA79b.v17") specified in the first column as 17 option 18 MA79b-linked "gumanitarnogo" antibodies (designated here "huMA79b.v18") specified in the first column as 18, option 28 MA79b-linked "gumanitarnogo" antibodies (designated here "huMA79b.v28") specified in the first column as 28, and option 32 MA79b-linked "gumanitarnogo" antibodies (designated here "huMA79b.v32") specified in the first column 32. Styling characteristic of the divalent binding is represented as Kd particular option MA79b-linked "gumanitarnogo" antibodies (marked "Kdvariant”)/Kd chimeric MA79b antibody (chMA79b) (marked “Kdchimera"); values under the column entitled "laying characteristic of the divalent linking represent Kdvariant/Kdchimera. Netdetective binding are marked in the figure as “NDB”.

In figures 13A-B (consensus frame variable region heavy chain (VH)) and figure 14 (a consensus of the frame variable region of the light chain (VL)are representative of the consensus frame acceptor sequence of the human antibody is used to implement the present invention with sequence identifiers, such as: (figures 13A-B) consensus framework region is to be human VH subgroup I without CDR according to Kabat (SEQ ID NO: 108), consensus framework region of human VH subgroup I without extended hypervariable regions (SEQ ID nos: 109-111), a consensus framework region of human VH subgroup II without CDR according to Kabat (SEQ ID NO: 112), a consensus framework region of human VH subgroup II without extended hypervariable regions (SEQ ID NO: 113-115), the consensus framework region of human VH subgroup III without CDR according to Kabat (SEQ ID NO: 116), a consensus framework region of human VH subgroup III without extended hypervariable regions (SEQ ID nos: 117-119), the acceptor framework region of human VH without CDR according to Kabat (SEQ ID NO: 120), the acceptor framework region of human VH without extended hypervariable regions (SEQ ID NO: 121-122), the acceptor framework region of human VH2 without CDR according to Kabat (SEQ ID NO: 123) and the acceptor framework region of human VH2 without extended hypervariable regions (SEQ ID NO: 124-26) and (figure 14) consensus framework region of human VL Kappa subgroup I (SEQ ID NO: 127), the consensus of the frame region of the human VL Kappa subgroup II (SEQ ID NO: 128), consensus the human frame region Kappa subgroup III (SEQ ID NO: 129) and the consensus human frame region Kappa subgroup IV (SEQ ID NO: 130).

In figures 15A (light chain) and 15B (heavy chain) represented by amino acid sequences of the antibodies according to the invention (huMA79bv17). In figure 15A (light chain) and 15B (heavy chain) shows the amino acid sequence of framework region (FR), hypervariable region (HVR), the first constant domain (CL or CH1) and Fc region (Fc) of one of the variants of the antibodies according to the invention (huMA79b.v17) (SEQ ID NO: 152 to 159 (figure 15A) and SEQ ID NOs: 160-168 (figure 15B)). Also presents full amino acid sequence (variable and constant region light and heavy chains huMA79b.v17 (SEQ ID NO: 303 (figure 15A) and 304 (figure 15B), respectively, with the underlined constant domains. Presents the amino acid sequence of the variable domain (SEQ ID NO: 169 (figure 15A for light chain) and SEQ ID NO: 170 (figure 15B for the heavy chain)).

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

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

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

The figure 19 shows the alignment of amino acid sequences of human CD79b (SEQ ID NO: 2), abacadabra monkey (cyno) (SEQ ID NO: 7) and mouse (SEQ ID NO: 8). Amino acid sequence of human CD79b and abacadabra monkeys identical to 85%. Also shows the signal sequence, the test peptide (epitope of 11 amino acids for MA79b, chMA79b and CD79b antibodies against abacadabra monkeys as described in example 1; ARSEDRYRNPK (SEQ ID NO: 12)), transmembrane (TM) domain and domain motif activation immunoreceptor-based tyrosine (ITAM). The area shown in a frame is an area CD79b, which is absent in splanirovannim version CD79b (as described in example 1).

The figure 20 shows a graph of the inhibition of tumor growth in vivo in a BJAB-luciferase xenograft models, where the specified graph shows that the introduction of the anti-CD79b antibody(a) chMA79b-SMCC-DM1, download the medicinal product is around 2.9 (table 9), and (b) huMA79bL2/H3-SMCC-DM1, download the medicinal product 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 shows a graph of the inhibition of tumor growth in vivo in xenograft models Granta-519 (human lymphoma cells of the cerebral cortex), where from the specified graph shows that the introduction of the anti-CD79b antibody(a) chMA79b-SMCC-DM1, download the medicinal product is approximately 3.6 (table 10), (b) huMA79b.v17-SMCC-DM1, download the medicinal product is approximately 3.4 (table 10), (c) huMA79b.v28-SMCC-DM1, download the medicinal product is around 3.3 or 3.4 (table 10), (d) huMA79b.v18-SMCC-DM1, download the medicinal product is approximately 3.4 (table 10), and (e) huMA79b.v32-SMCC-DM1, download the medicinal product is around 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). Figure 21B shows a graph of the percentage change in mass of the studied mice with xenograft Granta-519 (figure 21A and table 10), indicating the absence of any significant changes in weight during the first 7 days of the ISS is adavani. "hu" means humanitariannet 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 drugs attached to engineered cysteine group in the light chain (LC-ADC); the heavy chain (HC-ADC) and Fc region (Fc-ADC).

Figure 23 illustrates the stages: (i) recovery of adducts of cysteine disulfides and Mirzayeva and noticablely disulfides in an anti-CD79b antibody, is developed on the basis of cysteine (ThioMab), a reducing agent TCEP (Tris hydrochloride-(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 re-oxidized antibodies with intermediate connection "drug-linker" with the formation of conjugate "cysteine anti-CD79b antibody-drug" (ADC).

The figure 24 shows (A) the sequence of the light chain (SEQ ID NO: 229) and (B) sequence of the heavy chain (SEQ ID NO: 228) gumanitarnogo anti-CD79b antibodies constructed on the basis of cysteine (thio-huMA79b.v17-HC-A118C)where alanine is present at position 118 in accordance with the European numbering system (the position of the alanine 118 in accordance with the laws the AI with sequential numbering system; the position Kabat - 114)in the heavy chain was replaced by a cysteine. The molecule drugs can be attached to the introduced cysteine group in the heavy chain. Each piece has 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 humanitariannet antibody.

The figure 25 shows (A) the sequence of the light chain (SEQ ID NO: 231) and (B) sequence of the heavy chain (SEQ ID NO: 230) gumanitarnogo anti-CD79b antibodies constructed on the basis of cysteine (thio-huMA79b.v18-HC-A118C)where alanine is present at position 118 in accordance with the European numbering system (the position of the alanine 118 in accordance with a sequential numbering system; the position Kabat - 114) in the heavy chain was replaced by a cysteine. The molecule drugs can be attached to the introduced cysteine group in the heavy chain. Each piece has 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 the hu" means humanitariannet antibody.

The figure 26 shows (A) the sequence of the light chain (SEQ ID NO: 233) and (B) sequence of the heavy chain (SEQ ID NO: 232) gumanitarnogo anti-CD79b antibodies constructed on the basis of cysteine (thio-huMA79b.v28-HC-A118C)where alanine is present at position 118 in accordance with the European numbering system (the position of the alanine 118 in accordance with a sequential numbering system; the position Kabat - 114) in the heavy chain was replaced by a cysteine. The molecule drugs can be attached to the introduced cysteine group in the heavy chain. Each piece has 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 humanitariannet antibody.

The figure 27 shows (A) the sequence of the light chain (SEQ ID NO: 235) and (B) sequence of the heavy chain (SEQ ID NO: 234) anti-CD79b antibodies constructed on the basis of cysteine (thio-huMA79b-LC-V205C), where valine at position 205 according to Kabat (position valine 209 in accordance with a sequential numbering system) light chain was replaced by a cysteine. The molecule drugs can be attached to the introduced cysteine group in the light chain. Each Phi is ur 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.

The figure 28 shows (A) the sequence of the light chain (SEQ ID NO: 237) and (B) sequence of the heavy chain (SEQ ID NO: 236), anti-CD79b antibodies constructed on the basis of cysteine (thio-huMA79b-HC-A118C)where alanine is present at position 118 in accordance with the European numbering system (the position of the alanine 118 in accordance with a sequential numbering system; the position Kabat - 114) in the heavy chain was replaced by a cysteine. The molecule drugs can be attached to the introduced cysteine group in the heavy chain. Each piece has 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-plots indicating that binding of conjugates of anti-CD79b antibody MAb-drug" (TDC) according to the invention with CD79b expressed on the surface of BJAB cells containing the luciferase, similar to the binding of the conjugated (A) options LC(V205C) thio-MAb and (B) variants HC(A118C) thio-MAb antibodies chA79b with MMAF. Detection was performed using mass spectrometry (MS) using PV-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody.

In figures 30A-D presents FACS-plots indicating that binding of conjugates of anti-CD79b antibody MAb-drug" (TDC) according to the invention with CD79b expressed on the surface of BJAB cells containing the luciferase, similar to the binding of (A) the "bare" (unconjugated) options HC (AS) thio-MAb huMA79b.v18 and conjugated variants HC (A118C) thio-MAb antibodies huMA79b.v18 with various specified drug conjugates (() MMAF, (C) MMAE and (D) DM1)). Detection was performed using mass spectrometry (MS) using PV-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

In figures 31A-D presents FACS-plots indicating that binding of conjugates of anti-CD79b antibody MAb-drug" (TDC) according to the invention with CD79b expressed on the surface of BJAB cells containing the luciferase, similar to the binding of (A) the "bare" (unconjugated) options HC (AS) thio-MAb huMA79b.v28 and conjugated variants HC (A118C) thio-MAb antibodies huMA79b.v28 with a variety of these conjugates Le is arctonyx means (C) MMAE, (C) DM1 and (D) MMAF)). Detection was performed using mass spectrometry (MS) using PV-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

In figures 32A-D presents FACS-plots indicating that binding of conjugates of the antibody against CD79b abacadabra monkeys thio-MAb-drug" (TDC) according to the invention with CD79b expressed on the surface of BJAB cells expressing CD79b abacadabra monkeys, similar to the binding of (A) the "bare" (unconjugated) options HLC(As) thio-Mab against CD79b abacadabra monkeys (ch10D10) and conjugated variants HC (A118C) thio-MAb against CD79b abacadabra monkeys (ch10D10) with different specified drug conjugates ((C) MMAE, (C) DM1 and (D) MMAF)). Detection was performed using mass spectrometry (MS) using PV-conjugated antibodies against human IgG. "Thio" means constructed on the basis of cysteine antibody.

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

Figure 34A shows a graph of the inhibition of tumor growth in vivo in xenograft models of BJAB cells containing the luciferase (Burkitt lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, download the cure the aqueous funds approximately 1,87 (table 12), thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1,85 (table 12), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means approximately 1,95 (table 12), found a significant inhibition of tumor growth during the study. As control was used a 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), 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 shows a graph of the percentage change in mass of the studied mice with xenograft BJAB-luciferase (figure 34A and table 12), indicating the absence of any significant changes in weight during the first 7 days of research. "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

Figure 35A shows a graph of the inhibition of tumor growth in vivo in xenograft models WSU-DLCL2 (both diffuse lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-MCvcAB-MMAE, downloading medicines approximately 1,87 (table 13), thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1,85 (table 13), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means approximately 1,95 (table 13), found a significant inhibition of tumor growth during the study. As control was used a 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), 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 shows a graph of the percentage change in mass of the studied mice with xenograft WSU-DLCL2 (figure 35A and table 13), indicating the absence of any significant changes in weight during the first 7 days of research. "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

The figure 36 shows a graph of the inhibition of tumor growth in vivo in xenograft models DOHH2 (follicular lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE)), SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Model xentra is spuntata, processed thio-huMA79b.v28-BMPEO-DM1 downloading medicines approximately 1,85 (table 14), thio-huMA79b.v28-MC-MMAF with drug loading means approximately 1,95 (table 14), or thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE) downloading medicines approximately 1,87 (table 14), found a significant inhibition of tumor growth during the study. As control was used a 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), 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 humanitariannet antibody.

Figure 37 shows a graph of the inhibition of tumor growth in vivo in xenograft models of BJAB cells containing the luciferase (Burkitt lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (MCvcPAB-MMAE, BMPEO-DM1 or MC-MMAF), and/or in different doses to SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 downloading medicines approximately 1,85 (table 15), thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, with a drug loading of media is TBA estimated 1.9 (table 15), or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means an estimated 1.9 (table 15), found a significant inhibition of tumor growth during the study. As control was used media (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), 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 humanitariannet antibody.

Figure 38A shows a graph of the inhibition of tumor growth in vivo in xenograft models Granta-519 (human lymphoma cells of the cerebral cortex, where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (BMPEO-DM1 or MC-MMAF), and/or in different doses to SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 downloading medicines approximately 1,85 (table 16) or thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means approximately 1,95 (table 16), found a significant inhibition of tumor growth during the study. As control was used a control anti-HER2 anti-Christ. ate (thio-hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio-hu-anti-HER2-HC(A118C)-MC-MMAF). Figure 38B shows a graph of the percentage change in mass of the studied mice with xenograft Granta-519 (figure 38A and table 16), indicating the absence of any significant changes in weight during the first 14 days of research. "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

Figure 39 shows a graph of the inhibition of tumor growth in vivo in xenograft models WSU-DLCL2 (both diffuse lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), and/or in different doses to SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1,85 (table 17), thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means an estimated 1.9 (table 17) or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, downloading medicines estimated 1.9 (table 17), found a significant inhibition of tumor growth during the study. As control was used media (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-ER2-HC(A118C)-MCvcPAB-MMAE). "Thio" means constructed on the basis of cysteine antibody, and "hu" means humanitariannet antibody.

Figure 40 shows a graph of the inhibition of tumor growth in vivo in xenograft models Granta-519 (human lymphoma cells of the cerebral cortex, where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (BMPEO-DM1 or MCvcPAB-MMAE), and/or in different doses to SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1 downloading medicines approximately 1,85 (table 18) or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, downloading medicines approximately 1,87 (table 18), found a significant inhibition of tumor growth during the study. As control was used a 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 humanitariannet antibody.

Figure 41 presents a graph 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 different 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 MME/Ab, (2) control antibody thio-hu-anti-gD-HC(A118C)-MC-MMAF, c loading 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, download 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 antibody, and “hu” means humanitariannet antibody. “gD” means glycoprotein D.

Figure 42 presents the nucleotide sequence of the cDNA (SEQ ID NO: 238) PRO283627, where SEQ ID NO: 235 is a clone designated “DNA548455” (also referred to here as “cyno CD79b”). The nucleotide sequence encodes CD79b abacadabra monkeys 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 presents the nucleotide sequence (SEQ ID NO: 240) light chain antibodies against CD79b abacadabra monkeys (ch10D10). The nucleotide sequence encodes the light chain of antibodies against CD79b abacadabra monkeys (ch10D10) with start and stop codons are indicated in bold and underlined.

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

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

Figure 47 presents the amino acid sequence (SEQ ID NO: 243), not containing the first 18 amino acids of the signal sequence and the last 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 sequence of the light chain (SEQ ID NO: 245) and (B) sequence of the heavy chain (SEQ ID NO: 244) CD79b antibodies against abacadabra monkeys, constructed on the basis of cysteine (thio-anti-cynoCD79b HC-A118C)where alanine is present at position 118 in accordance with the European numbering system (the position of the alanine 118 in accordance with a sequential numbering system; the position Kabat - 114)in the heavy chain was replaced by a 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 an E Molecule drugs can be attached to the introduced cysteine group in the heavy chain. Each piece has 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 sequence of the light chain (SEQ ID NO: 300) and (B) sequence of the heavy chain (SEQ ID NO: 299) CD79b antibodies against abacadabra monkeys, constructed on the basis of cysteine (thio-anti-cynoCD79b-LC-V205C), where valine at position 205 according to Kabat (position valine 209 in accordance with a sequential numbering system) in the light chain was replaced by a cysteine. Amino acid D at position 6 in accordance with the European numbering system (shaded in the figure) in the heavy chain may be an alternative that is, the Molecule drugs can be attached to the introduced cysteine group of the heavy chain. Each piece has 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 shows a graph of the inhibition of tumor growth in vivo in xenograft models BJAB-cynoCD79b (BJAB cells, expressyou the e cynoCD79b) (Burkitt lymphoma), where it is shown that the introduction of anti-CD79b TDC conjugated with various molecules "linker-drug" (BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), SCID mice having human b-cell tumors, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1,85 (table 19), thio-huMA79b.v28-HC(A118C)-MC-MMAF with drug loading means an estimated 1.9 (table 19), or thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE, downloading medicines estimated 1.9 (table 19), thio anti-cyno-CD79b (ch10D10)-HC(A118C)-BMPEO-DM1, drug loading funds approximately 1.8 (table 19), thio anti-cyno-CD79b (ch10D10)-HC(A118C)-MC-MMAF with drug loading means an estimated 1.9 (table 19), or thio anti-cyno-CD79b (ch10D10)-HC(A118C)-MCvcPAB-MMAE, downloading medicines approximately 1,86 (table 19), found a significant inhibition of tumor growth during the study. As control was used a 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 humanitariannet antibody.

Figure 51 shows a graph of the inhibition of tumor growth in vivo in xenograft models BJAB-cynoCD79b (BJAB cells expressing cynoCD79b) (limf what we Burkitt), 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, led to a significant inhibition of tumor growth. Xenograft models treated with thio-huMA79b.v28-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1,85 (table 20), or thio anti-cyno (ch10D10)-HC(A118C)-BMPEO-DM1, downloading medicines approximately 1.8 (table 20), found a significant inhibition of tumor growth during the study. As control was used a 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 humanitariannet antibody.

A detailed description of the preferred variants of the invention

The present invention relates to methods, compositions, kits and industrial products used to identify the compositions used for the treatment of hematopoietic tumor in mammals and to methods of applying such compositions according to the invention for the purpose specified.

A detailed description of these methods, compositions, kits and industrial products is n the same.

I. General methods

The present invention can be implemented, if it is not specifically mentioned, 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 following are definitions of terms used in this description, if it is not specifically mentioned, it is understood that the terms used in the singular can mean the nouns in the plural, and Vice versa. If the definition of any term in this description there are contradictions with the definition contained in any document entered into the present description by reference, they can be settled in accordance with the description below.

Used herein, the term “marker-glue the internal surface” or “antigen b-cell surface” refers to the antigen, expressed on the surface of b cells, which can be directed antagonist, binding to this cell, including, but not limited to, antibodies against the antigen At the cell surface or soluble form of the antigen At the cell surface, having the ability to inhibit the binding of the ligand with 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 (see description in the publication “The Leukocyte Antigen Facts Book, 2nd Edition, 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, at btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287. The marker At the cell surface, of particular interest, is expressed primarily on b cells, in contrast to other non-b-cell tissues of a mammal, and can be expressed as b-cell precursors and Mature b cells.

Used herein, the term "CD79b" means any natural CD79b, derived from any vertebrate, including mammals such as primates (e.g., people, abacadabra monkey (cyno) and rodents (e.g. mice and rats), unless otherwise specified particularly. Human CD79b also is indicated here "PRO36249" (SEQ ID NO: 2) and is encoded by the nucleotide sequence (SEQ ID NO: 1), also called here "DNA225786". CD79b abacadabra monkeys is also indicated here "cyno-CD79b" or "PRO283627" (SEQ ID NO: 239) and is encoded by the nucleotide sequence (SEQ ID NO: 238), also called here "DNA548455". The term "CD79b" encompasses "full-size" reprezentirovanii CD79b, as well as any form CD79b, which results from processing in the cell. The term also covers natural ways CD79b, for example splanirowannya variants, allelic variants and isoforms. Described here CD79b polypeptide can be isolated from various sources, such as from human tissue or from other sources, or they can be obtained by recombinant methods or methods of synthesis. "Native sequence of the CD79b polypeptide" includes a polypeptide having the same amino acid sequence as the corresponding natural CD79b polypeptide. Such a CD79b polypeptide with a native sequence can be isolated from a natural source, or they can be obtained by recombinant methods or methods of synthesis. The term "CD79b polypeptide with native sequence", in particular, covers the natural truncated or secreted forms of the specific CD79b polypeptide (for example, the sequence of the extracellular domain), natural variants (e.g., alternative splanirowannya forms and natural allelic variants of the polypeptide. In some embodiments of the invention described here, a CD79b polypeptide with native sequence represent the Mature polypeptide or a full-sized polypeptides with native sequence containing the full amino acid sequence represented in the description of graphical material. In the description of graphic materials, start - and stop-codons (if specified) is shown in bold and underlined. Residues of nucleic acids labeled “N” in the description of graphical material, are any remnants of nucleic acids. Although the CD79b polypeptide, referred to in the description of graphical material, begin with methioninol residues designated herein as amino acid position 1, however, it is possible that as the starting amino acid residue for CD79b polypeptide can be used, and other methionine remains above or below the position of amino acids 1, as shown in the description of graphical material.

Used herein, the terms "MA79b" or "murine anti-CD79b antibody or murine antibody against CD79b, in particular, the mean mouse monoclonal anti-CD79b antibody, which 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). Mouse monoclonal anti-anti-CD79b the ate can 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 can be isolated from hybridoma clone 3A2-2E7, deposited in the American type culture collection (ATCC) under the Depository number HB11413 assigned ATCC July 20, 1993

Used herein, the term "chMA79b" or "chimeric MA79b antibody, in particular, means a chimeric antibody against human CD79b (previously described in application for U.S. patent No. 11/462336, filed August 3, 2006), where the aforementioned chimeric anti-CD79b antibody includes a light chain of 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 NO: 6 also contains a variable domain SEQ ID NO: 14 (figures 8A-B) and a constant domain of the heavy chain of human IgG1.

Used herein, the term "anti-cynoCD79b" or "antibody against CD79b abacadabra monkeys" refers to antibodies that bind to CD79b abacadabra monkeys (SEQ ID NO: 239 figure 43) (as described earlier in the application for U.S. patent No. 11/462336, filed August 3, 2006). Used herein, the term "anti-cynoCD79b (ch10D10)" or "ch10D10" means a chimeric antibody against CD79b abacadabra about what Asian (previously described in application for U.S. patent No. 11/462336, filed August 3, 2006), which binds to CD79b abacadabra monkeys (SEQ ID NO: 239 figure 43). Anti-cynoCD79b(ch10D10) or ch10D10 is a chimeric antibody against CD79b abacadabra monkeys, 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).

Used herein, the term "MA79b-hybrid" or "MA79b-associated humanitariannet antibody" or "huMA79b-hybrid", in particular, means a hybrid obtained by attaching hypervariable regions, derived from the murine anti-CD79b antibody (MA79b)to acceptor sequence, 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 herein, the term "modification" amino acid residue/position means replacing the primary amino acid sequence compared to the original amino acid sequence, where this replacement is the result of a modification of the sequence that includes the specified amino acid residues/positions. So, for example, a typical modifications are the replacement of the remainder (or residue at the specified position) other amino acid residue (e.g., conservative or non-conservative substitution), but the function of one or more (usually less than 5 or 3) amino acids, adjacent to the specified residue/position, and the deletion of the above balances/provisions. The term "amino acid substitution" or its variant means the replacement of an 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 that contains the original amino acid sequence (or the sequence of wild type"). For example, in the case of antibodies, modified physico-biochemical activity may be the affinity binding molecule-target, the ability to bind to the molecule-target and/or influence on the binding molecule-target.

Used herein, the term “antibody” is used in its broadest sense and specifically covers single monoclonal anti-CD79b antibodies (including agonist, antagonist, and neutralizing antibodies, full-size monoclonal antibodies or intact monoclonal antibodies), the composition of anti-CD79b antibodies possessing polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (for example, bespecifically antibodies, provided that they possess n is mportant biological activity), formed from at least two intact antibodies, single-chain anti-CD79b antibodies and fragments of anti-CD79b antibody (see below), including Fab, Fab'and F(ab')2and Fv fragments, dianthicola, single domain antibodies (sdAbs), provided that they possess the desired biological or immunological activity. Used herein, the terms "immunoglobulin (Ig)" and "antibody" are synonyms. The antibody can 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 communicate with CD79b with an affinity sufficient for use of this antibody as a diagnostic and/or therapeutic agent aimed at CD79b. Preferably the level of binding of anti-CD79b antibodies with an unrelated protein, i.e. protein, 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 to bind to CD79b, has a dissociation constant (Kd), which is ≤1 ám ≤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 conserved in CD79b different types.

"Isolated" antibody is an antibody, whichwas identified and isolated and/or purified from components of its natural environment. Contaminant components of its natural environment are materials that can adversely affect therapeutic efficacy of antibodies, and such components may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments of the invention, the specified antibody may be cleaned: (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 antibodies, (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 a sequencing machine, equipped with a centrifuge vessel, or (3) to homogeneity, which can be confirmed by electrophoresis in LTO-page in reducing or non conditions with staining of Kumasi blue or, preferably, silver. The term “isolated antibody includes the antibody in situ within recombinant cells, if there is at least one natural component of this antibody. However, usually, the selected antibody can be obtained, at least in one stage of cleaning.

The basic 4-chain molecule antibody is heterotetrameric glycoprotein consisting of two identical light (L) chains and two Ident is cnyh heavy (H) chains (IgM antibody consists of 5 main heterotetrameric molecules together with an additional polypeptide, called J chain, and therefore it contains 10 antigenspecific sites, while secreted IgA antibodies can polymerise to form polyvalent structures containing 2-5 basic 4-chain molecules with J-chain). In the case of IgG 4-chain molecule mainly has a size of approximately 150,000 daltons. Each L-chain is associated with the H-chain by one covalent disulfide bond and two N-chains are linked together by one or more disulfide bonds, depending on the isotype N-chain. Each H and L chain also has regularly spaced noticeplease disulfide bridges. Each H chain has at its N-terminal variable domain (VH)followed by three constant domains (CHfor each of the α and γ chains and four CHdomains for μ and ε isotypes. Each L chain has at its N-terminal variable domain (VL), followed by a constant domain (CL) at its other end. VLis in 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 border region between the variable domains of the light chain and heavy chain. Mating VHand VLleads to the formation of one antigennegative site. Structure and properties of antibodies of different CLA the owls described, for example, in the publication of Basic 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-circuit occurring antibodies from any vertebrate species can belong to one of two clearly differentiated 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 small differences in the sequences and functions of CHfor example, in humans, is expressed immunoglobulins following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term "variable region" or "variable domain" antibody means an amino-terminal domains of the heavy or light chain antibodies. 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 generally the most variable parts of the antibodies and contain antigennegative sites.

The term “variable” refers to certain segments of variable house is new, which have significant differences in the sequences of different antibodies. Domain V mediates binding to the antigen and determines the specificity of a particular antibody to a specific antigen. However, the variability is not evenly distributed across all variable domains, consisting of 110 amino acids. But usually region V consist of 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 connected by three hypervariable regions, which form loops connecting, and in some cases forming part of β-folded structure. Hypervariable region in each chain are held in close proximity to each other through FR and, with the hypervariable regions of the other chain, involved in the formation antigennegative site of antibodies (see Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but have different effectormemory, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

“Intact” antibody is an antibody containing antigennegative variable region, and CLand at least the constant domains of the heavy chain, CH1, CH2 and CH3. The constant domains may represent a constant domains of the native sequence (e.g., the constant domains of human native sequence) or variants of the amino acid sequences. The intact antibody preferably has one or more effector functions.

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

"Antibody fragments" contain some of the intact antibody, and preferably antigennegative or variable region of the intact antibody. Examples of fragments of antibodies are Fab-, Fab'and F(ab')2and Fv fragments; dianthicola; linear antibodies (see U.S. patent No. 5641870, example 2; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-stranded molecules of the antibody; and multispecific antibodies formed from fragments of antibodies. In one embodiment of the invention, the antibody fragment contains antigennegative site of the intact antibody, and therefore it preserves the t its ability to bind to the antigen.

As a result of hydrolysis of the antibody with papain formed two identical antigenspecific fragment, called “Fab”fragments, and one remaining “Fc”fragment, whose name refers to his ability to easily crystallize. Fab-fragment consists of a full-sized L-chain together with the domain variable regions of the H-chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is a monovalent respect to binding to the antigen, i.e. it has one antigennegative website. As a result of processing of antibodies with pepsin to form one large F(ab')2the fragment, which corresponds roughly to the two linked by a disulfide bond Fab-fragments with divalent antigennegative activity and ability to cross-contact with the antigen. Fab'fragments differ from Fab fragments by the presence of several additional residues at the carboxy-end of the domain CH1, including one or more cysteines, derived from the hinge region of the antibody. Fab'-SH used in this application is a Fab'in which the cysteine(C) residue(TCA) constant domains have a free thiol group. F(ab')2-fragments of the antibodies were initially obtained as pairs of Fab'-fragments, which are located between the hinge qi is teeny. Experts also other known methods of chemical binding fragments of antibodies.

Fc-fragment contains a carboxy-terminal part of both N-chains connected by disulfide bonds. Effector functions of an antibody is determined by the sequence Fc region, which are also part recognized by Fc receptors (FcR), located on the cells of some types.

"Fv" is the minimum antibody fragment that contains a full-sized antigen-recognizing site and antigennegative website. 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 linked to a flexible peptide linker, resulting in a light and heavy chain may be associated with one another with formation of a "dimeric" structure analogous to the structure of double-stranded Fv. After laying these two domains formed 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 tell the antibody binding specificity to the antigen. However, even a single variable domain (or p is Lovina Fv, containing only three CDRs specific for an antigen) has the ability to recognize the antigen and to contact him, although with lower affinity than the entire binding site.

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

The term “dianthicola” means fragments of antibodies with two antihistamine sites, where these fragments include the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). Small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between domains VHand VLso that was achieved megamachine, but not intrachain, the mating domain V, with the formation of bivalent fragment, i.e. a fragment having two antigennegative is their website. Dianthicola can be divalent or bespecifically. Bespecifically venticelli are heterodimeric two "cross-linked" scFv fragments, in which the domains of the VHand VLtwo 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). Tranchitella 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, the antibodies included in this population and are identical except for possible natural mutations that may be present in small quantities. Monoclonal antibodies are highly specific and are directed against the same antigenic determinants. In addition, unlike drugs 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 “mono is lonely” does not mean, this antibody should be produced to any particular method. For example, the monoclonal antibodies according to the invention can be produced using hybridoma technology, first described by Kohler et al. (1975) Nature 256:495, or they can be obtained by the methods of recombinant DNA in bacterial cells, eukaryotic organisms or plants (see U.S. patent No. 4816567). “Monoclonal antibodies” may also be isolated from phage libraries of antibodies by methods described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

Used here monoclonal antibodies include, in particular, “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, and the other(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 of antibody, and include fragments of such antibodies, provided that they possess the desired biological activity (see U.S. patent No. 4816567 and the publication of Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Interest chimeric antibodies according to the invention are primaryservername the antibodies, containing antigennegative sequence of the variable domains derived from the sequences of the constant region of primates, non-human (e.g., monkeys, apes, and so on), and humans.

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

Used herein, the term "thio" or "thio" refers to the antibody, is developed on the basis of cysteine, as used herein, the term "hu" refers to humanitarianlaw antibody.

"Human antibody" is an antibody having an amino acid sequence that corresponds to amino acid sequences of the antibodies produced in humans and/or received by any of the methods of producing human antibodies described in this application. This definition of the human is ski antibodies in particular, excludes humanitariannet antibody containing antigennegative remains of nonhuman antibodies. Human antibodies can be obtained by various methods known in the art, including the use of phage libraries view. 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 the methods described in the publication 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 injecting the antigen transgenic animal that has been modified in order to produce such antibodies in response to antigenic stimulation, but which were blocked endogenous loci, for example, we immunized mice with xenograft (see, for example, U.S. patent No. 6075181 and 6150584 related to the XENOMOUSE technologyTM). See, also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)relating to the human antibodies obtained using the technology of human b-cell hybridomas.

Used herein, the term “hypervariable region”, “HVR” or “HV” means the area of the variable domain of the antibody, which are in sequence hypervariable and/or education the comfort of a loop of a certain structure. In General, these antibodies contain six hypervariable regions; three areas in VH (H1, H2, H3) and three areas in VL (L1, L2, L3). In this application uses a number of hypervariable regions, which are included in the scope of the present invention. Hypervariable region (complementarity-determining region (CDR)according to the Kabat possess 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 defining hypervariable regions according to Kabat, Chothia 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 CDR-H1 of Cotia, with numbering according to Kabat numbering varies between the provisions H32 and H34 depending on the length of the loop (this is because in accordance with the numbering scheme according to the Kabat insertions located at positions H35A and H35B; if not present 35A or 35B, the loop ends at position 32; if there is only 35A, the loop ends at position 33; and if present 35A and 35B, the loop ends at position 34). The definition of hypervariable regions AbM represents a compromise between the “CDR” by Kabat and “structural loops” of CATIA, and such definitions are used in the computer program for simulation of antibodies Oxford Molecular's AbM. "Contact" hypervariable region determine, based on analysis of existing complex crystal structures. The remnants of each of these hypervariable regions are given below.

LoopAccording to the KabatAbMOn CoteContact area
L1L24-L34L24-L34L26-L32L30-L36
L2L50-L56L50-L56L50-L56L46-L55
L3L89-L97L89-L97L89-L97L89-L96
H1N-NWN-NWN-NN-NW
(Numbering according to Kabat)
H1N-NN-NN-NN-N
(The numbering of CATIA)
H2Uw50-NUw50-NN-NN-N
H3N-NN-NN-NN-N

The term “hypervariable region” may include “extra long hypervariable region”as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL; and 26-35V (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The remains of the variable domains for each of these definitions are numbered according to Kabat, and others, see above.

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

Used herein, the term “balance of the variable domain are numbered according to the Kabat” or “amino acid position is numbered according to the Kabat” and its variants mean the system used for numbering the variable domains of the heavy chain or the variable domains of the light chain of the antibody described in 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 system the St-numbering, the actual primary amino acid sequence may contain fewer or additional amino acids corresponding shortened or elongated FR or CDR region, a variable domain. For example, the variable domain of the heavy chain can include the insertion of one amino acid (residue 52a in accordance with the numbering of Kabat) after residue 52 of H2, and residues (e.g. residues a, 82b and C etc., according to Kabat numbering), built after the balance 82 FR heavy chain. The numbering of residues of this antibody according to Kabat can be done after the alignment of its sequences in the regions of homology with the “standard” sequence, numbered according to Kabat.

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

“Affinity matured” 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 which does not have such a(s) modification(s). Preferred affinity Mature antibodies have nanomolar or even picomolar affinity to the target antigen. Affine Mature antibodies to be made by methods known to experts. The publication Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by permutation domains VH and VL. Nonspecific mutagenesis HVR and/or frame residues described in 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. Pre is respectful blocking antibodies or antibody antagonist mostly or completely inhibit the biological activity of the antigen.

Used herein, the term "antibody agonist" means an antibody that mimics at least one of the functional activities of interest polypeptide.

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

The term “binding affinity of p which means essentially force total non-covalent interactions of a single binding site of a molecule (for example, antibodies) with its binding partner (e.g., antigen). If it is not specifically mentioned, it is used here, the term “binding affinity of” means natural affinity binding, in which there is interaction of 1:1 between members of binding pairs (e.g., antibodies to the antigen). The binding affinity of the molecule X with its partner Y, in General terms, may be called the dissociation constant (Kd). The affinity can be determined by standard methods known in the art, including methods described here. Low-affinity antibodies typically bind to the antigen at a slower rate and have a tendency to easily dissociates, whereas high-affinity antibodies typically bind to the antigen with greater speed and tend to stay connected for a longer period of time. Professionals in this field are known various methods for measuring the affinity of binding, and for the implementation of the present invention can be applied to any of these methods. Specific representative embodiments of the invention described below.

Used here is the definition of "or above", 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 is the definition of "or above"if the but is used in this application, means stronger binding, defined less by the numerical value of Kd. For example, an antibody that has the binding affinity of the antigen "of 0.6 nm or higher"means an antibody that has the binding affinity of the antigen <0,6 nm, that is, of 0.59 nm, of 0.58 nm, of 0.57 nm, etc. or any value less than 0.6 nm.

In one embodiment of the invention “Kd” or “Kd value” according to the invention is determined using an analysis on the binding of radioactively labelled antigen (RIA)performed with the use of Fab-variant interest of the antibody and its antigen as described below, where the specified analysis allows you to measure the binding affinity of Fab with antigen in solution by balancing Fab minimum concentration (125I)-labeled antigen in the presence of titrating set its antigens, with subsequent immobilization associated antigen on the tablet sensitized with antibody against Fab-fragment (Chen et al. (1999) J. Mol. Biol. 293:865-881). In order to create appropriate conditions for the analysis of microtiter plates (Dynex) sensibiliser during the night of 5 µg/ml of binding anti-Fab antibody (Cappel Labs) in 50 mm sodium carbonate (pH 9,6), and then blocked with 2% (wt./about.) albumin bovine serum in PBS for 2-5 hours at room temperature (approximately 23°C). In readsorbing is ensete (Nunc # 269620), 100 PM or 26 PM [125I]-antigen are mixed with serial dilutions of interest Fab (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al. (1997) Cancer Res. 57:4593-4599). Then interest Fab incubated overnight, however, to ensure a balance, the incubation can be carried out over a longer period of time (for example, 65 hours). After this mixture is transferred into a tablet for immobilization and incubated at room temperature (for example, within one hour). Then the solution is removed and the plate washed eight times with 0.1% Tween-20 in PBS. After drying, the tablets add 150 μl/well scintillation fluid (MicroScint-20; Packard), and the tablets are counted on a gamma counter Topcount (Packard) for ten minutes. The concentration of each Fab, which constitute 20% or less of maximum binding, selected for their use in the analysis of competitive binding. In accordance with another variant of the invention Kd or Kd value is measured in the assays, surface plasmon resonance using BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using chips SM with immobilized them by antigen when the value of the response units (RU), amounting to ~10. Briefly, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate Hydra is chloride N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to 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) 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 injected injection twofold serial dilutions of 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 dissociation rate (koff) is calculated using a simple langourously model binding 1:1 (BIAcore Evaluation Software version 3.2) by simultaneous building sensorgram 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-1· with-1as defined above by means of surface plasmon resonance, the velocity of the Association may 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 of 7.2, in the presence of increasing concentrations of antigen, ka is measured on the spectrometer, such as a spectrophotometer, equipped with a flow limiter (Aviv Instrumtnts), or on the 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 can also be determined by the method described above surface plasmon resonance using BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ).

Used herein, the terms “substantially similar” or “essentially the same” means a sufficiently high degree of similarity between two numeric values (generally between one value corresponding to the antibody according to the invention, and another value corresponding to the reference/comparison to the antibody), and it is this degree of similarity, which would allow the specialist in this area to consider the difference between these two values is insignificant or biologically and/or statistically insignificant in relation to their biological properties, identified by the specified values (e.g., Kd values). The difference 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 IU is her than about 10% compared to values of reference/comparison of antibodies.

Used herein, the terms "essentially reduced" or "essentially 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 reference/comparison to the antibody), and it is this degree of difference, which would allow the specialist in this area to consider is the difference between these two values is statistically significant in relation to their biological properties, identified by the specified value (for example, the values of Kd, HAMA response). The difference 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 predetermined antigen, which can be selectively contacted by the antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other natural or synthetic compound. Preferred target antigen is a polypeptide.

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

"Human consensus framework region" is a framework region that includes the most frequently occurring amino acid residues, and is used when the selection frame sequence VL or VH of a human immunoglobulin. Usually the sequence VL or VH of a human immunoglobulin selected from a subset of sequences of variable domains. Basically, the subgroup of sequences is determined by the Kabat and others In one embodiment of the invention, for VL, such a subgroup is a subgroup Kappa I Kabat, etc. In one embodiment of the invention, for VH, such a subgroup is a subgroup III Kabat and other

"The consensus frame region VH subgroup III contains a consensus sequence selected from the amino acid sequences of heavy chain subgroup III Kabat, etc. In one embodiment of the invention the amino acid sequence of the consensus frame region VH subgroup III contains at least a portion of each of the following sequences or all of these 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 sequence of the variable light chain Kappa subgroup I in Kabat, etc. In one embodiment of the invention the amino acid sequence of the consensus frame region VL subgroup I contains at least part of each of the following sequences or all of these after the euteleostei: 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 framework region" is a human frame region having the same amino acid sequence as acceptor human framework region, for example, it does not replace human amino acids at inhuman amino acids, as in the acceptor human framework region.

Used herein, the term "modified hypervariable region" means a hypervariable region that contains one or more (e.g., from one and up to about 16) amino acid substitutions.

Used herein, the term "unmodified hypervariable region" means a hypervariable region having the same amino acid sequence as the sequence of the non-human antibody from which it is, then there is a sequence that does not contain one or more amino acid substitutions.

An antibody that “binds” with interest antigen, for example, tumor-associated polypeptide antigen target is antithical, bind to the antigen with an affinity that is sufficient for this antibody can be used as a therapeutic agent for delivery to a cell or tissue expressing the antigen, and that the s this antibody has no significant cross-reactivity with other proteins. In these embodiments of the invention the binding of an antibody to a protein which is not a target that is less than about 10% of the level of binding of the antibody with its specific protein target, as determined by analysis conducted with the use of cell sorting with activation of fluorescence (FACS) or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to the molecule target, the term "specifically bind" 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 such binding of antibody significantly different from non-specific interactions. Specific binding can be measured, for example, by determining the level of binding of the molecule compared to binding reference molecule, which is essentially a molecule with a similar structure, but does not have binding activity. For example, specific binding can be determined by assessing the competitive binding reference molecule that is similar to this target, for example, by excess unlabeled target. In this case, specific binding is detected, it is when the binding of labeled target probe competitive inhibited by excess unlabeled target. Used herein, the term "specifically bind" 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 may, for example, to indicate that this molecule has a Kd with respect to the target 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 over xpressway appropriate CD79b polypeptide. The CD79b polypeptide may be a transmembrane polypeptide expressed on the surface of cancer cells, or it can be a polypeptide that is produced and the secretory cancer cells. Preferred growth-inhibiting anti-CD79b antibodies inhibit the growth of CD79b-expressing tumor cells by 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 cells not treated with the test antibody. In one embodiment of the invention, growth inhibition can be measured at the antibody concentration of approximately 0.1 to 30 μg/ml or about 0.5 nm to 200 nm in cell culture, where the specified growth inhibition determined in 1-10 days after treatment of tumor cells with antibody. Inhibition of growth of tumor cells in vivo can be determined by various methods described below in the section "Experimental 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 proliferation of tumor cells during the period of time from about 5 days to 3 months, and preferably from about 5 to 30 days after the first injection of antibodies.

An antibody that “induces apoptosis”is an antibody that induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, the shrinkage of the cells, the expansion of the endoplasmic reticulum, fragmentation of cells and/or formation of membrane vesicles (called apoptotic bodies). Such a cell is typically a cell, sverkhekspressiya CD79b polypeptide. Preferred cells are tumor cells, such as 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 of annexin; DNA fragmentation can be evaluated by the formation of DNA ladder; and condensation cores/chromatin with DNA fragmentation can be evaluated by any increase hypodiploidy cells. Preferably the antibody inducing apoptosis, is an antibody that causes approximately 2-50-fold, preferably about 5-50-fold, and most preferably about 10-50-fold induction of binding to annexin compared with untreated cells in the analysis of binding an what axinom.

An antibody that "induces cell death"is an antibody that makes viable cells from nonviable cells. Such cells are cells expressing CD79b polypeptide, and cells of a particular type, which specifically Express or sverkhekspressiya CD79b polypeptide. These cells may be cancerous or 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 that is produced and the secretory cancer cells. These cells can be cancer cells, for example, b-cells or T-cells. Cell death in vitro can be determined in the absence of complement and immune effector cells to identify cell death induced antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, the analysis of cell death can be carried out using thermoinactivation serum (i.e. in the absence of complement), and in the absence of immune effector cells. To determine whether antibody to induce cell death, can be estimated loss of membrane integrity by absorption of iodide of propecia (PI), Trypanosoma blue (see Moore et al. Cytotechnology 17:1-11 (1995) or 7AAD compared to untreated cells. Preferred antibodies that induce cell death, are antibodies that induce PI uptake in the analysis of PI uptake in BT474 cells.

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

Used herein, the term "Fc region" means the C-terminal region of the heavy chain of immunoglobulin, including Fc-region with a native sequence Fc region with altered sequence. Although the boundaries of the Fc region of the heavy chain of immunoglobulin may vary, but the Fc region of the heavy chain of human IgG is usually defined as a fragment extending from amino acid residue in position Cys226, or from Pro230, to the carboxy-end. C-terminal lysine (residue 447 according to the European numbering system) of the Fc region may be removed, for example, the R, in the process of production or purification of antibodies, or by recombinant construct nucleic acid that encodes a heavy chain antibodies. Accordingly, the composition of intact antibodies may include populations of antibodies with all remote residues K447, populations of antibodies with the remnants K that have not been removed, and populations of antibodies with a mixture of antibodies that are present or missing the K447 residue.

"Functional Fc-region has an "effector function" Fc-region with the native sequence. Representative effector functions are binding to C1q; CDC; binding to an Fc receptor; ADCC; phagocytosis; inhibition of the function of cell surface receptors (e.g. B cell receptor; BCR), etc. To achieve effector functions typically requires Fc-region was combined with a binding domain (e.g., variable domain antibodies), and such effector functions can be assessed using various assays described, for example, in the section "Definitions" of this application.

"Fc-region with the native sequence contains an amino acid sequence identical to the amino acid sequence of natural Fc-region. Human Fc-areas with native sequence are the Fc-region of a native sequence h is human IgG1 (not-A and A-allotype); Fc-region of a native sequence human IgG2; Fc-region of a native sequence human IgG3 Fc region of native sequence human IgG4, as well as their natural options.

"Variant Fc region" contains an amino acid sequence that differs from the native sequence Fc region of at least one amino acid modification, and preferably one or more amino acid substitutions. Preferably the variant Fc region has at 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 the native sequence Fc region or Fc region of the parent polypeptide. Variant Fc region described in this application, preferably at least about 80%, more preferably at least about 90%and most preferably at least about 95% homologous to the native sequence Fc region and/or the 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), stoichiometric the sediments, and macrophages), what allows these cytotoxic effector cells to specifically bind with the antigen-bearing target cells, and thereby to destroy these target cells under the action of cytotoxins. These antibodies arm cytotoxic cells and are absolutely necessary for such cytolysis. Primary cells mediating ADCC, that is, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The data obtained for the FcR expression on hematopoietic cells, systematized in table 3 on page 464 of Ravetch & Kinet Annu. Rev. Immunol. (1991) 9:457-92. To assess ADCC activity of interest molecules can be evaluated ADCC in vitro, such as analysis, described in U.S. patent No. 5500362 or 5821337. Effector cells suitable for such analyses are mononuclear cells of peripheral blood (MCPC) 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 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. Preferred FcR is a human FcR with the native sequence. Moreover, a preferred FcR FcR is that with azeveda with IgG antibody (a gamma receptor), and receptor subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternative splanirowannya forms of these receptors. Receptors FcγRII 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 activating motif immunoreceptor-based tyrosine (ITAM). Inhibiting receptor FcγRIIB contains in its cytoplasmic domain inhibiting motif immunoreceptor-based tyrosine (ITIM). (see review M. Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcR described in 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). Used herein, the term “FcR” also covers other FcR, including FcR, which will be identified in the future. This term also includes the receptor is 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 that bind to human FcRn with high affinity binding can be analyzed, for example, in transgenic mice or in transfected human cell line is, expressing human FcRn, or primates, which were introduced polypeptides with a variant Fc region. In WO 2000/42072 (Presta) described variants of antibodies with increased or decreased 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 expressing one or more FcR and with effector functions. Preferably, these cells Express at least FcγRIII and have ADCC effector function. Examples of human leukocytes mediating ADCC, are mononuclear cells of peripheral blood (MCPC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; however, preferred are MCPC and NK-cells. The effector cells may be isolated from their natural source, for example from the blood.

"Complement-dependent cytotoxicity" or "CDC" means the lysis of target cells in the presence of complement. The classical pathway of activation of complement is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass)that are associated with their cognatum antigen. To assess activation of complement can be evaluated by the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants having the e modified amino acid sequence of Fc-region (polypeptides with a variant Fc region and has increased or decreased 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 Fc-region" means an antibody comprising an 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 construct nucleic acid that encodes the antibody. Accordingly, a composition comprising an antibody having an Fc region according to the invention may contain the antibody with the K447 residue; antibody, where all K447 have been removed; or a mixture of antibodies containing and not containing the K447 residue.

"The extracellular domain of the polypeptide CD79b or "ECD" is a form of CD79b polypeptide, generally do not contain 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 the CD79b polypeptide according to the invention, identified in accordance with the criteria usually used by experts to identify the hydrophobic domain of this type. The exact boundaries of the transmembrane domain may vary, but most likely they are not bluechem about 5 amino acids at either end of the originally identified domain. Therefore, the extracellular domain of the CD79b polypeptide may include, but not necessarily, about 5 or fewer amino acids on either side of the border area "transmembrane domain/extracellular domain, identified as described in the examples or in the description of the present application, and such polypeptides, whether or not containing attached thereto, signal peptide, and nucleic acid encoding these polypeptides are included in the scope of the present invention.

The proposed localization signal peptides described here CD79b polypeptide may be specified in the description of this application and/or in the description of graphical material. However, it should be noted that the C-terminal boundary of the signal peptide may vary, but most likely it is not more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide originally identified in this application where indicated C-terminal boundary of the signal peptide may be identified in accordance with the criteria usually used by experts to identify the item 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 the signal sequence from Sekretareva the nogo polypeptide is not completely uniform, which leads to the formation of more than one Sekretareva molecules. Such Mature polypeptides, in which the signal peptide is cleaved within not more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide identified in this application, and polynucleotides encoding such polypeptides, are included in the scope of the present 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 the full sequence of native CD79b polypeptide, described in this application; sequence CD79b polypeptide that does not contain a signal peptide described in this application; the sequence of the extracellular domain of the polypeptide CD79b, whether or not containing signal peptide described in this application; or the sequence of any other fragment of a full-sized CD79b polypeptide, described in this application (such as a polypeptide encoded by a nucleic acid which is only part of the full sequence that encodes a full-CD79b polypeptide). Such variants of the CD79b polypeptide is, for example, a CD79b polypeptide, in which the one or more amino acid residues are added or deleterow N - or C-end full-sized native amino acid sequence. Usually variant of the CD79b polypeptide has an amino acid sequence that is at least about 80%, and the alternative of 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 amino acid sequence of a full-sized native CD79b polypeptide, described in this application; sequence CD79b polypeptide that does not contain a signal peptide described in this application; the sequence of the extracellular domain of the polypeptide CD79b, whether or not containing signal peptide described in this application; or the sequence of any other specifically defined fragment of a full-sized CD79b polypeptide, described in this application. Usually variants 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 polypeptide have, but not necessarily, no more than one conservative substitution and alternative of not more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared with the native sequence of the CD79b polypeptide.

"Percent (%) amino acid identity, the placenta is valnontey" peptides or polypeptides, that is, sequences of CD79b polypeptide identified in this 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 sequence, i.e. the sequence of CD79b polypeptide, after aligning the sequences and introducing them as “gaps”, if necessary, to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of identical sequences. The alignment is performed in order 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 along the entire length of the compared sequences. However, for the purposes of the present invention, the % identity of amino acid sequences is calculated using a computer program ALIGN-2, used for comparison PEFC is guatelmala, where the full source code of the program ALIGN-2 is shown below in table 1. Computer program ALIGN-2, is used to compare the sequences was developed by Genentech, Inc., and the source code is presented below in table 1, was registered in the user documentation in the copyright Office of the United States, Washington, D.C., 20559, under registration number U.S. Copyright Registration no TXU510087. The program ALIGN-2 is a public program, supplied by the firm Genentech, Inc., South San Francisco, California, or it can be based on the source code presented below in table 1. The program ALIGN-2 should be compiled for use on a UNIX operating system, and preferably in the UNIX V4.0D. All the parameters of comparison sequences were installed in the program ALIGN-2 was not changed.

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

100 • X/Y,

where X represents the number of amino acid residues that were OC is tive as fully appropriate when aligning sequences using the program ALIGN-2, which were compared to the sequences a and b, and where Y represents the total number of amino acid residues in the sequence C. it should be noted that if the length of the amino acid sequence is not equal to the length of amino acid sequence B, the % amino acid sequence identity And amino acid sequence does not equal the % amino acid sequence identity with the amino acid sequence of A.

The term "variant of polynucleotide CD79b" or "nucleic acid sequence variant CD79b" means a nucleic acid molecule encoding the CD79b polypeptide, preferably an active CD79b polypeptide, as defined in the present description and having the 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 a signal peptide described in this application; the extracellular domain of the polypeptide CD79b, whether or not containing signal peptide described in this application; or any other sequence fragment of a full-sized CD79b polypeptide, described in this application (takagaki polypeptide, encoded by a nucleic acid that represents only part of the full sequence that encodes a full-CD79b polypeptide). Usually the option of polynucleotide CD79b has a nucleic acid sequence that is at least about 80%, and the alternative of 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 sequence of native CD79b polypeptide that does not contain a signal peptide described in this application; the extracellular domain of the polypeptide CD79b, whether or not containing a signal sequence, as described in this application; or the sequence of any other fragment of a full-sized CD79b polypeptide, described in this application. These options do not contain native nucleotide sequence.

Typically, polynucleotide variants CD79b have a length of at least about 5 nucleotides, and the alternative of 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, 53, 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" means the length of a given nucleotide sequence ±10% of the length of the sequence.

"Percent (%) identity of nucleic acid sequences, i.e. sequences of nucleic acid that encodes a CD79b identified in this application, is defined as the percentage of nucleotides in the sequence is the candidate that are identical to nucleotides in the interest nucleic acid sequence CD79b, after aligning the sequences and introducing them as "gaps", if necessary, to achieve the maximum percent identity of the sequences. The alignment is performed in order to determine the percent identity of the sequences of the nucleic acid can 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, the % identity of nucleic acid sequences is calculated using a computer program ALIGN-2, used for comparison is posledovatelnostei, where the full source code of the program ALIGN-2 is shown below in table 1. Computer program ALIGN-2, is used to compare the sequences was developed by Genentech, Inc., and the source code is presented below in table 1, was registered in the user documentation in the copyright Office of the United States, Washington, D.C., 20559, under registration number U.S. Copyright Registration no TXU510087. The program ALIGN-2 is a public program, supplied by the firm Genentech, Inc., South San Francisco, California, or it can be based on the source code presented below in table 1. The program ALIGN-2 should be compiled for use on a UNIX operating system, and preferably in the UNIX V4.0D. All the parameters of comparison sequences were installed in the program ALIGN-2 was not changed.

In the case when comparing the sequences of nucleic acid is used, the program ALIGN-2, the % identity of a given nucleic acid sequence With the nucleic acid sequence D (which alternatively can be called the sequence of nucleic acid, with or constituent specific % identity with the nucleic acid sequence D) is calculated as follows:

100 • W/Z,

where W represents the number of nucleotides to the e were evaluated, as identical when aligning sequences using the program ALIGN-2, which were compared to the 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 nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % sequence identity of the nucleic acid with the nucleic acid sequence D is not equal to the % identity of the nucleic acid sequence of nucleic acid D. If it is not specifically mentioned, all values used here % identity to the nucleic acid sequences given as described in the previous paragraph using a 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 stringent conditions of hybridization and washing, with nucleotide sequences encoding described here full-CD79b polypeptide. Variants of the CD79b polypeptide can be a polypeptide encoded by a polynucleotide variant CD79b.

The term "full-coding region", if it refers to nucleic what islote, coding the CD79b polypeptide, means a nucleotide sequence encoding a full-CD79b polypeptide according to the invention (which is localized between the start and stop codons, inclusive, as this is often illustrated in the graphic material). The term "full-coding region"when he refers to the nucleic acid deposited in the ATCC, means the part of the cDNA encoding the polypeptide CD79b and integrated into the vector deposited with the ATCC (which is localized between the start and stop codons, inclusive, as this is often illustrated in the graphic material (where the start and stop codons shown in bold and underlined)).

The term "isolated"if it refers to the various described here CD79b polypeptide, means polypeptide that has been identified and isolated and/or purified from components of its natural environment. Contaminant components of its natural environment are materials that have a negative impact on therapeutic efficacy of the polypeptide, and such components may include 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 sufficient for the introduction of at least 15 residues of N-terminal or internal part of the amino acid serial is a major, using a sequencing machine, equipped with a centrifuge vessel, or (2) to homogeneity, which can be confirmed by electrophoresis in LTO-page in reducing or non conditions with staining of Kumasi blue or, preferably, silver. The term "isolated polypeptide includes polypeptide in situ within recombinant cells, if there is at least one component of the CD79b polypeptide present in its natural environment. However, usually, the selected polypeptide may be obtained, at least in one stage of cleaning.

"Isolated" nucleic acid encoding a polypeptide CD79b, or other nucleic acid encoding a polypeptide, is a nucleic acid molecule that is identified and separated from at least one impurity molecules of nucleic acid with which it is normally associated in the natural source of the nucleic acid that encodes a polypeptide. The selected nucleic acid molecule encoding a polypeptide that has a shape or structure that is different from the form or structure of the natural molecule. Therefore, the selected nucleic acid molecule encoding a polypeptide that is different from the specific nucleic acid molecule that encodes a polypeptide and is present in natural cells. However, selected the second nucleic acid molecule, encoding a polypeptide that comprises the 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 necessary for the expression of functionally attached to the coding sequence in a particular organism, the host. Regulatory sequences that are suitable for prokaryotes, for example, are the promoter, optional operator sequence and the site of binding to the ribosome. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is functionally connected"if it is in functional relationship with another nucleic acid molecule. For example, DNA for proposedvalue or secretory leader sequence is functionally attached to the DNA of the polypeptide if it is expressed as preblock that participates in the secretion of the polypeptide; a promoter or enhancer functionally attached to the coding sequence, if they affect the transcription of the sequence and the site of binding to the ribosome is functionally attached to the coding sequence, if he is in a position conducive to the broadcast. Generally speaking, the term "functionally connected" means that the DNA sequences are related to each other are adjacent, as in the case of a secretory leader sequence they are adjacent and are in the same reading frame. However, enhancers do not have to be adjacent. Linking is performed by ligating the appropriate restriction sites. If such sites are not available, such 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 the average person skilled in the art, and typically it is calculated empirically depending on the length of the probe, the temperature of the wash and the salt concentration. Basically, the longer the probe, the higher must be the temperature of hybridization, and the shorter the probe, the lower the temperature should be. Hybridization generally depends on the ability of denatured DNA re-annealing, if the complementary chain present in the environment whose temperature is below the melting temperature. The higher the degree of desired homology between the probe and hybridizing sequence, the higher should be the relative temperature. From this it follows that bol is e high 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).

"Stiffness terms" or "terms " high rigidity"defined in the present invention, can be set as conditions, which include (1) washing at low ionic strength and high temperature, such as leaching of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) use in hybridization 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× denhardt's solution, processed by the 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 under conditions of high hardness, consisting of 0.1×SSC containing EDTA at 55°C.

"Conditions of moderate hardness can be determined as described in R. the leadership Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of less severe 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% of formamide, 5×SSC (150 mm NaCl, 15 mm triacrylate), 50 mm sodium phosphate (pH of 7.6), 5× denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured fragmented sperm DNA, salmon, followed by washing the filters in 1×SSC at about 37-50°C. If you want adaptiroval these conditions to the relevant parameters, such as probe length and the like, the temperature, ionic strength, etc. can to be adjusted by methods known to experts.

Used herein, the term "labeled epitope" means a chimeric polypeptide containing a CD79b polypeptide or anti-CD79b antibody attached to the polypeptide-label". Polypeptide-tag must have a certain number of residues sufficient to create epitope against which may be sent to the antibody, and should be short enough, i.e. such that it does not affect the activity of the polypeptide to which it is attached. In addition, preferably, such a polypeptide is the label was sufficiently unique so that the antibody is not about Adela significant cross-reactivity with other epeople. 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).

Used herein, the terms "active" or "activity" refers to form(s) of the CD79b polypeptide that retains the biological and/or immunological activity of natural or native CD79b, where the term "biological activity" refers to biological function (inhibitory or stimulatory)reported native or natural CD79b, except for the ability to induce the production of antibodies against the antigenic epitope located on native or natural CD79b, and the term "immunological activity" means the ability to induce the production of antibodies against the 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 the native polypeptide CD79b. Similarly, the term "agonist" is used here in its broadest sense and includes any molecule that partially or fully mimics the biological activity of the native polypeptide CD79b. Suitable molecules are agonists or as is tagonists, in particular, antibodies are agonists or antagonists or antibody fragments, 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 can include contacting the polypeptide with CD79b molecule agonist or antagonist, used as a candidate, and determining the detected changes in one or more biological activities normally associated with the 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.

"Isolated" 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 in the natural environment. The selected nucleic acid molecule includes a nucleic acid molecule contained in cells that normally Express this molecule of nucleic acid, but this nucleic acid molecule is present outside of the chromosome or chromosome position that is different from its natural position in the chromosome.

Used herein, the term “vector” means a nucleic acid molecule that can carry another nucleic acid to which it is attached. One type of vector is a “plasmid”, which is 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, into which additional DNA segments can be legirovanyh viral genome. Certain vectors are capable of Autonomous replicated in the cell host, in which they were entered (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 their introduction in the specified cell of the host, so that they can be replicated together with the genome of the host. Moreover, certain vectors are capable of regulating the expression of genes to which they are functionally connected. Such vectors are referred to here as “recombinant expression vectors” (or simply “recombinant vectors”). In General, expression vectors commonly used in the methods of recombinant DNA, often have the form of plasmids. In the present described and the terms “plasmid” and “vector” can be used interchangeably, because the plasmid is the most common form of the vector.

Used herein, the terms “polynucleotide” or “nucleic acid” are used interchangeably and mean a polymer of any length consisting of nucleotides, and such polymers are DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase, or by synthesis reaction. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If necessary, modification of the nucleotide structure may be performed before or after Assembly of the polymer. The nucleotide sequence may be interrupted dinucleotide components. Polynucleotide can be further modified after synthesis, for example, by conjugation with a label. Modifications of other types are, for example, “caps”, substitution of one or more natural nucleotides and their analogs; mezhnukleotidnyh modifications such as, for example, modification by introduction of uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphamidon, carbamates and the like), charged linkages (e.g., phosphothioate, phosphodithioate is in and so on); modification containing side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine and the like); modifications contain intercalating agents (e.g., acridine, psoralen, etc.); modification containing hepatoblastoma agents (e.g., metals, radioactive metals, boron, metals, oxidizing agents, and the like); modification containing alkylating agents; modifying containing modified linkages (e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotide(s). In addition, any hydroxyl group, usually present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, protected by standard protective groups; or activated with the formation of additional bonds additional nucleotides; or they can be conjugated to a solid or semi-solid media. 5'- and 3'-end IT can be phosphorylated or substituted amines or organic “kapinowski” groups of 1-20 carbon atoms. Other hydroxyl can also be derivatization 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-methyl-ribose, 2'-O-Allie is a ribose; 2'-fluoro - or 2'-isidoros; carbocyclic analogues of sugars; alpha-anomeric sugar; epimeria sugars such as arabinose, xylose or lyxose; sugar pyranose, sugar furanose; sedoheptulose; acyclic analogs and ό 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”), P(O)R, P(O)OR', CO or CH2(formatall”), where each of R or R' independently represents H or substituted or unsubstituted alkyl (C1-20), optionally containing an ether linkage (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not all communication polynucleotide must be identical. The above description applies to all used here polynucleotides, including RNA and DNA.

Used herein, the term “oligonucleotide”generally means a short, mostly single-stranded, usually synthetic polynucleotide, the length of which is, mainly, but not necessarily, less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. Vicepresidencia description of polynucleotides mo is no equally and fully to treat oligonucleotides.

Used herein, the terms “cancer” and “cancerous” refer to the physiological status of mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, cancer of the hematopoietic system or blood cancer, such as lymphoma, leukemia, myeloma or lymphoma malignant diseases as well as cancer of the spleen, cancer of the lymph nodes, carcinoma, blastoma and sarcoma. More particular examples of such cancers are b-cell cancer, including, for example, vysokokachestvennyy, srednekagesstroy and nizkolegirovannuju lymphoma (including b-cell lymphoma, such as b-cell lymphoma lymphoid tissue of the mucous membrane and non-Hodgkin's lymphoma (NHL), the lymphoma cells of the cortex of the brain, Burkitt's lymphoma, small cell lymphocytic lymphoma, lymphoma marginal zone, both diffuse lymphoma, follicular lymphoma, jackinsky lymphoma and T-cell lymphoma and leukemia (including secondary leukemia, chronic lymphocytic leukemia (CLL), such as b-cell leukemia (CD5+-b cells), 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 RA is inhibiting tumor. In the present invention also includes cancers of other hematopoietic cells, including polymorphically white blood cells, such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, red blood cells and natural killer cells. In the present invention also includes cancerous b-cell proliferative disorder selected from the following diseases, such as lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 memory cells in the marginal zone, follicular lymphoma and diffuse both 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's lymphoma of razvivaetsa centroblasts in the dark zone of the germinal centers. Fabrics that include hematopoietic cells and are called here "fabrics geopoetika cells are thymus and bone marrow and peripheral lymphoid tissues such as spleen and lymph nodes, lymphoid tissue associated with the mucous membrane, such as lymphoid tissue of the intestine, tonsil, Peyer's patches and the Appendix, and the lymphoid tissue associated with other mucosal sites, such as the lining of the bronchi. Other particular examples of such cancers are squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer and colon, carcinoma of the endometrium or uterine carcinoma, salivary glands, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, carcinoma liver, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

Used herein, the term "b-cell malignant tumor" encompasses nahodkinskuju lymphoma (NHL), including nizkolegirovannuju/follicular NHL, small cell of limp Citaro (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 cortex of the brain, lymphoma associated with AIDS and macroglobulinemia waldenstrom; nahodkinskuju lymphoma (NHL), lymphocytic predominantly Hodgkin's disease (LPBK), small cell lymphocytic lymphoma (MLL), chronic lymphocytic 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 malignancies. Such malignant diseases can be treated with antibodies against markers of b-cell surface, such as CD79b. Such diseases are treated here as diseases that can be treated by injecting antibodies against the marker At the cell surface, such as CD79b, where said treatment includes the introduction of unconjugated ("bare") antibody or antibodies, anywhereman the CSOs with a cytotoxic agent, described in this application. Such diseases are also treated here as a disease that can be treated by the methods of combination therapy, including the introduction of anti-CD79b antibody or conjugate anti-CD79b antibody-drug" according to the invention in combination with the introduction of other antibodies or other conjugate the antibody-drug", or other cytotoxic funds with radiation therapy or other therapy, carried out simultaneously 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 can be carried out simultaneously or sequentially. Anti-CD20 antibody may be "naked" antibody or conjugate antibody-drug". In the embodiment, combination therapy of anti-CD79b antibody is an antibody according to the invention, and anti-CD20 antibody is Rituxan® (rituximab).

Used herein, the term "non-Hodgkin's lymphoma or NHL" means a cancer of the lymphatic system, with the exception of Hodgkin lymphoma. In General, Hodgkin's lymphoma and non-Hodgkin lymphoma can vary the fact that in Hodgkin lymphoma cells are present reed-Sternberg and neh is jkinsey lymphoma these cells are absent. Examples of non-Hodgkin lymphoma covered used here, the term, are all lymphomas, which can be identified by the person skilled in the art (e.g., oncologist or pathologist) in accordance with known classification schemes, such as the Revised Euro-American classification scheme lymphoma (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, in particular, the lists presented on Fig, 11.58 and 11.59. More specific examples of lymphomas include, but are not limited to, relapsed or resistant to treatment NHL; border nizkozameshhennoj NHL first line; NHL stage III/IV; NHL resistant to chemotherapy; lymphoblastic leukemia and/or lymphoma, containing B-cell precursors; small cell lymphocytic lymphoma; b-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small cell lymphocytic lymphoma; b-cell prolymphocyte lymphoma; immunocytoma and/or lymphoplasmacytic lymphoma; lymphoplasmacytic lymphoma; b-cell lymphoma marginal zone lymphoma marginal zone spleen; lymphoma extranodal marginal zone MALT; nodal lymphoma marginal zone; reticuloendotheliosis; plasmacytoma and/or plasmacytoma myeloma; nizkozameshhennoj/follicular lymphoma; srednestaticheskaya/follicular NHL; lymphoma cortex; follicular-centralita lymphoma (follicular); srednestaticheskaya diffuse NHL; both diffuse b-cell lymphoma, aggressive NHL (including aggressive edge and aggressive relapsing NHL); NHL, relapsed after transplantation of autologous stem cells, or NHL, resistant to such transplantation; primary mediastinal both b-cell lymphoma; primary affeziona lymphoma; vysokokachestvennaya immunoblastic NHL; vysokokachestvennaya lymphoblastic NHL; vysokokachestvennaya small cell undifferentiated NHL; generalized NHL; Burkitt's lymphoma; both granulocyte leukemia precursor cells (peripheral); mushroom avium and/or syndrome Cesari; lymphoma of the skin (cutaneous lymphoma); both anaplastic lymphoma and angiocentricity lymphoma.

The term "disorder" is any condition that is susceptible to treatment with a substance/molecule or method according to the invention. These disorders are chronic and acute disorders or diseases including pathological conditions that create this mammal is predisposed to rassm trilemma disorder. Non-limiting examples described herein disorders, being treated, are cancers, such as malignant and benign tumors; non-leukemia and lymphoid malignancies; destruction of neurons, glial cells, astrocytes, hypothalamus and other disorders of the endocrine system, macrophages, epithelium, stroma and blastocele; inflammatory and immune disorders and other disorders associated with angiogenesis. In addition, such disorders are 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 is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

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

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

Used herein, the term “autoimmune disease” means a disease or disorder caused by the reaction produced by the body against its own tissues or organs, or co-segregation or the manifestation of these disorders or their associated state. With many of these autoimmune and inflammatory disorders can present a certain number clinical laboratory markers, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, the precipitation of the complex antigen-antibody" in the tissues, the beneficial effect of treatment with corticosteroids or immunosuppressants, and aggregates of lymphoid cells in the affected tissues. Not limited to any particular theory regarding b-cell autoimmune disease, we can say that when human autoimmune diseases, b cells demonstrate their pathogenic effects by multiple mechanisms, including the production of autoantibodies, the formation of immune complexes and activation of dendritic cells and T cells, the synthesis of cytokines that direct the release of chemokines and the appearance of ectopic focus of politogenesis. Each of timehorizon in varying degrees may participate in the development of autoimmune diseases.

"Autoimmune disease" may be organ-specific disease (i.e., the immune response is specifically directed to a system bodies, such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal tract and liver, and renal system, thyroid gland, ears, neuromuscular system, the Central nervous system and the like) or a systemic disease that can affect a system of many organs (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, and so on). Preferably such diseases are autoimmune rheumatologic disorders (such as rheumatoid arthritis, Sjogren syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia syndrome antiphospholipid antibodies and psoriatic arthritis), autoimmune diseases of the gastrointestinal tract and liver diseases (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 coeliac disease), vasculitis (such as, for example, ANCA-negative vasculitis, and ANCA-associated vasculitis, including vasculitis charge-Strauss, Wegener is egenera and microscopic polyangiitis), autoimmune neurological disorders (such as multiple sclerosis, syndrome, dancing eye, severe myasthenia gravis, neuromyelitis optic nerve, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), kidney disease (such as, for example, glomerulonephritis syndrome? and illness Berger), autoimmune skin diseases (such as, for example, psoriasis, urticaria, rash, vulgar disease, bullous pemphigoid and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, platelet thrombocytopenic purpura, purpura occurring after blood transfusion, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune diseases of the auditory pathways (such as, for example, a disease of the inner ear and hearing loss), Behcet's disease, Raynaud's syndrome, a disease associated with organ transplantation, and autoimmune diseases of the endocrine system (such as, for example, autoimmune diseases associated with diabetes, such as insulin-dependent diabetes mellitus (IDDM), Addison disease and autoimmune thyroid disease (e.g., graves ' disease and thyroiditis)). Of these diseases is more preferable are, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren syndrome, graves ' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Specific examples of other autoimmune diseases referred to in this application, which in some cases cover the above listed diseases, include, but are not limited to, arthritis (acute and chronic arthritis, rheumatoid 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, the disease still, the vertebrae arthritis, osteoarthritis, progredient chronic arthritis, arthritis deformans, 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 psoriasis of the nails, atopy including atopic diseases such as hay fever and syndrome Jobe; dermatitis, including contact dermatitis, Hranice the cue contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, urticaria, dermatitis herpetiformis, coins dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis and atopic dermatitis; coupled with the X-chromosome Hyper-IgM syndrome, allergic intraocular inflammatory 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 RS, accompanied by dysfunction of the spinal cord and organs of sight, primary progressive MS (PPRs) and relapsing-remitirse RS (RRRS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple (multiple) sclerosis, atoxicity sclerosis, neuromyelitis optic nerve (SN), inflammatory bowel disease (IBD)(for example, Crohn's disease, autoimmune gastrointestinal disorders, inflammation of the gastrointestinal tract, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenosis colitis, poly is religious colitis, necrotorous enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease; colitis, pyoderma gangrenosum, nodular erythema, primary sclerosing cholangitis, respiratory distress syndrome, including respiratory distress syndrome in adults (rdsw) or acute respiratory distress syndrome, meningitis, inflammation of the entire uveal tract or part thereof, iritis, hareidit, an autoimmune hematological disorder, graft-versus-host, angioedema, such as hereditary angioedema, lesion of cranial nerve, as in meningitis, herpes pregnant, pemphigoid pregnant, itching in the scrotum, autoimmune premature the 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 encephalitis and encephalitis with lesions of the extremities and/or brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatosis 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, immunobased the p 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 pluriglandular endocrine failure, balanitis, including plasmacytosis around balanitis, balanoposthitis, annular centrifugal erythema, ashy dermatosis, erythema multiforme, annular granuloma, brilliant zoster, sclerotic atrophic lichen, simple chronic zoster, thorn vitiligo, lichen planus, lamellar ichthyosis, epidermolizei hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, 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, disgestrotical eczema and bubble palmoplantar eczema; asthma, such as bronchial asthma and autoimmune asthma; condition caused by infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens, such as antigens of blood groups a-b-O fruit produced when b is of pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, insufficient adhesion of leukocytes; lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, vnepochechny erythematosus, discoid lupus, discoid lupus erythematosus and lupus alopecia; systemic lupus erythematosus (SLE)such as cutaneous SLE or subacute cutaneous SLE, lupus syndrome of newborns (HRV) and disseminated lupus erythematosus; juvenile diabetes (type I), including child insulin-dependent diabetes mellitus (IDDM)diabetes mellitus in adults (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large-artery disease; immune responses associated with acute delayed-type hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, Wegener, including lymphomatoid Wegener, agranulocytosis; vasculitides (including vasculitis, large blood vessels, such as polymyalgia rheumatica and giant cell arteritis diagnostics ('s arteritis, vasculitis secondary to blood vessels, such as disease Kawasaki and nodular polyarteritis/periarteritis nodosa, immunovaccine, CNS vasculitis, cutaneous vasculitis, allergic Vasco is it necrotorous vasculitis, such as fibrinoid necrotorous vasculitis and systemic necrotorous vasculitis, ANCA-negative vasculitis, ANCA-associated vasculitis, such as the syndrome charge-Strauss (SCS), Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis diagnostics, aplastic anemia, autoimmune aplastic anemia, positive anemia of Coombs, anemia diamond-Blackfan, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIG), pernicious anemia (pernicious anemia, Addison disease, true red cell anemia or aplasia (IEA), the lack of factor VIII, And hemophilia, autoimmune neutropenia, cytopenia, such as pancytopenia, leukopenia, diseases, leading to diabetes leukocytes, inflammatory Central nervous system disorders, Alzheimer's disease, Parkinson's disease, syndrome lesions multiple organs, such as secondary syndrome associated with sepsis, trauma, or hemorrhage; diseases, mediated by complex formation of the antigen-antibody”, the disease glomerular basement membranes catalyzed reaction of the antibody-antigen, antiphospholipid syndrome, narit motor nerve, allergic narit, disease/syndrome behceta, syndrome Castellana syndrome?, Raynaud's syndrome, syndrome Segren syndrome Stevens-D. the Onsong, pemphigoid or disease, such as bullous pemphigoid, cicatrizing pemphigoid (mucosa), skin pemphigoid, bladderwort vulgaris, paraneoplastic disease, leaf bladderwort, pemphigoid mucous membranes-the membranous pemphigoid and bladderwort erythematous acquired bullous bullosa, eye inflammation, preferably 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 syndrome, Reiter, burn injury caused by an autoimmune disease, pre-eclampsia, a disease of immune complexes, such as jade immune complexes, antibody-mediated jade, neirolepticalkie disorder, polyneuropathy, 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), post-transfusion purple (PTP), heparin-induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purple (ITP)including chronic or acute ITP; scleritis, takoyaki idiopathic keratoscleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidic, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidic, graves ' disease, eye disease, graves ophthalmopathy or ophthalmopathy associated with lesions of the thyroid gland), pluriglandular syndromes such as autoimmune pluriglandular syndromes, for example, type I (or syndromes pluriglandular endocrinopathies), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as myasthenic syndrome Lambert-Eaton or the syndrome of Eaton-Lambert syndrome, stiff person”, encephalomyelitis such as allergic encephalomyelitis (or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), severe myasthenia, such as severe myasthenia gravis associated with thymoma, degeneration of the cerebellum, neuromyotonia, needs to be or syndrome “dancing eyes” (LNG) and neuropathy senses, multifocal motor neuropathy systems, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupus is hepatic, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), obliterative bronchiolitis (not transmitted, unlike NSIP); Guillain-Barre syndrome, a disease Berger (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient contritione dermatitis, cirrhosis, such as primary biliary cirrhosis and pneumocytes syndrome autoimmune enteropathy, bowel disease or celiac disease, intestinal sprue (gluten enteropathy)is not curable sprue, idiopathic sprue, cryoglobulinemia, such as mixed cryoglobulinemia, amyotrophies lateral sclerosis (ABS; disease Louis Gehrig's disease (als), coronary heart disease; autoimmune ear disease such as autoimmune disease of the inner ear (CBA); autoimmune hearing loss; polyhedra, such as untreatable or recurrent polyhedra; pulmonary alveolar proteins, keratitis, such as the syndrome Kogan/recipricocity interstitial keratitis, bell's palsy, a disease/sweet syndrome, rosacea autoimmune, pain associated with shingles, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, including the monoclinic crystal is national b-cell lymphocytosis (e.g., benign monoclonal gammopathy and the monoclonal gammopathy unclear etiology, MGUS); peripheral neuropathy, paraneoplastic syndrome; “kalapati”, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and kalapati” of the CNS, autism, inflammatory myopathy, and focal or segmental or focal segmental glomerulosclerosis (USGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine failure, syndrome Schmidt, adrenalin, atrophy of the stomach, presenilny dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinizing polyneuropathy, Dressler syndrome, alopecia alopecia, total alopecia, CREST syndrome (calcinosis, phenomenon, Raynaud's disease, dysmotility of the esophagus, sclerodactyly and telangiectasia), autoimmune infertility in men and women, for example, caused by antibodies against sperm, mixed disease of connective tissue, Chagas disease, rheumatic fever, habitual miscarriage, lung disease, farmers, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, a disease of the lungs birdwatchers, allergic granulomatous of anyit, dobrock the quality of lymphocytic anghit, syndrome Alport, alveolitis such as allergic alveolitis and fibrosis alveolitis, interstitial lung disease, transfusion disease, leprosy, malaria, parasitic diseases such as leishmaniasis, typanosoma, schistosomiasis, Ascaris, aspergillosis, syndrome Santera syndrome Kaplan, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, fibrous mediastinal, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophallic, erythema lofty persistent, erythroblasts fruit, eosinophilic fasciitis, syndrome, Shulman syndrome, still's, flares, cycle, such as chronic cycle, heterochronies cycle, iridocyclitis (acute or chronic) or cycle Fuchs, purpura's disease-Seleina, infection caused by the human immunodeficiency virus (HIV), severe combined immunodeficiency (TCID), acquired immunodeficiency syndrome (AIDS), infections caused by echoviruses; sepsis (systemic syndrome associated with inflammatory response); groove toxins; pancreatitis; Tiresias; infections caused by parvovirus; infection caused by a virus; rubella virus; a syndrome that develops after vaccination; hereditary infection caused by the virus; rubella virus; infection caused by the Epstein-BA is RA; mumps, Evans syndrome, autoimmune gonadal failure, chorea of Sydenham, post-streptococcal nephritis, obliterating thromboangiitis, thyrotoxicosis, tabes, horida, giant cell rheumatica, chronic allergic pneumonitis, conjunctivitis, such as vernal allergic conjunctivitis, keratoconjunctivitis sicca, epidermal keratoconjunctivitis syndrome idiopathic nephritis, nephropathy, characterized by minimal changes in renal tissue, benign hereditary and caused by ischemia reperfusion disorders, reperfusion injury in organ transplantation, autoimmune disease of the retina, inflammation of the joints, bronchitis, chronic obstructive airway disease/lung, silicosis, atty, 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 lepromatosis erythema, idiopathic facial palsy, chronic fatigue syndrome, rheumatic fever syndrome Hamman-rich; sensorineural hearing loss, paroxysmal what I hemoglobinuria, hypogonadism, regional REIT, radiation, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia (sympathetic ophthalmic), ophthalmic newborns, narit optic nerve, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, quervain's thyroiditis, acquired atrophy of the spinal cord, non-malignant thymoma, lymphovascular limit, vitiligo, toxic shock syndrome, food poisoning, conditions caused by 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 multiple organs, diseases, mediated by complex formation of the antigen-antibody”, the disease glomerular basement membranes catalyzed reaction “antigen-antibody”, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed disease of connective tissue, nephrotic syndrome, insult, polyendocrine failure, autoimmune pluriglandular syndromes, including pluriglandular syndrome the IPA I, idiopathic hypoparathyroidism adults (VVM), cardiomyopathy such as congestive (dilated) cardiomyopathy, such as congestive cardiomyopathy, acquired bullous bullosa (PBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or negniy sinusitis, acute or chronic sinusitis; ethmoid sinusitis, frontal sinusitis, maxillary sinusitis or sphenoidal; allergic sinusitis, eosinophilic disorder such as eosinophilia, pulmonary infiltrates eosinophilia syndrome eosinophilia-myalgia syndrome Leffler, 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 Muco-cutaneous candidiasis, Bruton syndrome, transient gipogammaglobulinemia in children, the syndrome Wiskott-Aldrich syndrome ataxia-telangiectasia, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, such as chronic arthroleptis, lymphadenitis, in response to reduction of blood pressure, vascular dysfunction, tissue injury, cardiovascular ischemia, g is paralyse, 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, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with components of acute inflammation, the 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 treated here as diseases that can be treated by introducing an antibody that binds to a marker of b-cell surface, such as CD79b, and such treatment includes the introduction of unconjugated ("bare") antibody or an antibody conjugated with a cytotoxic agent, as described in this application. Such diseases are also systems which are here as disease, which can be treated by combined modality therapy, including the introduction of anti-CD79b antibody or conjugate anti-CD79b antibody-drug" according to the invention in combination with another antibody or conjugate antibody-drug", with another cytotoxic agent, as well as with radiation therapy or other treatment method carried out simultaneously or sequentially.

The terms "treatment", "therapy" or "relieving symptoms" refers to therapeutic treatment and prophylactic or preventative measures, which are aimed at preventing or slowing down (weakening) of the undesired physiological change or disorder in an individual. The individual in need of treatment, is the individual who already has a specified disorder, and the individual who has a predisposition to develop such disorders, or an individual who needs preventive measures aimed at prevention of such disorders. Treatment of cancer in an individual or mammal, in tumors which expressed the CD79b polypeptide, is considered successful if after administration of a therapeutic amount of an anti-CD79b antibodies by methods according to the invention in a given patient is observed visible and/or measurable reduction in the number of cancer cells or otsutstvie; reduction in tumor size; inhibition (that is, slowing to some extent, preferably stopping) infiltration of cancer cells into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (that is, slowing to some extent, preferably stopping) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or weakening to some extent 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 inhibit 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 to evaluate the effectiveness of treatment and positive dynamics of the disease can be easily identified using routine procedures known to the doctor. With regard to the treatment of cancer, the treatment efficiency can be estimated, for example, by determining the elapsed time to disease progression (TTP), and/or determine the speed of response (RR). Metastases can be identified through analysis at the stage of the development of tumors and by scanning the bones, and with the help of analyses on the levels of calcium and enzymes to determine the distribution of tumor in the bone. May also be undertaken scanning methods computed tomography (CT) for the detection of tumor spread in the pelvis and lymph nodes. For the detection of tumor metastases in the lungs and liver, respectively, can be made by chest x-ray and measurements of 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 for determining the progression of this disease include cytological analysis of urine under the cystoscope, monitoring the presence of blood in the urine, visualization urothelial tract by conducting ultrasound or intravenous pyelography, computed tomography (CT) and visualization method of magnetic resonance imaging (MRI). The presence of peripheral metastases can be estimated using abdominal CT, chest x-ray or radionuclide imaging of the skeleton.

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

The term "individual" 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), animals, participating in sports, animal companion (such as cats, dogs and horses), primates, mice and rats. In some embodiments of the invention specified mammal is man.

"Mammal", which may be subject to treat or reduce 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 animal companions such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferred mammal is a human.

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

Used herein, the term "media" includes pharmaceutically acceptable carriers, fillers or stabilizatio is s, which is nontoxic to the cells in which they are administered, or for mammals, which impose these carriers, excipients or stabilizers used in the doses and concentrations. In most cases, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; a polypeptide with a 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; the spirits of a number 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 may be caused or to which can be attached to the antibody according to the invention. Examples of solid phases considered in this proposal are those of the solid phase obtained frequent is a rule or entirely of glass (e.g., glass with adjustable 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 be a well analytical tablet, and in other embodiments of the invention, a solid phase is a column for purification (for example, a column for affinity chromatography). This term also includes a heterogeneous solid phase consisting of discrete particles, such as phase, described in U.S. patent No. 4275149.

"Liposome" is a small vesicles composed of lipids of different types of 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, agricultural the animals (such as cows), animals participating in sports, animal companion (such as cats, dogs and horses), primates, mice and rats. In some embodiments of the invention specified mammal is man.

The term "pharmaceutical composition" means a drug which, when it is cooking in this form provides effective biological action of the active ingredient and does not contain other components, which can be very toxic for the individual who administered the specified drug. Such a drug may be sterile.

Sterile product is aseptic, i.e. it does not contain living microorganisms and their spores.

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

The term "therapeutically effective amount" means the amount of antibodies or other drug effective to "treat" a disease or disorder in an individual or mammal. In the case of such therapeutically effective quantity of a drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to slow down to some of the second degree, and preferably stop) infiltration of cancer cells into peripheral organs; inhibit (i.e., to slow to some extent and preferably stop) the development of tumor metastasis; inhibition, to some extent, tumor growth; and/or alleviate to some extent one or more symptoms associated with cancer. Cm. the definition of "treatment". The drug, to some extent, can prevent the growth of cancer cells and/or to destroy cancer cells, and such medicinal products may be cytostatic and/or cytotoxic effect. The term "prophylactically effective amount" means the amount that, in enacting it at the right dose or within a reasonable period of time, is effective to achieve the desired prophylactic result. Because prophylactic dose is administered to the individual prior to disease development or at the early stage of its 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, especially tumor, e.g., cancer cell, either in vitro or in vivo. "Growth inhibitory amount" of an anti-CD79b antibody is sportwave for inhibiting the growth of tumor cells, can 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, especially tumor, e.g., cancer cell, either in vitro or in vivo. "Cytotoxic amount" of an anti-CD79b antibodies used for inhibiting the growth of tumor cells, can be determined empirically and in a routine way.

"CD79b-expressing cell" is a cell which expresses an endogenous or transfetsirovannyh CD79b polypeptide, or a cell surface or in Sekretareva form. "Cancer expressing CD79b" is a cancer containing cells, which have on their surface a CD79b polypeptide or produce and secrete the CD79b polypeptide. "Cancer expressing CD79b" produces, but not necessarily sufficient levels of CD79b polypeptide on the cell surface, allowing the anti-CD79b antibody to contact this polypeptide and have a therapeutic effect on cancer. In another embodiment of the invention the cancer expressing CD79b" produces and secretes, but not necessarily sufficient levels of CD79b polypeptide that allows anti-CD79b antibody-antagonist to contact this polypeptide and provide therapeutic action is a cancerous disease. In the latter case, the specified antagonist may be an antisense oligonucleotide that decreases, reduces, inhibits or prevents the production and secretion of the secreted polypeptide CD79b tumor cells. Cancer, which "sverkhekspressiya" CD79b polypeptide is a tumor with significantly higher levels of CD79b polypeptide on the cell surface, or produces and secretes such higher levels compared to non-cancer cells of the tissues of the same type. Such overexpression may be caused by gene amplification or increased level of transcription or translation. Overexpression of CD79b polypeptide can be determined by detecting or prognostic analysis by evaluating increased levels CD79b protein 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 can be obtained by the methods of recombinant DNA from the selected nucleic acid that encodes a CD79b polypeptide; FACS analysis, etc). Alternative or additionally, the levels of nucleic acids or mRNA that encodes a CD79b polypeptide, in cells can be measured by fluorescent in situ hybridization using nuclein vakalatnama probe, the corresponding 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 real-time PCR (RT-PCR). Can be also analyzed for overexpression of CD79b polypeptide by measuring the level of "shedding" 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. Methods 132:73-80 (1990)). In addition to the above analyses, the experts in this field can be carried out various analyses in vivo. For example, cells in the body of the patient can be subjected to contact with the antibody, which is labeled, but not necessarily, a detectable label, e.g. a radioactive isotope, and the binding of an antibody to a cell of the patient can be assessed, for example, by external scanning for radioactivity or by analysis of the biopsy specimen taken from the patient who was introduced this antibody.

Used herein, the term "immunoadhesin" means an antibody-like molecules which have binding specificity heterolo the ranks of the protein ("adhesin") in combination with effector functions constant domains of immunoglobulin. Its structure immunoadhesin contain the hybrid amino acid sequence with the desired binding specificity, but are not antigen-recognizing sequence and antigennegative site of antibodies (i.e., is "heterologous"), and the sequence of the constant domain of immunoglobulin. Adhesiva part of the 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 may 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.

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

Used herein, the term “cytotoxic agent” means a substance that inhibits or prevent the AET functioning of cells and/or causes destruction of cells. This term shall include radioactive isotopes (e.g.,211At,131I125I90Y186Re,188Re, 153Sm,212Bi32R and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, 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 various anti-tumor and anti-cancer agents, as described below. Other cytotoxic agents are described below. Ofwholesale means causes the 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 the mechanism of its action may be used to treat cancer. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, Vinca alkaloids spindle poisonous plants, cytotoxic/antitumor antibiotics, and hibitory topoisomerase, antibodies, photosensitizing agents and inhibitors of the kinase. Chemotherapeutics are compounds used in therapy "directional" and standard chemotherapy. Examples of chemotherapeutic agents are 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-pentacapital-[4.3.0]Nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbuta-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD and rapamycin.

Other examples of chemotherapeutic agents are oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), of imatinib mesilate (GLEEVEC®, Novartis), XL-518 (a 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 (a PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinovaya acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (does not contain cremophor), constructed on the basis of albumin composition of nanoparticles of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (aurothiomalate, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478 effect, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), infospeed (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylaniline; acetogenin (in particular, bullatacin, 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 the synthetic analogues, KW-2189 and SW-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard gas analogues, such as chlorambucil, chlornaphazine, chloroformed, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, ur the silt similar mustard gas; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, and in particular calicheamicin gamma II and calicheamicin omega II (see, e.g., 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 antibiotics-chromophores), aclacinomycin, actinomycin, astromicin, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, morphosyntactical, cyanomethaemoglobin, 2-pyrroline doxorubicin and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate 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 analogues such as fludarabine, 6-mercaptopurine, timipre and tioguanin; pyrimidine analogs, such as ancitabine, azasite is in, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane and testolactone; antiadrainergicakimi tools such as aminoglutethimide, mitotane, trilostane; means supplying the deficiency of folic acid, such as prolinnova acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid, such as mitanin and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenotomy acid; triaziquone; 2,2',2”-trihlortrietilamin; trichothecenes (in particular, the toxin T-2, verrucarin And, roridin and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine, etoposide (VP-16); ifosfamide mitoxantrone; vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of all the above compounds.

In the definition of "chemotherapeutic agent" also includes: (i) antihormone money, 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 cortex of the adrenal gland, such as, for example, 4(5)-imidazoles, aminoglutetimid, MEGASE® (acetate megestrol), AROMASIN® (exemestane, Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole, AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide and goserelin; as well as troxacitabine (nucleoside casinoby similar 1,3-dioxolane); (iv) inhibitors of protein kinases, such as MEK inhibitors (WO 2007/044515); (v) inhibitors of the lipid kinase; (vi) antism slowie oligonucleotides, in particular oligonucleotides that inhibit expression of genes in signal transduction pathways involved in proliferation of undesirable cells, such as, for example, PKC-alpha, Ralf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii)ribozymes such as inhibitors of the 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®; inhibitors of topoisomerase 1, such as LURTOTECAN®; rmRH ABARELIX®; (ix) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of all of the above 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 the antibody-drug", gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Used herein, the term “growth inhibitory agent” means a compound or composition, inhibiting growth of cells, particularly cancer cells expressing CD79b, either in vitro or in vivo. Thus, by means of inhibiting the growth of cells, can be the means of greatly syrause the percentage of cells expressing CD79b, in phase S. Examples of means, inhibiting the growth of cells, means blocking the passage of the cell cycle (in the other phase except phase S), such as a means of inducing blocking the G1 phase and M phase, using conventional means, the blocking phase Meters are vinylchloride (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Means blocking 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 publication The 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 the 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 tubulinovykh dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis of the cells.

“On carubicin” is an anthracycline antibiotic. Doxorubicin has full chemical name is (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 generic term that refers to proteins that are released by one cell population and which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and standard 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 lactogenic; tumor necrosis factor-α and-β; Millerovskiy inhibitory factor; peptide associated with mouse gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors tissues, such as NGF-β; platelet-growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like factor is the OST-I and-II; erythropoietin (EPO); the factors that induce osteogenesis; interferons such as interferon-α, -β and-γ; colony stimulating factors (CSFs)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-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and ligand it (KL). Used herein, the term “cytokine” includes proteins derived from natural sources or from recombinant cell culture and biologically active equivalents of the cytokines with the native sequence.

Used herein, the term “liner in packaging” means instructions, which are usually invested in available-for-sale packaging of therapeutic products and in which there is information concerning the indications, route of administration, dose, method of administration, contraindications and/or warnings concerning the use of such therapeutic products.

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

The terms "split inside the cells and intracellular cleavage" refers to the process of metabolism or reactions conjugate the antibody-drug" (ADC) inside the cells where under the influence of such processes or reactions rupture of covalent bonds, that is, the linker between the molecule drug (D) and antibody (Ab), which leads to the formation of free medicines, dissociating of antibodies inside the cells. Thus, atmasamyama groups ADC are intracellular metabolites.

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

The term "cytotoxic activity" means a cytotoxic, cytostatic, or growth-inhibitory effect of the ADC, or an intracellular metabolite of the ADC. Cytotoxic activity can be expressed as the value of the IC50, which represents the concentration (molar or mass per unit volume), which survives 50% of the cells.

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 not necessarily, independently substituted by one or more substituents described below. In another embodiment of the invention the alkyl moiety has 1-8 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, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1 pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-until (-CH(CH 3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)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)CH2CH3), 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)(CH2CH3)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)C(CH3)3), 1-heptyl, 1-octyl etc.

The term "alkenyl" means a straight or branched monovalent hydrocarbon radical, consisting of 2-8 carbon atoms (C2-C8at least one unsaturated bond, i.e. a carbon-carbon bond and sp2is a double bond, where alkanniny radical can be, but not necessarily, independently substituted by one or more substituent and, described in this application, and includes radicals having "CIS"and "TRANS"orientations, or alternatively, "E"and "Z"orientations. Examples include, but are not limited to, ethylenic or vinyl (-CH=CH2), allyl (-CH2CH=CH2and so on

The term "quinil" means a straight or branched monovalent hydrocarbon radical, consisting of 2-8 carbon atoms (C2-C8at least one unsaturated bond, i.e. a carbon-carbon bond and sp triple bond, where alkynylaryl radical can be, but not necessarily, 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" means a monovalent non-aromatic saturated or partially unsaturated ring having 3 to 12 carbon atoms (C3-C12as the monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycle having 7-12 atoms can be located, for example, in the form of bicyclo-[4,5]-, -[5,5]-, -[5,6]- or[6,6]system, and bicyclic carbocycle having 9 or 10 carbon atoms in the ring may be located, for example, in the form of bicyclo-[5,6]or[6,6]system, or as m is Stolovich systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonan. 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, cyclohexadienyl, cycloheptyl, cyclooctyl, cycloneii, cyclodecyl, cyclodecyl, cyclododecyl etc.

The term "aryl" means a monovalent aromatic hydrocarbon radical consisting of 6-20 carbon atoms (C6-C20and resulting from removal of one hydrogen atom from a single carbon atom of the original aromatic system. Some of the aryl group in the representative structures designated as “Ar”. Typical Allami are bicyclic radicals containing aromatic ring condensed with a saturated, partially saturated 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 and the like, Aryl groups independently substituted, but not necessarily, one or more substituents described in this application.

The terms "heterocycle", "heterocy the Lil" and "heterocyclic ring" are used interchangeably and mean 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 selected from nitrogen atoms, oxygen, phosphorus and sulfur and the rest of the atoms on the ring atoms are C, where one or more atoms on the ring of which option replaced, but not necessarily, one or more substituents described below. A heterocycle may be a monocycle having 3 to 7 members in the ring (2-6 carbon atoms and 1-4 heteroatoms selected from N, O, P and S), or Bicycle having from 7 to 10 members on 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; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), 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, dihydrofurane, tetrahydrothieno, tetrahydropyranyl, dihydropyran, tetrahydrothiopyran, piperidine, morpholine, Timo is folino, tixall, piperazinil, homopiperazine, azetidine, oxetane, titanyl, homopiperazine, oxetanyl, tepanil, oxazepines, diazepines, thiazepines, 2-pyrrolyl, 3-pyrrolyl, indolyl, 2H-pyranyl, 4H-pyranyl, dioxanes, 1,3-DIOXOLANYL, pyrazolines, dithienyl, dithiolane, dihydropyran, dehydration, dihydrofurane, pyrazolopyrimidines, imidazolidinyl, 3-azabicyclo[3.1.0]hexenyl, 3-azabicyclo[4.1.0]heptanes, azabicyclo[2.2.2]hexanal, 3H-indolyl, finalizing and N-pyridylamine. In the scope of this definition also includes Spiro-molecules. Examples of the heterocyclic group in which 2 carbon atoms in ring substituted by an oxo (=O) groups are pyrimidinones and 1,1-dioxo-thiomorpholine. Described here heterocyclic group is independently substituted, but not necessarily, 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, piramidi the sludge (including, for example, 4-hydroxypyrimidine), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolin, pyrrolyl, chinoline, ethenolysis, indolyl, benzimidazolyl, benzofuranyl, indolinyl, indazoles, indolizinyl, phthalazine, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinol, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furutani, benzofurazanyl, benzothiophene, benzothiazole, benzoxazole, hintline, honokalani, naphthyridines and properidine. Heteroaryl groups are independently substituted, but not necessarily, one or more substituents described in this application.

Heterocyclic or heteroaryl groups may be linked, where possible, through the carbon atom (connected with the carbon atom or through a nitrogen atom (bound 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 pyrazine, in 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 osiride is a, 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 isoquinoline.

Non-limiting examples linked through nitrogen heterocycles or heteroaryl are heterocycles or heteroaryl connected to 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 a isoindole, or isoindoline in position 4 of the research and in position 9 carbazole or β-carboline.

“Alkylene” represents a saturated, branched-chain or cyclic hydrocarbon moiety consisting of 1 to 18 carbon atoms and having two monovalent radical center formed by removing two hydrogen atoms from two identical or different carbon atoms of the original alkane. Typical alkionovymi radicals include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2and so on

"C1-C10alkylene" is a straight saturated hydrocarbon group of the formula -(CH2)1-10-. Examples1-C10alkylenes are methyl is h, ethylene, propylene, butylene, pentile, hexylen, reptile, octiles, Nonlin and decalin.

“Albaniles” represents an unsaturated, branched-chain or cyclic hydrocarbon moiety consisting of 2-18 carbon atoms, and having two monovalent radical center formed by removing two hydrogen atoms from two identical or different carbon atoms of the original alkene. Typical alkenylamine radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

“Akinyan” represents an unsaturated, branched-chain or cyclic hydrocarbon moiety consisting of 2-18 carbon atoms, and having two monovalent radical center formed by removing two hydrogen atoms from two identical or different carbon atoms of the original alkyne. Typical alkenylamine radicals include, but are not limited to, acetylene (-C°), propargyl (-CH2C°C-4-pentenyl (-CH2CH2CH2C°SN-).

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

where the phenyl group can be unsubstituted or may be substituted by 1-4 groups in the 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)other', -C(O)N(R')2-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 a terminal carbon atom or sp3-carbon atom, substituted 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, alkenylphenol or alkenylphenol 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 a terminal carbon atom or sp3-carbon atom, substituted heteroaryl radical. Typical heteroarylboronic groups include, but are not limited to, 2-benzimidazolylthio, 2-purolater etc. Heteroallyl group had the t from 6 to 20 carbon atoms; so, for example, alkyl part, including albanello, alkenylphenol or alkenylphenol part heteroallyl group has 1-6 carbon atoms, and the heteroaryl portion has 5 to 14 carbon atoms and 1-3 heteroatoms selected from N, O, P and S. the Heteroaryl part 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 on the ring (4-9 carbon atoms and 1-3 heteroatoms 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, which, in comparison 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; thiophosphoramide prodrugs; sulfadimethoxine prodrugs; peptideatlas prodrugs; prodrugs, modified the s D-amino acid; glycosylated prodrugs; β-lactosidase prodrugs; prodrugs containing optionally substituted phenoxyacetamide; or prodrugs containing optionally substituted phenylacetamide; 5-fortitudinous and other 5-ptoluidine prodrugs that can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivationally obtaining proletarienne forms for use in the present invention include, but are not limited to, the compounds according to the invention and the chemotherapeutic agents described above.

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

"Liposome" is a small vesicles composed of lipids of different types of phospholipids and/or surfactant that can be used to deliver the drug to a 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 covalent bond or a chain of atoms that covalently bind the antibody to the molecule drugs. In various embodiments of the invention the linker is a divalent radical, such as alkerdeel, areldil, heteroaryl, groups such as -(CR2)nO(CR2)n-, repeating units of alkyloxy (for example, polietilene, PEG, polymethylenes), alkylamino (for example, polyethylenimine, JeffamineTM), and dibasic ester and amides including succinate, succinamide, diglycolate, malonate and caproamide.

The term “chiral” refers to molecules that do not align with their mirrored by similar, while the term “achiral” refers to molecules that are aligned with their mirrored tabraani equivalent.

The term “stereoisomers” refers to compounds which have identical chemical structure but differ in the spatial arrangement of atoms or groups.

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

The term “enantiomers” means two stereoisomer of the compound, mirroring which do not align with each other.

Definitions and notation used here stereochemical terms in General are 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 of plane-polarized 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 (-) used throughout the world is to designate the sign of rotation of plane-polarized light by this connection, a (-) or l means that the connection is levogyrate. The connection to the prefix (+) or d is Pervouralsk. For a given chemical structure of these stereoisomers are identical, except that they are mirror images of each other. Specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mix of enantiomers is called a racemic mixture or a racemate, which can be formed in the case, if a chemical reaction or chemical process there is no stereoselectivity or stereospecificity. The terms “racemic mixture” and “racemate” refers to an equimolar mixture of two enantiomeric molecules that do not possess optical activity.

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

Used the first here, the term “pharmaceutically acceptable salt” means a 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, saharat, formate, benzoate, glutamate, methanesulfonate "mesilate", aconsultant, bansilalpet, p-toluensulfonate, pamoate (i.e. 1,1'-methylene-bis-(2-hydroxy-3-aftout)). The term “pharmaceutically acceptable salt” may include another molecule, such as acetate ion, succinate ion, or other counterion. This counterion can be any organic or inorganic molecule that stabilizes the charge on the source connection. In addition, pharmaceutically acceptable salt can have in its structure more than one charged atom. If many of charged atoms is part of a pharmaceutically acceptable salt, this salt, there can be many 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 for the m specialists for example, by treating the free base of an inorganic acid, such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, methanesulfonate acid, phosphoric acid, etc. or organic acid, such as acetic acid, triperoxonane 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-hydroxycitrate, such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluensulfonate acid or econsultancy acid or the like

If the connection according to the invention is an acid, the desired pharmaceutically acceptable salt may be obtained by any suitable method, for example by treating the free acid with an inorganic or organic base such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide or alkali earth metal hydroxide, or other Representative examples of suitable salts include, but are not on reicived them 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 that make up this composition, and/or with the body of a mammal, which enter the composition.

The term "MES" means an Association or complex of one or more solvent molecules and compounds according to the invention. Examples of solvents that form the solvate include, 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, which is typically used to block or protect certain functional groups reacting with another functional group on the compound. For example, "aminosidine group" represents a Deputy, attached to the amino group that blocks or protects the options is the national amino group in this connection. Suitable aminosidine groups are acetyl, TRIFLUOROACETYL, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethoxycarbonyl (Fmoc). Similarly, "hidroxizina group" means the Deputy hydroxy-group that blocks or protects the functional hydroxy-group. Suitable protective groups are acetyl and silyl. "Carboxyamide group" means the Deputy carboxypropyl that blocks or protects the functional carboxypropyl. Standard carboxyamide 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 these groups include, but are not limited to, a halide (e.g. chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluensulfonyl (tosyl), trifloromethyl (triplet) and triftormetilfullerenov.

Designation

Linker components:

MC = 6-maleimidomethyl

Vl-Cit or vc” = valine-citrulline (representative of the dipeptide in the linker, otseplena protease)

The citrulline = 2-amino-5-breedopedia 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 zisteinom antibodies)

Cytotoxic drugs:

MMAE = monomethylaniline E (molecular weight (MW) 718)

MMAF = option auristatin E (MMAE) with a phenylalanine at the C-Terminus of the medicinal product (MW 731,5)

MMAF-DMAEA = MMAF with DMAEA (diethylaminoethylamine)linked by an amide bond to a C-terminal phenylalanine (MW 801,5)

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

MMAF-NtBu = N-tert-butyl associated with the C-end of MMAF amide bond

DM1 = N(2')-deazetil-N(2')-(3-mercapto-1-oxopropyl)-maytansine

DM3 = N(2')-deazetil-N2-(4-mercapto-1-oxobutyl)-maytansine

DM4 = N(2')-deazetil-N2-(4-mercapto-4-methyl-1-oxobutyl)-maytansine

Other used herein, the abbreviations have the following meanings: AE means auristatin E, Vos means N-(tert-butoxycarbonyl), cit is citrulline, dap means dalapon, DCC means 1,3-dicyclohexylcarbodiimide, DCM (DHM) means dichloromethane, DEA means diethylamin, DEAD means diethylester oxalat, DEPC means diethylphosphoramidite, DIAD means diisopropylsalicylic, 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 dimethyl sulfoxide, doe means draftin, dov means N,N-dimethylamine, DTNB means 5,5'-dithiobis(2-nitrobenzoic acid), DTPA means diethylenetriaminopentaacetic acid, DTT means dithiothreitol, EDCl mean hydrochloride 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EEDQ means 2 ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline, ES-MS stands for mass spectrometry by elektrorazpredelenie, EtOAc means ethyl acetate, Fmoc means N-(9-fluorenylmethoxycarbonyl), means gly glycine, HATU means hexaphosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea, HOBt is 1-hydroxybenzotriazole, HPLC (ghvd) means liquid chromatography high pressure, ile means isoleucine, lys means lysine, MeCN (CH3CN) means acetonitrile, MeOH means methanol, Mtr means 4-insidefamily (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, Pnp means p-nitrophenyl, MS means the 6th is lepidosaphes, phe means L-phenylamine, PyBrop means hexaphosphate bromo-Tris-pyrrolidinone, SEC means exclusion chromatography, Su means succinimide, TFA means triperoxonane acid, TLC (TLC means thin layer chromatography, UV means ultraviolet radiation, and val denotes valine.

Free amino acid cysteine" refers to 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 “value thiol reactivity” means a quantitative measure of the reactivity of free cysteine amino acid residues. The amount of thiol reactivity represents the percentage of free cysteine amino acid residues in constructed on the basis of the cysteine antibody that reacts with the reagent, interacting with thiol, with the maximum value of this reactance is equal to 1. For example, a free cysteine amino acid in engineered on the basis of the cysteine antibody that reacts with interacting with a thiol reagent such as Biotin-maleimide reagent with 100% yield, with the formation of labeled Biotin antibodies, has a value of thiol reactive the STI, component of 1.0. Another cysteine amino acid is introduced at the same or another parent antibody that reacts with interacting with thiol reagent, with 80% yield, is the amount of thiol reactivity, which is 0.8. Another cysteine amino acid is introduced at the same or another parent antibody, which just does not react with interacting with thiol reagent, has a value of thiol reactivity, component 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 substituted by one or more cysteine residues. The parent antibody may contain native sequence or the sequence of the wild type. The parent antibody may have existing modifications of the amino acid sequence (such as additions, deletions and/or substitutions)in comparison with other native antibodies, antibody wild-type or modified forms of antibodies. The parent antibody may be directed against the pre is representing the interest of the target antigen, for example, important from the biological point of view polypeptide. 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 the method of their use for the treatment of hematopoietic tumors.

In one of its aspects the present invention relates to an antibody that binds, preferably specifically, to any of the above or below mentioned polypeptides. This antibody is, but not necessarily, a monoclonal antibody, a fragment of antibodies, including Fab, Fab'and F(ab')2 and Fv-fragment, dentice is about, a single domain antibody, a chimeric antibody, humanitariannet antibody, single-chain antibody or antibody that competitively inhibits the binding of antibodies against the CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be, but not necessarily, conjugated to a 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 other Antibodies according to the invention can be, but not necessarily, produced in the cells of SNO or bacterial cells and preferably induce death of cells, with which they are associated. Antibodies according to the invention, used for detection, can be detektirano labeled, attached to a solid carrier or the like

In one of its aspects the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of monovalent antibodies against CD79b (e.g., affinity of the antibody used as a Fab fragment against CD79b) is essentially the same as the monovalent affinity of murine antibody (e.g. affinity of a murine antibody, used as a Fab fragment against CD79b)or a chimeric antibody (e.g. affinity is ti chimeric antibodies, used as a Fab fragment against CD79b), containing 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 humanitarianlaw anti-CD79b antibody, where the affinity of monovalent antibodies against CD79b (e.g., affinity of the antibody used as a 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-fold lower affinity monovalent murine antibody (e.g. affinity of a murine antibody, used as a Fab fragment against CD79b)or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab fragment against CD79b), containing 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 humanitarianlaw anti-CD79b antibody, where the affinity of monovalent antibodies against CD79b (e.g., affinity of the antibody used as a Fab fragment against CD79b), for example, less is th least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher than the monovalent affinity of murine antibody (e.g. affinity of a murine antibody, used as a Fab fragment against CD79b)or a chimeric antibody (e.g. affinity of the chimeric antibody, used as a Fab fragment against CD79b), containing 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 one of its aspects the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of the specified anti-CD79b antibody in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is essentially the same as the affinity of a murine antibody (e.g. affinity of the antibody used as IgG against 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 comprising or essentially consisting of the specified 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 humanitarianlaw anti-CD79b and is the antibodies, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG 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-fold lower than the affinity of a murine antibody (e.g. affinity of the antibody used as IgG against 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 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 humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against 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 a murine antibody, used as IgG against CD79b)or a chimeric antibody (e.g. affinity of the chimeric antibody is 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 status is asego or essentially consisting of a specified 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 humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.4 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.4 nm ± 0,04.

In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.3 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.32 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.36 n is or higher. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.4 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is of 0.44 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.48 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.5 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.3 nm to 0.5 nm. In another aspect of the present invention relative to the tsya to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) (0.32-of 0.48 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.36 to 0.44 nm.

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

In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.1 nm or higher. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (in the example, the affinity of the antibodies used as IgG against CD79b) is 0.12 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.14 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.16 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.18 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.2 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as a gG against CD79b) is 0.22 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is of 0.24 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) 0,26 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is of 0.28 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0,30 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.1-0.3 nm. In another aspect the present invention relates what I humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.12 to 0.28 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.14-0,26 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0,16-0,24 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0,18-0,22 nm.

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

In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.4 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.5 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.6 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.7 nm or above. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) sostavljaet,3-0,7 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.4 to 0.6 nm. In another aspect the present invention relates to humanitarianlaw anti-CD79b antibody, where the affinity of antibodies against CD79b in its bivalent form (e.g., affinity of the antibody used as IgG against CD79b) is 0.5 to 0.55 nm.

In one aspect of the invention, the monovalent affinity of murine antibodies against CD79b is essentially the same as the affinity of binding of 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 murine antibodies against CD79b is essentially the same as the affinity of binding of Fab-fragment containing sequences of variable domains of antibodies derived from hybridoma deposited in the ATCC under No. HB11413 July 20, 1993, or 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 in the art, the binding affinity of the ligand to the receptor can be determined using any of the various analyses and are expressed in VI is the number of quantitative variables. Accordingly, in one embodiment of the invention the affinity of binding is expressed in the values of Kd and defines natural affinity binding (for example, with minimized effects of avidity). Generally and preferably, the binding affinity of antibodies measured in vitro, regardless of whether it is in the extracellular or cell-associated environment. As described in detail in this application, the fold difference in the affinity of binding can be quantified as the ratio of the magnitude of the affinity of monovalent binding gumanitarnogo antibodies (e.g., Fab-form) and amount of the monovalent affinity of binding of the reference/comparison of antibodies (e.g., Fab-form) (for example, mouse antibody with the donor sequence hypervariable region), where the magnitude of the affinity of binding determined in a similar situation analysis. Thus, in one embodiment of the invention fold difference in the affinity of binding is defined as the ratio of Kd gumanitarnogo antibody Fab-form and the specified reference/comparison Fab-antibodies. For example, in one embodiment of the invention, if the antibody according to the invention (A) has an affinity that is "3 times below the affinity of a reference antibody (M), then if the Kd value for A is equal to 3x, led the rank Kd for M should be 1x, and Kd for A:Kd for M" should be 3:1. Conversely, in one embodiment of the invention, if the antibody according to the invention (C) has an affinity that are 3 times stronger than the affinity of a reference antibody (R), then if the Kd value for C equal to 1x, the value of Kd for R should be 3x and Kd for:Kd for R should be 1:3. To determine the affinity of binding can be applied a number of different assays known in the art, including the assays described in this application, such as, for example, Biacore analysis, radioimmunoassays (RIA) and ELISA.

In one of its aspects the present invention relates to antibodies that bind 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 the 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 (SEQID NO: 136).

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

In one of its aspects the present invention relates to antibodies that bind 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 the 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 the Oh 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 this variant HVR sequence is modified by at least one residue sequence represented in SEQ ID NO: 131, 132, 133, 134, 135 or 136. In one embodiment of the invention HVR-L1 antibodies according to the invention contains a sequence of SEQ ID NO: 131. In one embodiment of the invention HVR-L2 antibodies according to the invention contains a sequence of SEQ ID NO: 132. In one embodiment of the invention HVR-L3 of the antibody according to the invention contains a sequence of SEQ ID NO: 133. In one embodiment of the invention HVR-H1 antibodies according to the invention contains a sequence of SEQ ID NO: 134. In one embodiment of the invention HVR-H2 antibodies according to the invention contains a sequence of SEQ ID NO: 135. In one embodiment of the invention HVR-H3 antibodies according to the invention contains a sequence of SEQ ID NO: 136. In one embodiment of the invention, the antibody according to the invention containing these sequences (in combination described in this application), is humanized or human.

Options HVR in the antibody according to the invention can have a modification of one or more residues in the HVR. In one embodiment of the invention a variant HVR-L1 contains a single substitution in the following positions: A4 (K), A9 (E or S) and A10 (A or S). In one embodiment of the invention the variant HVR-L2 contains 1-5 (1, 2, 3, 4 or 5) substitutions in any one position or in combination with, 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 a variant HVR-L3 contains 1-4 (1, 2, 3, or 4) substitutions in any one position or in combination with, the following provisions: 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 a variant HVR-H1 contains 1-7(1, 2, 3, 4, 5, 6 or 7) substitutions in any one position or in combination mark the affected 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 contains 1-3 (1, 2 or 3) substitutions in any one position or in combination with, 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(ut) representative replacement amino acid; and how it will be obvious to the person skilled in the art given the context of the present description, the validity of the substitution of other amino acids can be assessed 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 a L. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H3, where F6 is a I, F7 represents R, and F8 is a L. In 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 represents R, and F8 is a L. In one embodiment of the invention A9 in a variant HVR-L1 is a S. In the bottom of the 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 represents R, and F8 is a L.

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 this 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 an 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 is an E or N, and/or C7 represents A. In some embodiments of the invention, these antibodies also contain a consensus sequence of a frame region of a human heavy chain subgroup III. In one embodiment, the antibody consensus framework 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 frame human light chain K1. In some embodiments, these antibodies consensus placenta is the frame sequence of the human frame is light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L, and/or position 47 is an F. In one embodiment, these antibodies consensus sequence frame 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 (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, position 67 represents a 69 position and represents F, position 71 is a, A, position 73 is a T, the position of 75 represents S, position 78 represents A, and/or position 80 is a M. In some embodiments of these antibodies, these antibodies also contain a consensus sequence frame human light chain K1. In one embodiment, these antibodies consensus sequence skeleton the human skeleton is light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position in the consensus sequence frame human light chain K1) 4 is a L and/or position 47 is a F.

In one embodiment of the invention is titulo according to the invention contains a variant HVR-L2, where B3 represents R, B4 represents 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 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 represents 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 N and/or C7 represents A. In some embodiments of the invention, these antibodies also contain a consensus sequence of a frame region of a human heavy chain subgroup III. In one embodiment, these antibodies consensus sequence frame region contains a substitution at position 71, 73 and/or 78. In some embodiments, these and the 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 the frame sequence of human light chain K1. In some embodiments, these antibodies consensus sequence frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is an F. In one embodiment, these antibodies consensus sequence frame 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 (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 represents 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 frame human light chain K1. In one embodiment, these antibodies consensus sequence frame human light chain K1 contains the ENU in position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is a 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 is a 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, 132, 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 N and/or C7 represents A. In some embodiments of the invention, these antibodies also contain a consensus sequence of a frame region of a human heavy chain subgroup III. In one embodiment, these antibodies consensus sequence frame region contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 submitted the 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 frame human light chain K1. In some embodiments, these antibodies consensus sequence frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is an F. In one embodiment, these antibodies consensus sequence frame 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 (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 represents 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 frame human light chain K1. In one embodiment, these antibodies consensus sequence frame human light chain K1 contains C the exchange in position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is a F.

In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H1, where D3 represents P, D5 represents 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 represents P, D5 represents 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, 132, 133, 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 N and/or C7 represents A. In some embodiments of the invention, these antibodies also contain a consensus sequence of a frame region of a human heavy chain subgroup III. In one embodiment, these antibodies of consensus the I 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 frame human light chain K1. In some embodiments, these antibodies consensus sequence frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is an F. In one embodiment, these antibodies consensus sequence frame 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 (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 represents 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 frame human light chain K1. In one embodiment of these antibodies, consensusprevailed frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is a 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 a L. In one embodiment of the invention, the antibody according to the invention contains a variant HVR-H3, where F6 is a I, F7 represents R, and F8 is a L. In some embodiments of the invention the antibody containing the specified variant HVR-H3, also contains HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H1, where each of them 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 N and/or C7 represents A. In some embodiments of the invention, these antibodies also contain a consensus sequence of a frame region of a human heavy chain subgroup III. In one embodiment, these antibodies consensus sequence frame human heavy chain p is gruppy III contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position (consensus sequence frame human heavy chain podgruppy III) 71 represents A, 73 is a T and/or 78 is A. In a one embodiment, the antibody consensus sequence frame 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 (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 represents 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 frame human light chain K1. In some embodiments, these antibodies consensus sequence frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is an F. In one embodiment, these antibodies consensus sequence frame of the human heavy chain subgroup III contains a substitution at position 48, 6, 69, 71, 73, 75, 78 and/or 80. In some embodiments of these antibodies, position (consensus sequence frame of the human heavy chain subgroup III) 48 represents I, 67 represents A 69 represents 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 frame human light chain K1. In one embodiment, these antibodies consensus sequence frame human light chain K1 contains a substitution at position 4 and/or 47. In some embodiments of these antibodies, position (consensus sequence frame human light chain K1) 4 is a L and/or 47 is a F.

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

Used therapeutic agent in his introduction to the host preferably causes a weak immune response or do not cause an immune response in the specified owner. In one of its variants the present invention relates to the specified tool. So, e.g. the, in one of its variants, the present invention relates to humanitarianlaw antibody, which produces and/or presumably produces a humoral response in humans against mouse antibodies (HAMA) at a much lower level compared with the antibody containing the sequence of SEQ ID NO: 10 and 14 of the individual owner. In another example, the present invention relates to humanitarianlaw antibody, which produces and/or presumably produces a response in a mammal or a mammal, not a person, against mouse antibodies (HAMA). In one example the antibody according to the invention produces a response against murine antibodies to clinically acceptable level or at a lower level.

Humanitariannet antibody according to the invention may contain one or more human and/or human consensus sequences negueruela region (for example, a frame region) in the variable domain of the 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 variable domain heavy chain antibodies according to the invention contains a human who forge a consensus frame sequence, which in one embodiment of the invention is a consensus frame sequence of subgroup III. In one embodiment of the invention, the antibody according to the invention contains a variant of the consensus framework sequence of subgroups III, modified in one position of amino acid residue. For example, in one embodiment of the invention the variant consensus framework sequence of subgroups III can contain a substitution at one or more positions selected from positions 71, 73 and/or 78. In one embodiment of the invention, such substitution is R71A, N73T and/or L78A, in any combination. For example, in one embodiment of the invention frame option 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 the specified replacement is V48I, F67A, I69F, R71A, N73T and/or L78A. For example, in one embodiment of the invention frame option 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 the specified replacement is V48I, F67A, I69F, R71A, N73T, K75S, L78A and/or L80M. In one embodiment of the invention the variable domain of the light chain of the antibodies according to the invention contains a human consensus framework sequence, to the which in one embodiment of the invention is a consensus frame sequence K1. In one embodiment of the invention, the antibody according to the invention contains a variant of the consensus framework sequence K1, modified by at least one amino acid position. For example, in one embodiment of the invention the variant consensus framework sequence K1 can contain a substitution at position 4. In one embodiment of the invention the specified replacement is on m4l. For example, in one embodiment of the invention the variant consensus framework sequence K1 can contain a substitution at position 4 and/or 47. In one embodiment of the invention the specified replacement is on m4l and/or L47F.

As known in the art, and as described in more detail below, the position of the amino acid/edge amino acid that defines the hypervariable region of the antibody can 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 hybrid hypervariable position, i.e., these provisions can, presumably, be in the hypervariable region in accordance with one set of criteria, and can reside outside of the hypervariable region in accordance with a different set of criteria. One or more of such provisions may is also be available in the 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, 57-65, 93, 94, 101-102 in the variable domain of the heavy chain. In one embodiment of the invention such hybrid hypervariable provisions are one or more provisions 24-29, 35-36, 46-49, 56, and 97 in the variable domain of the light chain. In one embodiment of the invention, the antibody according to the invention contains a variant of a human consensus framework sequence of 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 the consensus framework sequence of the human subgroup III, modified in one or more 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 contains replacement 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 soda the inhabitants 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 the consensus framework sequence of human subgroup I Kappa modified in one or more 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 replacement 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 the consensus framework sequence of the human subgroup III, modified 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 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 coderit option consensus framework sequence of subgroups III, modified provisions 48, 67, 69, 71, 73, 75, 78 and/or 80. In one embodiment of the invention the specified 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 the consensus human frame sequence Kappa subgroup I, modified in positions 1, 2, 3, 4, 5 or all of the provisions of 24, 27-29, 56, and 97. In one embodiment of the invention the specified 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 framework sequence K1, modified in position 4 and/or 47. In one embodiment of the invention the specified replacement is on m4l and/or L47F.

The antibody according to the invention may contain any suitable human or a human consensus framework sequence of the light chain, provided that this antibody will possess the desired biological properties (for example, the desired binding affinity of). 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 sequence). In one embodiment of the invention, the antibody according to the invention contains at least part of the frame of the consensus sequence of the human subgroup I Kappa (or all of the sequence).

In one aspect of the invention, the antibody according to the invention contains a variable domain of the 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 aspect of the invention, the antibody according to the invention is humanitariannet anti-CD79b antibody conjugated with a cytotoxic agent. In one aspect of the invention humanitariannet anti-CD79b antibody conjugated with a cytotoxic agent that inhibits progressirovanie tumor xenografts.

In one aspect of the invention humanitariannet antibody and chimeric antibody is monovalent. In one embodiment of the invention humanitariannet and chimeric antibody contains one Fab-region associated with the Fc-region. In one embodiment of the invention reference chimeric antibody contains sequences of the variable domains, are shown in figures 7A-B (SEQ ID NO: 10) and figures 8A-B (SEQ ID NO: 14) and associated with a human Fc region. In one embodiment of the invention the 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 the antibody containing the variable domain of the heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 15 (SEQ ID NO: 164-166). In one embodiment of the invention variable domain contains a sequence of FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 15 (SEQ ID NO: 160-163). In one aspect of the invention, the antibody contains a sequence of CH1 and/or Fc presented on figure 15 (SEQ ID NO: 167, and/or 168). In one embodiment of the invention, the antibody according to the invention contains a variable domain of a heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164-166), and the sequence FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 15, SEQ ID NO: 160-163). In one embodiment of the invention antitelomerase invention contains a variable domain of the heavy chain, including the sequence of HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164-166), 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 a heavy chain consisting of the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 15, SEQ ID NO: 164-166), and the sequence FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 15, SEQ ID NO: 160-163), 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 the antibody containing the variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 15 (SEQ ID NO: 156-158). In one embodiment of the invention variable domain contains a sequence of 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 of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence 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), shown in figure 15. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising a follower of the awn HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 156-158), and the sequence CL1 (SEQ ID NO: 159), shown in figure 15. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence 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), shown in figure 15, and the sequence CL1 presented on figure 15 (SEQ ID NO: 159).

In one of its aspects the present invention relates to the antibody containing the variable domain of the heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 16 (SEQ ID NO: 183-185). In one embodiment of the invention variable domain contains a sequence of 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 contains a variable domain of a heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 183-185), and the sequence 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 a heavy chain consisting of the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 183-185), and sledovatelnot CH1 and/or Fc, presented in 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 a heavy chain consisting of the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 16, SEQ ID NO: 183-185), and the sequence 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 the antibody containing the variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 16 (SEQ ID NO: 175-177). In one embodiment of the invention variable domain contains a sequence of 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 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), shown in figure 16. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 175-177), and the sequence CL1 (SEQ ID NO: 178), shown in figure 16. In one the m variant of the invention, the antibody according to the invention contains a variable domain of light chain, including 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), shown in figure 16, and the sequence CL1 presented on figure 16 (SEQ ID NO: 178).

In one of its aspects the present invention relates to the antibody containing the variable domain of the heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 17 (SEQ ID NO: 202-204). In one embodiment of the invention variable domain contains a sequence of 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 a heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC (figure 17, SEQ ID NO: 202-204), and the sequence 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 a heavy chain consisting of the sequence of the 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, including the settlement of egovernance HVR1-HC, HVR2-HC and/or HVR3-HC (figure 17, SEQ ID NO: 202-204), and the sequence FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 17, SEQ ID NO: 198-201), 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 the antibody containing the variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 17 (SEQ ID NO: 194-196). In one embodiment of the invention variable domain contains a sequence of 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 HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 194-196), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 190-193), shown in figure 17. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 194-196), and the sequence CL1 (SEQ ID NO: 197), shown in figure 17. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 194-196), and the sequence FR1-C, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 190-193), shown in figure 17, and the sequence CL1 presented on figure 17 (SEQ ID NO: 197).

In one of its aspects the present invention relates to the antibody containing the variable domain of the heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 18 (SEQ ID NO: 221-223). In one embodiment of the invention variable domain contains a sequence of FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 18 (SEQ ID NO: 217 to 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 a heavy chain containing a sequence HVR1-HC, HVR2-HC and/or HVR3-HC (figure 18, SEQ ID NO: 221-223), and the sequence FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 18, SEQ ID NO: 217 to 220). In one embodiment of the invention, the antibody according to the invention contains a variable domain of a heavy chain consisting of the sequence of the 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 a heavy chain consisting of the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 18, SEQ ID NO: 221-223), and the sequence FR1-HC, FR2-HC, FR-HC and/or FR4-HC (figure 18, SEQ ID NO: 217 to 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 the antibody containing the variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 18 (SEQ ID NO: 213-215). In one embodiment of the invention variable domain contains a sequence of 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 HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213-215), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 209-212), shown in figure 18. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213-215), and the sequence CL1 (SEQ ID NO: 216), shown in figure 18. In one embodiment of the invention, the antibody according to the invention contains a variable domain light chain comprising the sequence HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO: 213-215), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 209-212), shown in figure 18, and the sequence CL1, before the purposes of the figure 18 (SEQ ID NO: 216).

In one aspect of the invention, the antibodies according to the invention are constructed on the basis of cysteine antibodies in which one or more amino acids of the parent antibody is 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 in its entirety are introduced in the present description by reference). Therefore, it may be constructed in any form of anti-CD79b antibodies, i.e. mutated antibody. So, for example, Fab-fragment of the parent antibody may be designed so that it was a constructed on the basis of cysteine Fab, shown here in "ThioFab". Similarly, can be constructed of the parent monoclonal antibody, representing "ThioMab". It should be noted that the mutation at one site leads to the inclusion of one cysteine residue in ThioFab and mutation in two sites leads to the inclusion of two cysteine residues in ThioMab, due to the dimeric nature of IgG antibodies. Constructed on the basis of cysteine anti-CD79b antibodies according to the invention are monoclonal antibodies, humanized or chimeric monoclonal antibodies, and antigennegative fragments of antibodies, hybrid polypeptides and analogs, which are mainly associated with cell-associirovat the governmental CD79b polypeptide. Constructed on the basis of cysteine antibody may alternatively include antibody containing cysteine described here in terms of the antibody or Fab, and this design can be obtained after constructing the sequence and/or selection of antibodies, without requiring modification of the parent antibody, where such construction is carried out by creating antibodies by the method of phage presentation and selection of such antibodies or by constructing a frame sequences and constant regions of light and/or heavy chain de novo. Constructed on the basis of cysteine antibody contains one or more free cysteine amino acids, with the amount of thiol reactivity in the range of 0.6 to 1.0; 0.7 to 1.0, or of 0.8-1.0. Free cysteine amino acid is a cysteine residue, which was introduced in the parent antibody and is not part of a disulfide bridge. Constructed on the basis of cysteine antibody can be used to attach the cytotoxic and/or imaging compounds in the cysteine through, for example, maleimide or halogenoacetyl. The nucleophilic reactivity of 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 b is the left main coronary artery, such as amino group of lysine residues or N-terminal amino group. Thiol-specific functional group in iodization and maleimide reagents may react with the amine 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 the position in the light chain numbered according to Kabat (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 Fc-region) (see Kabat et al. (1991), see above), where the constant region of light chain, presented and highlighted in figures 24A, 25A, 26A, 27A, 28, 48A and 49A, begins with the provisions 109 (numbering according to Kabat), and the constant region of the heavy chain represented and highlighted in figures 24B, 25B, 26B, 27B, 28B, 48B and 49B, begins with the provisions 118 (in accordance with the European numbering system (EU)). This position may be the position in the sequential numbering of the amino acids of a full-sized light chain or the heavy chain shown in figures 24-28, 48 and 49. In one embodiment of the invention the anti-CD79b antibody content is tons of cysteine, introduced in LC-V205C (Kabat: Val 205; number 209 on the figure 27A and means 49A Cys entered in this position). Cysteine is introduced into 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 in HC-A118C (EU number: Ala 118; Kabat 114; number 118 in figure 24B, 25B, 26B, 28B, or 48B means Cys entered in this position). Cysteine is introduced into the heavy chain shown in bold and double underlined below in figures 24B, 25B, 26B, 28B, or 48B. In one embodiment of the invention the anti-CD79b antibody contains a cysteine introduced in the Fc-S400C (EU number: Ser 400; Kabat 396; sequence number 400 on the figures 24B, 25B, 26B, 28B, or 48B means Cys entered in this position). In other embodiments of the invention, the cysteine introduced in the heavy chain (including Fc-region), is present in any one of the following provisions (in accordance with the numbering according to Kabat, and in brackets in accordance with the EU numbering): 5, 23, 84, 112, 114 (the number of EU 118), 116 (EU-120), 278 (EU 282), 371 (EU 375) or 396 (EU 400). Thus, modifications of amino acids in these positions for a parent gumanitarnogo anti-CD79b antibodies according to the invention are: V5C, A23C, A84C, S112C, A114C (EU A118C), T116C (EU T120C), V278C (EU V282C), S371C (EU S375C) or S396C (EU S400C). That is they way modifications of amino acids in these positions for a parent chimeric anti-CD79b antibody according to the invention are: Q5C, K23C, S84C, S112C, A114C (EU A118C), T116C (EU T120C), V278C (EU V282C), S371C (EU S375C) or S396C (EU S400C). Thus, modifications of amino acids in these positions for a parent CD79b antibodies against abacadabra monkeys (anti-cynoCD79b) according to the invention are: Q5C, T23C, S84C, S112C, A114C (EU A118C), T116C (EU T120C), V278C (EU V282C), S371C (EU S375C) or S396C (EU S400C). In other embodiments of the invention a cysteine introduced in the light chain, is a cysteine in any of the following provisions: (in accordance with the numbering according to Kabat): 15, 110, 114, 121, 127, 168, 205. Thus, modifications of amino acids in these positions for a parent gumanitarnogo anti-CD79b antibodies 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 antibodies according to the invention are: L15C, V110C, S114C, S121C, S127C, S168C or V205C.

In one of its aspects the present invention includes constructed on the basis of cysteine anti-CD79b antibody containing the E. one or more free cysteine amino acids, where indicated, constructed on the basis of cysteine anti-CD79b antibody binds to CD79b polypeptide, and where the specified antibody produced by the 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 the 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%, 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 the scone is trueromance on the basis of cysteine antibodies having full sequence described in this application, or amino acid sequences designed on the basis of cysteine antibodies that do not contain a signal peptide and described in this application.

In yet another aspect the present invention relates to selected constructed on the basis of cysteine anti-CD79b antibody containing the amino acid sequence that is encoded by the nucleotide sequence for hybridization with the complement of the DNA molecule that encodes (a) constructed on the basis of cysteine antibody having the full amino acid sequence described in this application, (b) amino acid sequence is constructed on the basis of cysteine antibodies that do not contain a signal peptide and described in this application, (c) extracellular domain transmembrane protein constructed on the basis of cysteine antibodies, or not containing the signal peptide, as described in this application, (d) amino acid sequence encoded by any of the sequences described here nucleic acid, or (e) any other specifically defined fragment of the full amino acid sequence is constructed on the basis of cysteine antibodies described in this application.

is their a particular aspect the present invention relates to selected constructed on the basis of cysteine anti-CD79b antibody, 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 this application. In this application also describes methods of making such antibodies, where these methods include culturing host cells containing a vector comprising the appropriate encoding nucleic acid molecule, under conditions suitable for expression of the specified constructed on the basis of cysteine antibodies, and the allocation constructed on the basis of cysteine antibodies from cell culture.

In another aspect the present invention relates to selected constructed on the basis of cysteine anti-CD79b antibody which is an antibody with deletionism transmembrane domain or inaktivirovannye transmembrane domain. In this application also describes methods of making such antibodies, where these methods include culturing host cells containing a vector comprising the appropriate encoding nucleic acid molecule, under conditions suitable for expression of the specified constructed on the basis of cysteine antibodies, and the allocation constructed on the basis of cysteine antibodies from cell culture.

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

Constructed on the basis of cysteine anti-CD79b antibody may be a monoclonal antibody, antibody fragment, chimeric antibody, humanitariannet antibody, single-chain antibody or antibody that competitively inhibits the binding of antibodies against the CD79b polypeptide with its respective antigenic epitope. Antibodies according to the invention can be, but not necessarily, conjugated to a 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 other Antibodies according to the invention can be, but not necessarily, produced in CHO cells or bacterial cells and preferably inhibit the growth or proliferation of cells, with which they are associated, or induce the death of such cells. Antibodies according to the invention, used in the diagnosis the practical purposes, can be detektirano labeled, attached to a solid carrier or the like

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

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

Constructed on the basis of cysteine antibodies according to the invention preferably retain antigennegative the ability of their 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 the cell surface receptors and other molecules on the cell surface, transmembrane proteins, signaling proteins, factors regulating survival of cells, factors that regulate proliferation of cells, molecules, associated with the development or differentiation of tissue (for example, molecules that are known or assumed to be involved in such development or differentiation), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in the formation of blood vessels, and molecules associated with angiogenesis (e.g., molecules that as it is known or assumed to be involved in TA is th angiogenesis). Tumor-associated antigen may be a factor in the differentiation of the cluster (i.e. protein CDS, including, but not limited to, CD79b). Constructed on the basis of cysteine anti-CD79b antibodies according to the invention retain antigennegative the ability of the parent analogues of the anti-CD79b antibody. Thus, constructed on the basis of cysteine anti-CD79b antibodies according to the invention have the ability to bind, preferably specifically, CD79b antigen, including isoforms beta and/or alpha-human anti-CD79b antibodies, if these antigens are expressed on the cell surface, including, but not limited to, b cells.

In one aspect of the invention, the antibodies according to the invention can be conjugated to any molecule with a tag that can be covalently linked to the antibody by means of reactive molecules, 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). Attached tag can have the following functions, namely: (i) to produce a detected signal; (ii) interact with a second label and thereby modify the detected signal from the first or vtoro the label, for example, through FRET (transfer fluorescence resonance energy); (iii) to stabilize the interaction, or to increase the affinity of binding to the antigen or ligand; (iv) affect mobility, e.g. electrophoretic mobility or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) to form an immobilized molecule and thereby modulate the affinity of the ligand, the binding of antibody to the antigen or the formation of ionic complexes.

Labeled antibodies constructed on the basis of cysteine, can be used in diagnostic assays, for example, to detect expression of interest antigen-specific cells, tissues, or serum. For use in diagnostic purposes specified antibody usually mark detectable molecule. There are various labels that can be essentially divided into groups in the following categories:

Radioisotopes (radionuclides), such as3H,11C,14C,18F,32R35S64Cu68Ga86Y99Tc111In123I124I125I131I133Xe177Lu,211At or213Bi. Radiolabelled antibodies can be used in experiments on visualization of receptor targets. The antibody can be labeled with reagents, ligands, which bind or form a chelate complex or any other complex metal radioisotope, where the specified reagent capable of reacting 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). Chelat forming ligands that can form a complex with metal ions, are DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). Radionuclides can be attached through education complex conjugates of the 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 through a reaction between aminobenzyl-DOTA with 4-maleimidomethyl acid (Fluka), activated isopropylcarbamate (Aldrich)according to the procedure described Axworthy et al. (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807). DOTA-maleimide reagents react with the free cysteine amino acid residues were constructed on the basis of cysteine antibodies and contribute to the formation of complex metal-ligand on the specified antibody (Lewis et al. (1998) Bioconj. Chem. 9:72-86). Reagents for labeling chelat forming linker, such as DOTA-NHS (mono-N-hydroxy-Succinimidyl l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraoxane sour is you), are commercially available (Macrocyclics, Dallas, TX). Visualization of receptor-target labeled with a radionuclide antibodies allows to identify 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 to radioactive metals may remain inside the cells after degradation lysosomes.

Chelate complexes with metal, suitable for labeling antibodies in experiments on visualization, 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. 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 chelates formed of rare earth metals (Hel is you, formed by europium), fluorescein 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 conjugated to antibodies by methods described, for example, the manual Current Protocols in Immunology, see above. Fluorescent dyes and fluorescent reagents tags are commercially available reagents supplied by firms Invitrogen/Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

Various label “enzyme substrate” are available or described in the literature (U.S. patent 4275149). The enzyme usually catalyzes chemical conversion 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 by a spectrophotometric method. An alternative enzyme can change the intensity of the fluorescence or chemiluminescence substrate. Methods to quantify the changes in fluorescence intensity described above. Chemiluminescent substrate undergoes electronic excitation under the influence of a chemical reaction, then it can emit light, which can then be measured (for example, on chemiluminometer), or to inform ene is the tech of the fluorescent acceptor. Examples of enzymatic labels include luciferase (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 (apase), β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactosidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides etc. Methods of conjugating enzymes to antibodies are described in 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 hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or hydrochloride 3,3',5,5'- tetramethylbenzidine (TMB));

(ii) alkaline phosphatase (apase) with a 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 enzyme-substra is”. General description can be found in U.S. patent 4275149 and 4318980.

The label can be directly conjugated with amino acid side chain, with an activated amino acid side chain, an antibody, is developed on the basis cysteine etc. So, for example, the antibody can be conjugated with Biotin and any of the labels above three broad categories can be conjugated with Avidya or streptavidin, or Vice versa. Biotin selectively binds with streptavidin, and, thus, this label may be indirectly conjugated with the antibody. Alternatively, for the implementation of indirect conjugation of the label with the polypeptide variant of such a polypeptide variant kongugiruut with a small hapten (e.g., digoxin)and one of the marks of the above-mentioned various types kongugiruut with antiJapanese polypeptide variant (e.g., antibody against digoxin). Thus, it 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 analyses on competitive binding, direct and indirect sandwich assays and analyses conducted by thus (Zola, (1987) Monoclonal Antibodies: A Manual of Technique, pp.147-158, CRC Press, Inc.).

Detected tag can be used to determine the localization, visualization and quantification of event binding or recognition. Labeled antibodies according to the invention can recognize receptors on the cell surface. Another application of detektirano labeled antibodies is the immune method of immobilization on spheres, including conjugation sphere with a fluorescently labeled antibody and detection of the fluorescent signal upon binding to ligand. Similar methods of detecting binding, for measuring and detecting interactions of the antibody with the antigen used effect of surface plasmon resonance (SPR).

Detected labels such as fluorescent dyes, 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 antibodies, preferably having the following properties, namely: (i) labeled antibody should produce a signal with a very high intensity with a low background signal, so that a small number of antibodies could be detected by highly sensitive method in cell-free or cellular analysis; and (ii) labeled antibody should be photostabilizer, so that the fluorescent signal could be detected, be observed and recorded without the use of a significant level of optical bleaching. In methods that use the binding of the labeled antibody with the cell membrane or cell surfaces, especially of live cells, the data label is preferably (iii) must have good solubility in water, which allows to achieve the effective concentration of the conjugate and high sensitivity detection, and (iv) must be non-toxic in relation to living cells in order to avoid disruption of normal metabolic processes in cells or premature cell death.

Direct quantifying the fluorescence intensity of cells and count events for fluorescent labeling, such as events binding of conjugates of the peptide-dye” with the cell surface, can be carried out on the system FMAT® 8100 HTS System (Applied Biosystems, Foster City, Calif.), which allows for automatic mixing and reading, and non-radioactive assays in 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 assays for binding to the receptor on the cell surface, tests with immune immobilization, fluorescent solid-phase assays (ELISA)analysis by resale the Oia by caspase (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis 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 the analysis of cytotoxicity. Fluorimetric method of analysis at the level of microvolumes may be used to identify positive or negative regulation under the action of molecules delivered to the cell surface (Swartzman, "A homogeneous and multiplexed 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 a variety of methods and technologies of Biomedicine and molecular imaging, such as (i) MRI imaging using magnetic resonance method); (ii) MicroCT (computed 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) an ultrasound examination. Immunoscintigraphy is an imaging technique in which antibodies labeled with radioactive material is administered to an animal or person, and the image determine the localization of antibodies in the animal or human body (U.S. patent 6528624). Visualisers the biomarkers can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic treatment. Biological markers can be markers of several types: tokens of type 0, which are natural long-known markers of this disease and linearly correlated with known clinical parameters, 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 a therapeutic treatment in accordance with the current mechanism, even if this mechanism may not be associated with clinical outcome; markers of type II, which act as “surrogate endpoints, where changes in the biomarker or change of a signal from a biomarker that can predict 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) treatment data concerning (i) the expression of the protein target, (ii) to bind a therapeutic agent to a protein target, i.e. selectivity, and (iii) the clearance and half-life; and pharmacokinetic data. The advantages of in vivo visualisers the x biomarkers compared with laboratory 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 analyses, that is, at different points in time, and the possibility of applying the results obtained in preclinical studies (for small animals), for clinical research (for a man). In some cases, biological visualization allows us not to conduct a series of experiments or to minimize the number of experiments on animals in preclinical studies.

Methods for 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) Bioconjugate Chem. 13:110-115; Mier et al. (2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labeled with two molecules of the fluorescent reporter and the quencher is in close proximity to each friend is, participate in the transfer of resonant energy fluorescence (FRET). Reporter groups are usually fluorescent dyes that are excited by light at specific wavelengths and transfer energy to the acceptor or quencher, that is, the group with the corresponding Stokes shift, providing radiation with maximum brightness. Fluorescent dyes are molecules with more explicit aromaticity, such as fluorescein and rhodamine and their derivatives. Fluorescent reporter may be partially or largely suppressed molecule quencher in an intact peptide. After cleavage of the peptide by peptidase or protease may be detective increase in 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 way labeled antibody immobilized on a solid phase, such as resin Sephadex or filter paper, using methods well known in the art. The immobilized antibody is subjected to contact with a sample 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 p is soedinyaet to the immobilized polypeptide variant. Finally, the carrier is washed with another suitable solvent, such as glycine buffer, pH 5.0, which releases the antigen polypeptide variant.

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

Representative reactive functional group is N-hydroxysuccinimidyl (NHS), in which carboxypropyl substituted detectable label, such as Biotin or a fluorescent dye. The NHS-ester of the specified labels can be pre-fetched, selected, cleaned and/or okharakterizovan, or it can be formed in situ and subjected to a reaction with the nucleophilic group of an antibody. Typically, the carboxyl form of the label act is Virut by the reaction of interaction with a specific combination of a carbodiimide reagent, for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, or uronium reagent, for example, TSTU (tetrafluoroborate O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea), HBTU (hexaflurophosphate (O-benzotriazol-1-yl)-N,N,N, N'-tetramethylurea) or HATU (hexaphosphate O-(7-asobancaria-l-yl)-N,N,N',N'-tetramethylurea); and an activator such as 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide, receiving NHS-ester label. In some cases, the label and the antibody can be connected by in situ activation of the label and the interaction with the antibody getting conjugate label-antibody” in one stage. Other activating and linking reagents are TBTU (hexaphosphate 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium), TFFH (2-forexceptional N,N',N",N'"-tetramethylurea), PyBOP (hexaphosphate benzotriazol-1-yl-oxy-Tris-pyrrolidinone), EEDQ (2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (l-(mesitylene-2-sulfonyl)-3-nitro-lH-l,2,4-triazole) and arylsulfonate, for example, triisopropylbenzenesulfonyl.

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

In one aspect of the invention, the antibody according to the invention attached to the albumin-binding protein. Binding to plasma protein can be an effective tool for the improvement of pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Peptides that bind to albumin serum (ATS), can alter the pharmacodynamics of hybrid proteins containing the active domains, for example to modify the absorptive capacity of the fabric, its permeability and diffusion. These pharmacodynamic parameters can be modulated by specific selection of the appropriate sequence of peptide binding to serum albumin (application for U.S. patent 2004/0001827). The number of albumin-binding peptides was identified by a screening method of phage display (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 in (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 NO: 9-22; and (iii) WO 01/45746, page 12-13: entered in the present description by reference. Albumin-binding (ABP)-Fab were constructed by attaching the albumin-binding peptide to the C-end of the heavy chain Fab in the stoichiometric ratio 1:1 (1 ABP/1 Fab). It was shown that the binding of these ABP-Fab with albumin increases the half-life of antibodies in rabbits and mice more than 25 times. Therefore, the above-described reactive Cys residues can be entered into this the ABP-Fab and used for site-specific conjugation with a cytotoxic drugs with subsequent 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

The conjugates of the antibody-drug"

In another aspect the present invention relates to immunoconjugates or conjugates of the antibody-drug" (ADC)containing antibody conjugated 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 applying such immunoconjugates. In one aspect of the invention immunoconjugate contains any of the above anti-CD79b antibody, covalently linked to a cytotoxic agent or detektivami 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 a different antibody against CD79b. In another embodiment of the invention the STI-CD79b antibody according to the invention binds to the same epitope on CD79b, associated with Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC under number HB11413 July 20, 1993, monoclonal antibody containing the variable domains of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or chimeric antibodies containing a variable domain antibodies derived from hybridomas HB11413 deposited in the ATCC on 20 July 1993, and the constant domains from IgG1, or the variable domains of monoclonal antibodies, comprising sequences of SEQ ID NO: 10 (figures 7A-B and SEQ ID NO: 14 (figures 8A-B). In another embodiment of the invention the 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 the anti-CD79b antibody according to the invention binds to an epitope on CD79b, different from the epitope is associated with other anti-CD79b antibody. In another embodiment of the invention the anti-CD79b antibody according to the invention binds to an epitope on CD79b, different from 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 antibody containing the variable domains of the EQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or chimeric antibodies containing a variable domain antibodies derived from hybridomas HB11413 deposited in the ATCC on 20 July 1993, and the constant domains from IgG1, or the variable domains of monoclonal antibodies, comprising sequences of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another embodiment of the invention the anti-CD79b antibody according to the invention binds to an epitope on CD79b, different from 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 the anti-CD79b antibody according to the invention differs from (i.e. is not them) Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC under number HB11413 July 20, 1993, monoclonal antibody containing the variable domains of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B), or chimeric antibodies containing a variable domain antibodies derived from hybridomas HB11413 deposited in the ATCC on 20 July 1993, and the constant domains from IgG1, or variable domains monoclonal antibodies, comprising sequences of SEQ ID NO: 10 (figures 7A-B) and SEQ ID NO: 14 (figures 8A-B). In another aspect of the invention the anti-CD79b antibody according to the invention differs from (i.e. is not them) Fab-is ragment 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), 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 does not specifically binds to CD79b animal of another species. In one embodiment of the invention the animal of the first species is human and/or Primate (e.g., abacadabra monkey), and the animal of the second species is an animal of a family of murine (e.g. a mouse) and/or the animal 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, abacadabra monkey. In one embodiment of the invention the animal of the second species is an animal of the family of the mouse, such as a mouse. In one embodiment of the invention the animal of the second species is the animal 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 invented the Y.

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 a 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 can be used in cell-owners 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, for example mammalian cells, such as cells of the Chinese hamster ovary (Cho).

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

In one of its aspects the present invention relates to industrial isdel the Yu, with a container and a composition contained in the container where the specified composition comprises one or more anti-CD79b antibodies according to the invention. In one embodiment of the invention, this composition contains a nucleic acid according to the invention. In one embodiment of the invention a composition comprising the antibody also contains the media that, in some embodiments of the invention that is pharmaceutically acceptable. In one embodiment of the invention an industrial product according to the invention also contains instructions for the introduction of the composition (e.g., antibodies) to the 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. In one embodiment of the invention a composition comprising the antibody antagonist, also includes the media, which, in some embodiments of the invention that is pharmaceutically acceptable. In one embodiment of the invention, the kit also contains instructions for the introduction of the composition (e.g., antibodies) to the individual.

In one of its aspects the present invention is applied to the Yu anti-CD79b antibodies according to the invention for the preparation of a medicinal product for therapeutic and/or preventive treatment of disease, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) limphoma 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 medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) 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 medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed basyntan the th NHL untreatable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or cell-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor and/or CL is for sure-proliferative disorder. In one embodiment of the invention, a cancer, a tumor and/or cell proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 the method includes contacting these cells with the antibody according to the invention, leading to growth inhibition of these cells. In one embodiment of the invention the antibody is conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a 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 containing cells expressing CD79b, where the method includes the introduction of the specified mammal a therapeutically effective amount of the antibody according to the invention, and thus effective treatment specified is recapitalise. In one embodiment of the invention, the specified antibody conjugated with zitotoksicskimi means. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of treating or preventing cell proliferative disorders associated with an increased level of expression of CD79b, where the method includes the administration to an individual in need of such treatment, an effective amount of the antibody according to the invention, thereby effectively treating or preventing a specified cell-proliferative disorders. In one embodiment of the invention the specified cell-proliferative disorder is cancer. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of inhibiting the growth of cells whose growth is, at least partially, depends on the growth-potentiating actions CD79b, where the method includes contacting these cells with an effective amount of the antibody according to the invention and thereby inhibit growth of these cells. In one embodiment, and is gaining the specified antibody conjugated with zitotoksicskimi means. In one embodiment of the invention, the specified antibody conjugated to a 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, at least partially, depends on the growth-potentiating actions CD79b, where the method includes contacting these cells with an effective amount of the antibody according to the invention and thus effective treatment of the indicated tumor. In one embodiment of the invention, the specified antibody conjugated with zitotoksicskimi means. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

In one of its aspects the present invention relates to a method of treatment of cancer, comprising the administration to a patient the pharmaceutical compositions containing the described here immunoconjugate acceptable diluent, carrier or filler. In one embodiment of the invention specified cancer is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES)astropolitical leukemia (ALL) and lymphoma cells of the cerebral cortex. In one embodiment of the invention, the patient is given a cytotoxic agent in combination with the compound-conjugate antibody-drug".

In one of its aspects the present invention relates to a method of inhibiting proliferation of b cells, comprising treating the cells with 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 the proliferation of b-cells, selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 of containing CD79b, where the method includes processing the specified is brazza antibody according to the invention and determining the level of binding of the indicated antibody to CD79b in the specified sample, where the level of binding of the indicated antibody to CD79b in the 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 collected from a mammal suffering from or suspected of suffering from b-cell disorder and/or b-cell-proliferative disorder, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, recurrent asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 cell proliferative disorder associated with increased numbers of cells, such as b cells expressing CD79b, where the method includes contacting the test cells in the biological sample with any of the above antibodies; determining the level of antibodies bound to test the alignment of the cells in the sample, by detecting binding of an antibody to CD79b; and comparing the level of antibodies bound to cells in a control sample, where the level of bound antibody normalize by the number of CD79b-expressing cells in the test and control samples, and where a higher level of bound antibody in the test sample compared to a control sample, indicates the presence of cell proliferative disorders associated with cells expressing CD79b.

In one of its aspects the present invention relates to a method of detecting soluble CD79b in blood or serum, where the method includes contacting the test sample of blood or serum obtained from a mammal, preferably suffering from b-cell-proliferative disorder, an anti-CD79b antibody according to the invention, and the detection of increased levels of soluble CD79b in the test sample compared to a control sample of blood or serum from a healthy mammal. In one embodiment of the invention, this 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 STRs is also binding antibodies according to the invention with the cell, expressing CD79b, where the method includes contacting the specified cell with the antibody according to the invention. In one embodiment of the invention, the specified antibody conjugated with a cytotoxic agent. In one embodiment of the invention, the specified antibody conjugated to a growth inhibitory agent.

The methods according to the invention can be applied to any suitable treatment of a pathological condition, such as a condition in which cells and/or tissues Express CD79b. In one embodiment of the invention, in the method according to the invention, the cell-target is a hematopoietic cell. For example, hematopoietic cell may be a cell selected from the group consisting of lymphocytes, leukocytes, platelets, red blood cells 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 can be cells selected from the group consisting of lymphoma, leukemia or myeloma.

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

As described in this application, CD79b is a signal component of the b-cell receptor. Accordingly, in one embodiment of the methods according to the invention specified by the cell-target (e.g., a cancer cell) is the cell in which is expressed CD79b, compared to a cell which is not expressed CD79b. In another embodiment of the invention specified by the cell-target is a cancer cell, in which there is an increased level of expression of CD79b, compared to normal non-cancerous 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 aspects the present invention relates to vectors containing DNA encoding any of the antibodies described here. The present invention also relates to cells of the host containing any such vector. So, for example, cells of the host may be CHO cells, E. coli cells, or yeast cells. The present invention also relates to a method for producing any of the antibodies described here, where the method includes culturing the host cells under conditions suitable for expression of the desired antibody, and separation of the desired antibody from the cell culture.

Even one who m his aspect the present invention relates to the composition, contains described here anti-CD79b antibody in combination with a carrier. The specified media is optional pharmaceutically acceptable carrier.

In another aspect the present invention relates to the use described herein antibodies against the CD79b polypeptide for the preparation of a medicinal product for the treatment of a condition that is susceptible to antibody against CD79b polypeptide.

Another aspect of the present invention is a composition comprising a mixture of compounds "antibody-drug 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, a mixture of compounds of the ADC of formula I or their pharmaceutically acceptable salt, or a solvate, pharmaceutically acceptable diluent, carrier or filler.

In another aspect the present invention relates to pharmaceutical combinations containing 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 MC is connected, the method includes treating the cells with a conjugate of the antibody-drug formula I or its pharmaceutically acceptable salt or MES in number, effective for preventing or inhibiting the proliferation of tumor or cancer cells.

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

In another aspect the present invention relates to industrial products, i.e. sets containing the conjugate antibody-drug", the container and the liner into the container or label with instructions for treatment.

In one of its aspects the present invention relates to a method for producing the compound-conjugate "antibody-drug formula I, where the method involves the following stages: (a) a reaction between cysteine group entered into constructed on the basis of cysteine antibody with a linker reagent, with the formation of intermediate compounds of the antibody-linker Ab-L; and (b) a reaction between Ab-L with activated molecule drugs D with the formation of conjugate "antibody-drug"; or stages: (c) a reaction between the nucleophilic group of the molecule drugs with linker reagent with the formation of intermediate compounds "drug-linker D-L; and (d) the reaction between D-L-cysteine gr is POI, introduced in constructed on the basis of cysteine antibody with the formation of conjugate "antibody-drug".

In one of its aspects the present invention relates to the analysis for the detection of cancer cells, comprising: (a) treating the cells with the conjugate constructed on the basis of cysteine anti-CD79b antibody-drug", and (b) determining the degree of binding of the compound-conjugate "is constructed on the basis of cysteine anti-CD79b antibody-drug" with these cells.

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. Pavlova.) injections of the relevant antigen and an adjuvant. They can be used for conjugation of the corresponding antigen (in particular, if you use synthetic peptides) with a protein which is immunogenic for species subjected to immunization. So, for example, the antigen may be anywhereman with hemocyanin lymph snails (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidophenylmethacrylates (conjugated through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic acid anhydride, SOCl2or R1N=C=NR, where R and R1represent different alkyl groups.

Animals subjected to immunization with the antigen, 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's adjuvant and by subcutaneous injection of a solution in many areas. A month later the animals repeatedly subjected to immunization in many areas by subcutaneous injection 1/5-1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant. After 7-14 days in animals blood sample and analyze the serum for antibody titer. Then animals again subjected to immunization before reaching the plateau of the title. The conjugates can also be obtained in recombinant cell culture as hybrid proteins. In addition, to enhance the immune response can also be used aggregating agents such as alum.

2. Monoclonal antibodies

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

To obtain a hybrid, mouse or other suitable animal host, such as a hamster, subjected to immunization, as described above, for generating them lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternative 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 the formation hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

Thus obtained hybridoma cells were seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival Nikitich parental myeloma cells (also called "merger partner"). For example, if the parental myeloma cells do not contain enzyme gipoksantin-guanine-phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium to obtain hybridomas typically includes gipoksantin, aminopterin and thymidine environment (NAT), that is, substances that prevent the growth of HGPRT-deficient CL is current.

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

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

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

After identification of hybridoma cells that produce antibodies with the desired specificity, affinity and/or activity, the clones may be subcloned by procedures limiting dilution and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media designed to achieve this purpose are, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells may be grown in vivo as ascitic tumors in animals, for example, by I.P. Pavlova. injection of these cells in mice.

Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascitic fluid, or serum by standard procedures purification of antibodies, such as, for example, affinity chromatography (e.g., protein a or G-protein-sepharose), or ionoobmennaya chromatography, chromatography on hydroxiapatite, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies can be readily isolated and sequenced according to standard procedures (e.g., by using oligonucleotide probes, to the which can specifically bind with genes, encoding the heavy and light chains of murine antibodies). A preferred source of such DNA are hybridoma cells. After selecting this DNA can be incorporated into expression vectors, which are then transferout in cell host such as E.coli cells, simian COS cells, cells of the Chinese hamster ovary (Cho or myeloma cells that in other cases not produce protein antibodies, resulting in these recombinant cells masters synthesized monoclonal antibodies. Discussion recombinant expression of the 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 antibody fragments 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 selection of mouse and human antibodies, respectively, using phage libraries. In more recent publications describe the production of high affinity (nm) human antibodies by replacing genes chains antibody (Marks et al. Bio/Technology, 10:779-783 (1992)), and by combined infection and recombination in vivo used as a strategy for constructing very large the x phage libraries (Waterhouse et al. Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these methods are an acceptable alternative to the traditional hybridoma technology for obtaining monoclonal antibodies used for isolation of monoclonal antibodies.

DNA encoding the antibody can be modified for the production of polypeptides of chimeric or hybrid antibodies, for 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 of the entire coding sequence (or portion 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 antigennegative site of an antibody to create a chimeric bivalent antibody containing one antigennegative site having specificity for a single antigen, and the other antigennegative site having specificity for a different antigen.

3. Human and humanized antibodies

Anti-CD79b antibodies according to the invention can which also include humanized antibodies or human antibodies. Humanized forms of nonhuman antibodies (e.g., murine) antibodies are chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigennegative subsequences of antibodies)which contain minimal sequence derived from nonhuman immunoglobulin. Humanitarianism antibodies are human immunoglobulins (recipient antibody)in which residues, derived from the hypervariable region (CDR) of the antibody-recipient, replaced by residues derived from the hypervariable region (CDR) nonhuman antibodies (donor antibody)such as mouse antibody, rat antibody, or rabbit antibody with 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 may also comprise residues that are not found in the recipient antibody or in the "import" the sequences of the CDR or framework region. In General, humanitariannet antibody can contain basically all or at least one, and typically two, variable domain, in which all or nearly all areas of the CDRs correspond to nechelovek the ski immunoglobulin, and all or nearly all FR represent the FR of a human immunoglobulin consensus sequence. Humanitariannet antibody also includes, but not necessarily, at least a portion of the constant region of immunoglobulin (Fc), typically a human immunoglobulin [Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1998) Nature 332:323-329 and Presta Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods of humanizing the inhuman antibodies are well known in the art. Generally speaking, humanitariannet antibody has one or more amino acid residues introduced into it from a source that is 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 employees (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)], by replacing sequences of 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 to the variable domain of the intact human antibody are replaced by the corresponding sequence from the wick is human antibodies. In fact, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues, substituted residues, derived from similar areas of rodent antibodies.

When creating humanized antibodies, in order to reduce the antigenicity and NAMA-response generating human artemisinin antibodies) in the use of this antibody for therapeutic treatment of humans, it is very important to choose the variable domains of both light and heavy chains of human antibodies. Reducing NAMA-response or predotvrasenie 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 were Humanitary in order to reduce or prevent HAMA response. Variants of these antibodies can also be obtained routine methods known in the art, some of which are described in detail below. In accordance with the so-called method of “fit”, the sequence of the variable domain of the antibody of the rodent with miniroot entire library of known sequences of the variable domains of a human antibody. Then identify the sequence of the V-domain of a human antibody, which is the most similar to the sequence of rodents, and take as the human framework region (FR) for creating gumanitarnogo antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). In another method uses a particular framework region derived from a consensus sequence of all human antibodies of a particular subgroup of light and 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 source (parent) sequence diversification sequence(s) of the frame region and/or a hypervariable region. The selected frame sequence, to which is attached the original hypervariable sequence, called here the acceptor human frame sequence. Acceptor human framework sequences can be obtained, or can occur from human immunoglobulin (areas VL and/or VH), preferably human acceptor framework sequence mo the ut to be obtained or can occur from a human consensus framework sequence, since such a frame sequence, as has been demonstrated, have minimal immunogenicity, or do not have immunogenicity in humans.

If the acceptor is derived from a human immunoglobulin, 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, available in the collection of the human frame sequences, and selection of the most homologous frame sequence as acceptor.

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

Thus, the acceptor human frame the 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, X2represents T or N, and X3represents A or L,

FR4 containing WGQGTLVTVSS (SEQ ID NO: 146).

Examples consensusseeking areas VH, are:

consensus framework region of human VH subgroup I minus CDR according to Kabat (SEQ ID NO: 108);

consensus framework region of human VH subgroup I minus extra long hypervariable region (SEQ ID nos: 109-111);

consensus framework region of human VH subgroup II minus CDR according to Kabat (SEQ ID NO: 112);

consensus framework region of human VH subgroup II minus extra long hypervariable region (SEQ ID NO: 113-115);

consensus framework region of human VH subgroup III minus CDR according to Kabat (SEQ ID NO: 116);

consensus framework region of human VH subgroup III minus extra long hypervariable region (SEQ ID nos: 117-119);

the acceptor framework region of human VH minus CDR according to Kabat (SEQ ID NO: 120);

the acceptor framework region of human VH minus extra long hypervariable region (SEQ ID NO: 121-122);

the acceptor framework region 2 human VH minus CDR according to Kabat (SEQ ID NO: 123); or

the acceptor framework region 2 human VH minus extra long hypervariable region (SEQ ID NO: 124-126).

In one embodiment of the invention the acceptor framework region of human VH contains 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).

The acceptor framework region of human VL may 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:

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

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

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

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

Although the sequence of the acceptor can be identical to the selected human framework sequence, regardless of whether it is from a human immunoglobulin or human consensus framework sequence, however, in the present invention is considered acceptor sequence, which, compared with the human sequence immunoglobulines human consensus frame sequence, may contain existing amino acid replacement. These existing replacement, compared with a human immunoglobulin sequence or consensus of the frame sequence, preferably are minimal, and usually differ only on four, three, two amino acid residue or one amino acid residue.

The remains of the hypervariable regions of human antibodies injected into the acceptor framework regions of human VL and/or VH. So, for example, can be included residues corresponding to residues of the CDR according to Kabat, the remnants of the hypervariable loops of CATIA, the remnants of the Abm and/or the contacting 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 of the hypervariable region is discussed in the description of the present invention, however, it should be noted that this introduction can be carried out by various methods, for example, nucleic acid encoding the desired amino acid sequence can be obtained by introducing mutations in the nucleic acid encoding the sequence of the murine variable domain, followed by the replacement frame residue remains on the acceptor human framework region, the sludge is by making mutations in the nucleic acid, the coding sequence of the human variable domain, followed by the replacement of residues of the hypervariable domain on the remains of nonhuman antibodies, or by synthesis of nucleic acid encoding the desired sequence, etc.

In the here described examples options with attached hypervariable region were obtained by the method of Kunkel mutagenesis nucleic acid that encodes a 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 into the frame and/or a hypervariable region of routine methods for correction and recovery of the desired interactions "hypervariable region-antigen".

Phage (famine) representation (also referred to here in some cases rahovym display) can be used as a convenient and rapid method for the production and screening of many different potential options antibodies in the library, obtained by randomization of the sequences. However, experts and other known methods of obtaining and screening of modified antibodies.

The technology of phage (fahmideh) represent an effective means for the production and selection of new proteins that bind to the ligand,such as an antigen. The technology of phage (fahmideh) display enables you to get large libraries of protein variants that can be quickly sorted sequences that are associated with the molecule-target with high affinity. Nucleic acids encoding polypeptide variants, usually attached to a nucleic acid sequence that encodes a viral envelope protein, such as protein encoded by gene III or protein encoded by gene VIII. Were developed monovalent system fahmideh view, in which the sequence of a nucleic acid encoding a protein or polypeptide attached to a nucleic acid sequence that encodes a portion of the protein encoded by gene III (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent system fahmideh view, the hybrid gene is expressed at a low level, and protein gene III wild type expressed so that the infectivity of the particles is maintained. Methods of obtaining peptide libraries and screening these libraries are described in many patents (for example, in 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 antigenspecific polypeptides were obtained by different methods, including modification of one the ene by embedding randomized DNA sequences or the cloning of a family of related genes. Methods antibodies or antigenspecific fragments using phage display technology resulting in described in U.S. patent№№ 5750373, 5733743, 5837242, 5969108, 6172197, 5580717 and 5658727. Then the library sceneroot for the presence of antibodies or antigenspecific proteins with desired properties.

Methods replace the selected amino acids at the level of the matrix nucleic acids are well known in the art, and some of them are described in this application. For example, the remains of the hypervariable region may be replaced by a method of Kunkele. 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 triplets are used to encode the desired amino acid variants. Sets of codons can be represented by the symbols designating a specific nucleotides or equimolar mixture of the nucleotides 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 Kodo is s DVK D can represent the nucleotides A or G or T; V can be a A or G or C, and K can be a G or T. This set of codons can 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. The set of oligonucleotides can be synthesized, for example, by the method of 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 the "degeneracy" of selected nucleotides at certain positions. These sets of nucleotides that have specific sets of codons, can be synthesized on commercially available nucleic acid synthesizers (supplied, for example, Applied Biosystems, Foster City, CA), or they can be purchased from suppliers (for example, from Life Technologies, Rockville, MD). Therefore, the set of synthesized oligonucleotides with a specific set of codons, usually includes a lot of oligonucleotides with different sequences, different set of codons in the full sequence. The oligonucleotides used in accordance with the present invention have the sequence, p is permitted to hybridisierung matrix nucleic acid of the variable domain, and may include restriction sites for cloning.

In one of these methods, the 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. Nucleic Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid sequence encoding variants of amino acids, is produced by hybridizing set of oligonucleotides, containing the sets of codons, with a matrix of DNA, where the specified matrix is a single-stranded form of a plasmid containing the sequence matrix nucleic acid variable region. After hybridization for the synthesis of full length second complementary chain matrix using DNA polymerase to incorporate the oligonucleotide primer, and such a circuit will contain sets of codons, provide a set of oligonucleotides.

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

DNA-matrix is obtained using vectors, vectors derived from bacteriophage M13 (suitable are 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, DNA is mutated, can be integrated into one of these vectors to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 manual Sambrook et al., see above.

For modification of the native DNA sequence of the oligonucleotide hybridized stranded matrix in appropriate hybridization conditions. Then, for the synthesis of a complementary chain of the matrix is carried out using the oligonucleotide as a primer, add DNA polimerizuet enzyme, usually DNA polymerase T7 or piece maple DNA polymerase I. the result is heteroduplex molecule, in which one strand of DNA encodes the mutated form of the gene 1, and the other strand (the original matrix) encodes the native unmodified sequence of gene 1. Then this heteroduplex molecule is transferred into a suitable cell host, usually in prokaryotes, such as E. coli JM101. After culturing cells ivysaur plates with agarose and sceneroot using oligonucleotide primer, radioactively labeled 32-phosphate in order to identify the bacterial colonies that contain the mutated DNA.

The just-described method may be modified in order to create homoduplexes molecules in which both strands of the plasmid containing the mutation(s). Such modification carried out as follows: single-stranded oligonucleotide hybridized stranded matrix, as described above. A mixture of three deoxyribonucleotides, namely desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTT), together with a modified timezonebias denoted dCTP-(aS) (which can be obtained from Amersham). This mixture was added to the complex matrix-oligonucleotide". After adding DNA polymerase to this mixture there is a chain of DNA that is 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 protect DNA from cleavage restricteduse the endonuclease. After the formation of the single-stranded gap in the chain-matrix of double-stranded heteroduplex under the action of the corresponding restricteduser enzyme, a chain-matrix can be hydrolysed by ExoIII nuclease or other appropriate nuclease in position behind the scope mutagenic(s) site(s). Then the reaction stopped, the result of which it forms a molecule, which is only single-stranded halfway. Then get a full-sized double-stranded DNA homoduplex using DNA polymerase in the presence of all four deoxyribonucleotide-triphosphates, ATP, and DNA ligase. Then this homoduplex molecule can be transferred in a suitable cell host.

As was shown above, the dialing sequence of the oligonucleotide has a length sufficient for hybridization with the matrix nucleic acid, and may also, but not necessarily, contain restriction sites. DNA-matrix can be obtained by using vectors that are derived from the vectors of 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, DNA is mutated, it should be integrated into one of these vectors to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 manual Sambrook et al., see above.

In accordance with another method of binding to the antigen can be recovered in the process of humanization of antibodies by selecting re-paired hypervariable regions (see application No. 11/061841, filed February 18, 2005). This method includes the introduction of inhuman hypervariable regions in the acceptor frame posledovatelno the ü, and then the introduction of one or more amino acid substitutions in one or more hypervariable regions without modification of the acceptor framework sequence. Alternate introduction of one or more amino acid substitutions may be accompanied by a modification in the acceptor framework sequence.

In accordance with another method of the library can be obtained by constructing sets of upstream and downstream oligonucleotides, where each of these sets is the set of oligonucleotides with different sequences, formed by a series of codons present in the sequence of the oligonucleotides. The sets of upstream and downstream oligonucleotides, together with a sequence of matrix nucleic acid of the variable domain may 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, such as viral envelope proteins and dimerization domains, using well-developed techniques of molecular biology.

The sequence of PCR primers comprises one or more engineered nab the ditch codons in positions solvent-accessible, and in various provisions of the wide range in the hypervariable region. As described above, the set of codons is a set of different sequences of nucleotide triplets are used to encode the desired options amino acids.

Suitable antibodies that meet the desired criteria, and selected by appropriate stages of the screening/selection can be isolated and cloned standard recombinant methods.

It is also important to humanized antibodies retained high affinity binding to the antigen and other desired biological properties. To achieve this goal, 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 models of immunoglobulin are public and known to experts. There are also computer programs to illustrate and represent the likely 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 balances in the functioning of the processes in the 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 so as to obtain an antibody with the desired properties, such as increased affinity for the antigen(s)target(s). Basically, the immediate and greatest impact on the binding of antigen have remnants of the hypervariable region.

Also examines various forms gumanitarnogo anti-CD79b antibody. For example, the humanized antibody may be an antibody fragment such as Fab, which kongugiruut, but not necessarily, 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. So, 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 produced immunoglobulin. So, for example, indicated that the homozygous deletion of the gene in the joint area between the heavy price and antibodies (J H) in chimeric mice and in mice with mutated germ line leads to a complete inhibition of the production of endogenous antibodies. The transfer of an array of genes of human immunoglobulin germline of these mice with the mutated 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 Measurement. 7:33 (1993); U.S. patent No. 5545806, 5569825, 5591669 (all from GenPharm); 5545807; and WO 97/17852.

Alternative technology of phage display (McCafferty et al., Nature 348:552-553 [1990]) can be used for producing human antibodies and fragments of antibodies in vitro from sets of genes variable domain of the immunoglobulin (V) from unimmunized donors. In accordance with this method, the genes of the domain V antibody clone with preservation of the reading frame in major or minor gene protein coat of filamentous phage, such as M13 or fd, and present on the surface ragovoy particles as functional antibody fragments. Because filamentous particle contains a copy of the single-stranded DNA genome of the phage, the selection made on the basis of the functional properties of antibodies, also allows you to select the gene encoding the antibody possessing these properties. Thus, the phage mimics some properties of b-cells. Phage display can be about what destlen in a variety of formats, described in publications Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). For phage display can be used several sources of V-gene segments. In the publication Clackson et al., Nature, 352:624-628 (1991) described the selection of diverse arrays of antibodies against oxazolone from a small randomized combinatorial library of V genes derived from the spleens of immunized mice. May be also designed a set of V genes from unimmunized human donors, and can be obtained antibodies against diverse array of antigens (including proteins), mainly the methods described in the publication 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 No. 5565332 and 5573905.

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

4. Antibody fragments

In some cases it is desirable to use a full-sized 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, various techniques have been developed. Traditionally, these fragments are formed as a result of proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can be 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 slices in a large number of, antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternative Fab'-SH fragments can be directly isolated from E. coli and chemically bonded with the formation of F(ab')2fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, F(ab')2fragments can be isolated directly from a culture of the recombinant host cell. Fab and F(ab')2a fragment with a longer half-life in vivo, containing residues that bind to the epitope of the receptor "salvation", is described in U.S. patent No. 5869046. Specialists and other known methods of obtaining fragments of antibodies. In other embodiments of the invention selected 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 the process of their application in vivo. Can be designed sFv proteins with obtaining hybrid effector protein at the amino - or carboxy-end of the sFv. Cm. Antibody Engineering, ed. Borrebaeck, see above. The antibody fragment may also be “single-chain antibody, such as antibody described in U.S. patent No. 5641870. Such single-chain antibody fragments can be monospecific or bespecifically.

5. Bespecifically antibodies

Bespecifically antibodies are antibodies that have binding specificity for at least two different epitopes. Representative bespecifically antibodies can bind to two different epitopes. Representative bespecifically antibodies can bind to two different epitopes of the protein CD79b described in this application. Other such antibodies may be a combination of CD79b-binding site with a binding site for other proteins. An alternative branch of antibodies against CD79b can be combined with the branch, which binds to a triggering molecule on a leukocyte such as a molecule of 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 were focused in CD79b-expressing cells and localized in these cells. Bespecifically antibodies can also be used to determine the localization of cytotoxic agents to cells expressing CD79b. These antibodies have CD79-connecting branch and branch, which is associated with a cytotoxic agent (such as, for example, saporin, antibody against interferon-α, vinylchloride, a chain of ricin a, methotrexate or hapten labeled with a radioactive isotope). Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically F(ab')2-antibodies).

In WO 96/16673 described bespecifically anti-ErbB2/anti-FcγRIII antibody, and in U.S. patent No. 5837234 described bespecifically anti-ErbB2/anti-FcγRI antibody. Bespecifically anti-ErbB2/Fcα antibody 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 co-expression 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 hybridoma (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 carrying out affinity chromatography is a certain work the spine and gives a low yield of product. A similar procedure described in WO 93/08829 and 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 of the antibody-antigen”) is attached to the sequence of the constant domain of immunoglobulin. Such attachment is preferably carried out with a constant domain of the heavy chain Ig, containing at least part of the hinge region, WithN2, and CN3. While it is preferable that this hybrid was first constant region of the heavy chain (CN1)containing the site necessary for binding to the light chain is present at least in one of the hybrids. DNA encoding the hybrid heavy chain immunoglobulin and, if necessary, the light chain immunoglobulin is inserted into separate expression vectors, and cotransfected in a suitable cell host. This provides a high degree of flexibility in the correction ratios of the three polypeptide fragments in those embodiments of the invention, in which unequal content of the three polypeptide chains used in the construction provide the optimum desired outputs especifismo antibodies. However, you can embed the coding sequences for two or all three polypeptide chains in one expression vector when the expression, hence, is her least the two polypeptide chains in equal ratio gives a high output, or if such relationship does not have a significant impact on the yield of the desired combination of chain.

In a preferred variant of this approach, bespecifically antibodies are composed of a hybrid heavy chain immunoglobulin having a first binding specificity in one branch, and hybrid pair 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 the desired especifismo connections from unwanted combinations of immunoglobulin chains, because the presence of the light chain of immunoglobulin in only one half of bespecifically molecules provides a simple way of highlighting it. 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 boundary preferably contains at least part of the domainN3. In this method, small side chains of one or several amino acids related issues in the primary region of the first antibody molecules replace the larger side chains (for example, tyrosine or tryptophan). In the edge region of the second antibody molecules create compensatory "cavities", having a size that is identical or similar size larger(s) side(s) chain(s), by replacing large side chains of amino acids smaller side chains (e.g., alanine or threonine). This allows to increase the output of heterodimers in relation to other unwanted end-products such as homodimers.

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 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). Antibody-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, 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) described procedures is, in which the intact antibody is subjected to proteolytic cleavage with the formation of F(ab')2-fragments. These fragments regenerate in the presence of the agent, forming diceology complex, such as sodium arsenite to stabilize adjacent dithioles 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 turned into Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB, resulting in a gain 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 related to education bespecifically antibodies. In the work Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe 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 education especifismo antibodies. Thus, the obtained bespecifically antibody has the ability with asiatica cells, sverkhekspressiya the ErbB2 receptor and normal human T cells and triggers the lytic activity of human cytotoxic lymphocytes against tumor target human breast cancer.

Were also described various methods of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, bespecifically antibodies were produced using “latinovich lightning”. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Peptides latinboy lightning originating from proteins Fos and Jun were added to the Fab'-parts of two other antibodies by ligating genes. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used for the production of homodimeric antibodies. Technology “dential”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 VLthrough a linker that is too short to create a pairing between the two domains on the same chain. In accordance with this VLand VHdomains of one fragment are forced to mate with a complementary V Land VH-domains of another fragment, thereby forming two antigenspecific site. Was also described another strategy to obtain fragments especifismo antibodies using single-chain Fv(sFv)dimers. Cm. Gruber et al., J. Immunol., 152:5368 (1994).

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

6. Heteroconjugate antibodies

Heteroconjugate antibodies are also included in the scope of the present invention. Heteroconjugate antibodies consist 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 infections [WO 91/00360, WO 92/200373 and EP 03089]. It is known that antibodies can be obtained in vitro methods for the synthesis of proteins, including methods using cross-linking agents. For example, immunotoxins can be constructed by carrying out the reaction of disulfide exchange or formation of thioester 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. Multivalent antibodies

P is ivalentine antibody may be faster internalizacao (and/or it may be faster catabolism), than bivalent antibody with antigen expression in the cell is bound to the antibody. Antibodies according to the invention can be multivalent antibodies (not belonging to the class IgM) with three or more antihistamine sites (for example, tetravalent antibodies), which can be easily produced through recombinant expression of nucleic acid that encodes a polypeptide chain of this antibody. Multivalent antibody may contain a dimerization domain, and three or more antigenspecific sites. The preferred dimerization domain contains (or consists of) Fc-region or a hinge region. In this case, the antibody may contain the Fc-region and three or more antigenspecific site located from amino end relative to the Fc-region. Described here is the preferred polyvalent antibody contains (or consists of) three to approximately eight antigenspecific sites, and preferably four antigenspecific site. Multivalent antibody comprises at least one polypeptide chain (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 the first 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 circuit "VH-CH1-VH-CH1-Fc region". Polyvalent antibody according to the invention also preferably contains at least two (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 containing the variable domain of the light chain as well, but not necessarily, CL domain.

8. The design of antibodies with effector functions

It may be desirable to modify the antibody according to the invention to give him effector functions, such as 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 antibodies. Alternative or additionally, cysteine(C) residue(s) may be entered(s) in the Fc-region that will bring the ü education miaocheng disulfide bonds in this area. Thus, the obtained homodimeric antibody may have an enhanced ability to internalize and/or increased ability to 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-linking agents described in the publication Wolff et al., Cancer Research 53:2560-2565 (1993). An alternative can be constructed antibody having two Fc-region, which may be enhanced complement lysis and increased ADCC. Cm. Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To extend the half-life of antibodies in the serum at the indicated antibody (in particular, in its fragment) may be entered epitope binding to the receptor "salvation", for example as described in U.S. patent No. 5739277. Used herein, the term "epitope to bind to the receptor "salvation" means an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the half-life of the IgG molecule in serum in vivo.

9. Immunoconjugate

The present invention also relates to immunoconjugates (which are synonyms of the terms "antibody conjugates-carstone tool" or "ADC"), comprising an antibody conjugated 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 animal origin, or fragments), or a radioactive isotope (i.e radioactive conjugate).

In some embodiments of the invention immunoconjugate contains the antibody and chemotherapeutic agent or other toxin. Chemotherapeutic agents used for obtaining immunoconjugates described above. Enzymatically active toxins and fragments, which can be used for these purposes are the A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin A-chain, exotoxin a (from Pseudomonas aeruginosa), A-chain of ricin, A-circuit abrina And-chain medecine, alpha sarcin, proteins Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, krotin, inhibitor Sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. For producing radio-conjugated antibodies can be used in a variety of radionuclides. Examples of such radionuclides are212Bi131I131In90Y and186Re. Conjugates of the antibody and cytotoxic drugs on ucaut using a variety of bifunctional protein-coupling agents, such as N-Succinimidyl-3-(2-pyridylthio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (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, the immunotoxin ricin can be obtained, as described in the publication Vitetta et al. Science, 238:1098 (1987).14C-labeled 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is representative chelat forming agent for conjugation of the 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 monomethylester (MAE) (a synthetic analogue of dolastatin), maytansinoid, such as DM1, trichoton and SS, and derivatives of these toxins that have a toxic effect.

Representative immunoconjugate - conjugates of the antibody-drug"

Immunoconjugate (or conjugate antibody-drug with adsto" ("ADC")) according to the invention can be immunoconjugate formula I, specified below, where the antibody is conjugated (i.e. covalently associated) with one or more molecules of the drug (D) through an optional linker (L). The ADC may include conjugates "thio-Mab-drug" ("TDC").

Ab-(L-D)pI

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

A. Representative linkers

The linker may contain one or more linker components. Representative of the linker components are 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe), p-aminobenzeneboronic ("PAB"), and the components which you the resulting conjugation with linker reagents: N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", also called here "MCC") and N-Succinimidyl-(4-iodates)aminobenzoate ("fairs are forthcoming-Siab"). Specialists known various linker components, some of which are described below.

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

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

-Aa-Ww-Yy-II

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

In some embodiments of the invention the linker component may contain "extending component which binds to the antibody with a different linker component or molecule drugs. A representative of the extension components are presented below (where the wavy line indicates the site of covalent binding with the antibody):

In some embodiments of the invention the linker component may contain amino acid component. In one of these variants of the invention the amino acid component provides the cleavage of linker-protease that facilitates the release of drugs 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). Amino acid component may contain natural amino acid residues, and small amino acids and unnatural Amin the acid analogues, such as citrulline. Amino acid components can be designed and optimized in their selectivity against fermentativnogo cleavage by specific enzymes, for example, tumor-associated protease, cathepsin B, C and D, or platinovoi protease.

In some embodiments of the invention the linker component may contain "spacer elements" component that binds the antibody molecule with drugs, either directly or through the extension component and/or amino acid component. Spacer elements component can be "carolinensis" or "nesamierinamais" component. "Nesamierinamais" spacer elements component is a component, where part of the spacer elements of the component or all of this component remain associated with the molecule drugs after enzymatic (e.g., proteolytic) splitting the ADC. Examples nesmolkayuschee spacer elements components include, but are not limited to, glycine spacer elements component and a glycine-glycine spacer elements component. Also consider other combinations of peptide spacers, sensitive to the sequence-specific enzymatic cleavage. So, for example, enzymatic cleavage ADC containing a glycine-glycine spacer elements component is, a protease associated with tumor cells, will lead to the release of molecules "glycine-glycine-drug" from the rest of the ADC. In one of such variants molecule glycine-glycine-drug" subjected to single-stage hydrolysis in cancer cells, which leads to the elimination of a glycine-glycine spacer elements component from molecule drugs.

"Carolinensis" spacer elements allows for the release of a molecule drugs without conducting single-stage hydrolysis. In some embodiments of the invention, the spacer elements component of the linker contains p-aminobenzyl group. In one of such variants of the p-aminobenzoyl alcohol attached to the amino acid component via amide linkages, carbamate, methylcarbamate or carbonate formed by the reaction between 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 spacer elements component is p-aminobenzeneboronic (PAB). In some embodiments of the invention fenelonov part of the p-aminobenzyl group substituted Qm, where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), halogen, nitro or cyano; m is an integer from 0 to 4. Other examples zamolim niroshima spacer elements components are but not limited to, aromatic compounds that their electronic properties similar p-aminobenzoylamino alcohol (see, for example, the application 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 be used the spacers that are subjected to 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 ring bicyclo[2.2.1]- bicyclo[2.2.2]system (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and amides of 2-aminophenylamino acid (Amsberry et al. (1990) J. Org. Chem. 55:5867). Examples carolinensis spacers used in the ADC are amino compounds of medicinal product substituted in the α-position of glycine (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

In one embodiment of the invention, the spacer elements specified item below is a branched bis(hydroxymethyl)styrene (BHMS), which can be used to activate and release of many 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 a number between 1 and about 20.

In the other embodiment of the invention the linker L may be a linker dendritic type, used for covalent binding of more than one molecule drugs with the antibody through a branching, multifunctional linker molecule (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 drug to antibody, i.e. 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, by dendritic linker can be attached to a large number of molecules of the drug.

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

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

b. A representative molecule drugs

(1) Maytansine and maytansinoids

In some embodiments of the invention immunoconjugate contains antibody conjugated with one or more molecules maytansinoid. Maytansinoid are mitotic inhibitors, which shall act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrews Maytenus serrata (U.S. patent No. 3896111). Then it was discovered that certain microbes also produce maytansinoid, such as maytansine and esters With-3-maytansine (U.S. patent No. 4151042). Synthetic maytansines and its derivatives and analogs are 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 maytansinoids 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 of functional groups suitable for conjugation by joining disulfide and ridiculing of linkers to the antibody, (iii) are stable in plasma, and (iv) are effective against various tumor cell lines.

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

Representative maytansinoid drugs drugs are having a modified aromatic ring, such as C-19-dechloro (U.S. patent 4256746) (obtained by recovery of ansamitocins P2 lithium aluminum hydride); C-20-hydroxy (or C-20-desmethyl) +/-C-19-Deshler (U.S. patent N No. 4361650 and 4307016) (obtained by demethylation using Streptomyces or Actinomyces, or by dechlorination using LAH); and C-20-dimetoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. patent No. 4294757) (obtained by acylation using acylchlorides), and molecules with modifications in other provisions.

Representative molecules maytansinoid drugs are molecules with modifications such as: C-9-SH (U.S. patent 4424219) (obtained by reaction between maytansine with H2S or P2S5); C-14-alkoxymethyl(dimetoxy/CH2OR)(US patent 4331598); C-14-hydroxymethyl 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 No. 4313946 and 4315929) (isolated from Trewia nudlflora); C-18-N-desmethyl (utenti U.S. No. 4362663 and 4322348) (obtained by demethylation maytansine under the action of Streptomyces); and 4,5-deoxy (U.S. patent 4371533) (obtained by recovering maytansine trichloride titanium/LAH).

It is known that many of the provisions in maytansinoid connections, depending on the type of link that can be used as the provisions of the accession. For example, for the formation of ester bonds, are suitable C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, C-15 position modified with 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 drugs are molecules having the structure:

,

where the wavy line indicates the covalent binding of the sulfur atom of the molecule maytansinoid drug linker-ADC. R may independently represent H or C1-C6alkyl. Alkalinous chain linking amide group with a 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 stereoisomers of molecules maytansinoid drug compounds according to the invention, i.e. any combination of R and S configurations at the chiral atoms is of glared D. In one embodiment of the invention, the molecule maytansinoids medicinal product has the following stereochemical structure:

Representative variants of molecules maytansinoids medicines are: DM1; DM3 and DM4, having the structure:

,

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

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

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

,

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; U.S. patent No. 5208020, 5416064, in the application US 2005/0276812 A1 and European patent EP V, to the verge in its entirety entered into this application by reference.

The conjugates of the antibody-maytansinoid” produced by chemical binding of an antibody to a molecule maytansinoid, where the specified binding does not lead to a significant reduction of the biological activity of the antibodies or molecules maytansinoid. 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 maytansines and analogues maytansine with modifications in the aromatic ring or in other positions of the molecule maytansine, such as various esters maytansine.

To create conjugates of the 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 the European patent EP V, and in the publication of Chari et al., Cancer Research 52:127-131 (1992) and in the patent application US 2005/016993 A1, which in its entirety are introduced in the present description by reference. The conjugates of the antibody-maytansinoid"containing linker component SMCC can be obtained, as described in the patent application S 2005/0276812 A1, in the section "the Conjugates of the antibody-drug and methods". Linker groups are disulfide groups, thioester groups, groups that are sensitive to the effects of acid photochemically unstable, band, sensitive to the action of peptidases, or group, sensitive to the action of esterase described in the aforementioned patents. Additional linker groups described and illustrated in this application.

The conjugates of the antibody-maytansinoid" can be obtained using a variety of bifunctional protein-coupling 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 dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine)derived bis -, page (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-succini Idil-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 bond can be formed by reaction with the hydroxyl group of the standard methods of binding. This reaction can be carried out in position C-3, having a hydroxyl group in position C-14, modified with hydroxymethyl, C-15, modified with hydroxyl group in position C-20, having a hydroxyl group. In one embodiment of the invention the bond is formed at the position C-3 maytansine or its equivalent.

(2) Auristatin and dolastatin

In some embodiments of the invention immunoconjugate contains antibody conjugated with dolastatin or with a peptide analogue of dolastatin or their derivatives, for example, auristatin (U.S. patent No. 5635483, 5780588). It was found that dolastatin and auristatin negatively affect the dynamics of the formation of microtubules, the hydrolysis of GTP 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 at the N-(amino)-end or near-(carboxy)-end of the molecule peptide drug is wow tools (WO 02/088172).

Representative variants auristatin are molecules medicines containing attached to the N-end of monomethylaniline, DE and DF (application US 2005/0238649 described Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004, and this application in its entirety is introduced into the present description by reference).

Peptide molecule drug may be selected from compounds of formulas DEand DFbelow:

,

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

and at each position, independently:

R2selected from H and C1-C8of alkyl;

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

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

R5selected from H and methyl;

or R4and R5 taken 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 from H and C1-C8of alkyl;

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

each of R8independently selected from H, OH, C1-C8of alkyl, C3-C8carbocycle and O-(C1-C8the alkyl);

R9selected from H and C1-C8of alkyl;

R10selected from aryl or C3-C8heterocycle;

Z represents O, S, NH or NR12where R12represents a 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 a 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)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 a sec-butyl.

In another embodiment of the invention, each of R2and R6represents methyl, and R9is a-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 R6represents methyl, R5represents-H, R7represents sec-butyl, R8in each case represents-OCH3and R9is a-H.

In one embodiment of the invention Z represents the t a-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-, R11represents-H, methyl or tert-butyl.

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

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

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

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

Other representative options are monomethylamine connection, it is either phenylalaninamide-modification at the C-end pentapeptidnogo auristatin medicines (WO 2007/008848), and monomethylamine connection with phenylalanine modification of the side chain at C-end pentapeptidnogo auristatin medicines (WO 2007/008603).

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

In one aspect of the invention, the hydrophilic groups are, but are not limited to, esters of triethylene glycol (TEG), presented above, which can be attached to a molecule drugs in R11. Not limited to any specific theory, it can be noted that the hydrophilic group improve the internalization of the molecule medicines and prevent agglomeration.

Representative variants of the ADC of formula I containing auristatin/dolastatin or their derivatives, as described in the application US 2005-0238649 and publishing Doronina et al. (2006) Bioconjugate Chem. 17:114-124, which in its entirety are introduced in the present description by reference. Representative variants of the ADC of formula I containing MMAE or MMAF and various linker components have the following structures and symbols (where Ab is an antibody; p is from 1 to primerno, “Val-Cit” or “vc” represents a dipeptide valine-citrulline, and “S” is a sulfur atom). It should be noted that in some descriptions of the structure of the ADC associated with the sulfur atom, the antibody is labeled with "Ab-S", which indicates only communication with the sulfur atom, but does not indicate what specific sulfur atom attached to more than one molecular linker-drug". In the following structures of the bracket on the left can also be placed to the left of the sulfur atom Ab and S, and such entry is equivalent to a formula of the ADC according to the invention, represented in the present description.

Representative variants of the ADC of formula I containing MMAF and various linker components also include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. It is interesting to note that immunoconjugate containing MMAF, attached to the antibody through a linker, which does not undergo proteolytic cleavage, have activity comparable to the activity immunoconjugates containing MMAF, attached to the antibody through proteoliticeski biodegradable linker. See, Doronina et al. (2006) Bioconjugate Chem. 17:114-124. In such cases, the release of drugs, obviously, is due to the decomposition of antibodies in the cell. Cm. below.

Typically, the peptide molecule drugs can be obtained through about what adowanie 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 and K. 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 of medicines can be obtained by the methods described in the application US 2005-0238649 A1; U.S. patent No. 5635483; No. 5780588; 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 formulas DFsuch as MMAF and their derivatives, can be obtained by the methods described in the application U.S. 2005-0238649 A1 and publishing Doronina et al. (2006) Bioconjugate Chem. 17:114-124. Molecules auristatin/dolastatin medicines formulas DEsuch as MMAE and their derivatives, can be obtained by the 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, for example, 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 conjugated with interest the antibody.

(3) Calicheamicin

In other embodiments, the zebrette immunoconjugate contains the antibody, conjugated with one or more molecules calicheamicin. Antibiotics collection calicheamicin capable of producing double-stranded DNA breaks in subpicomolar concentrations. Description get conjugates collection calicheamicin can be found in U.S. patents№№ 5712374, 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 conjugated to the antibody, is a means QFA, which is antifolate. Calicheamicin and QFA have intracellular active sites and have difficulty passing through the plasma membrane. Therefore, the absorption of these agents cell by internalization mediated antibody leads to a significant increase in their cytotoxic effects.

C. Other cytotoxic tools

Other anticancer drugs, which can be conjugated with the antibody, are BCNU, streptozocin, vincristine and 5-torural, the family of agents known collectively as complex LL-E described in U.S. patent No. 5053394, 5770710 and espiramicina (U.S. patent No. 5877296).

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

The present invention also considered immunoconjugate formed by the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Dnazol).

In some embodiments of the invention immunoconjugate may contain highly radioactive atom. For producing radio-conjugated antibodies can be used in a variety of radioactive isotopes. Examples of such radionuclides are211At,131I, 125I90Y186Re,188Re,153Sm212Bi32R212Pb and radioactive isotopes of Lu. E. what if the specified immunoconjugate is used for detection, it might contain a radioactive atom for scintigraphic studies, for example,99mTc or123I, or a spin label for imaging method for nuclear magnetic resonance (NMR) (also known as Mr imaging, MRI), 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 the method of chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 instead of hydrogen. Labels such as99mTc or123I186Re,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 may be applied 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 Immunoscintigraphy" (Chatal, CRC Press 1989).

In some embodiments of the invention immunoconjugate may contain antibody conjugated with a prodrug-activating enzyme which converts a prodrug (e.g., peptidase chemotherapeutic agent, see WO 81/01145) in active Lek is a significant tool, such as anticancer drug. Such immunoconjugate can be used in antibody-dependent mediated by enzymes proletarienne therapy (ADEPT). Enzymes that can be conjugated with the antibody include, but are not limited to, alkaline phosphatase, which can be used for converting phosphate-containing prodrugs into free drugs; Ukrainian, which can be used to turn sulfidogenic prodrugs into free drugs; sitoindosides, which can be used for converting non-toxic 5-fertilizin in anticancer drug, namely 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which can be used to turn petitcodiac prodrugs into free drugs; D-alanismorissette, which can be used for converting prodrugs that contain D-amino acid substituents; carbohydrate-splitting enzymes, such as β-galactosidase and neuraminidase, which can be used for converting glycosylated prodrugs into free drugs; β-lactamase, which can be used for the reversine medicines, derivatizing β-lactams, into free drugs; and penicillin-amidase, such as penicillin V-amidase or penicillin G-amidase, which can be used for making medicines, derivatizing of nitrogen atoms amine phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Enzymes can be covalently attached to the antibody by methods of recombinant DNA, are well known in the art. (see, e.g., Neuberger et al., Nature, 312:604-608 (1984)).

d. Download drugs

Download medicinal product is denoted by p, i.e. the average number of drug molecules per antibody in a molecule of formula I. the Loaded drug can be from 1 to 20 molecules of the drug (D) antibody. The conjugates of the 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 the drug to the antibody in preparations of the ADC, resulting from conjugation reactions may be characterized by standard means such as mass spectroscopy, ELISA analysis and HPLC. Can also be determined quantitative distribution of ADC, expressed as "p". In some cases, a section is a group of purification and characterization of homogeneous ADC, where p is a certain value obtained for the ADC with another download of the medicinal product, can be carried out using reverse-phase HPLC or electrophoresis. So, for example, pharmaceutical compositions "antibody-drug formula I may be a heterogeneous mixture of such 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 restricted by the number of binding sites on the antibody. For example, if the attachment comes through thiol of cysteine, as in the representative embodiments described above, the antibody may have one or more thiol groups of cysteine, or it may have only one or few enough reactive thiol groups, through which may be joined by the linker. In some embodiments of the invention a higher loading of the medicinal product, 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 the loading of the drug to the ADC according to the invention is between 1 and the ome to 8, from about 2 to about 6 or from 3 to 5. Indeed, it was shown that for some ADC optimal ratio of molecules of the drug to the antibody may be less than 8, and may be approximately from 2 to 5. Cm. the application of the U.S. 2005-0238649 A1.

In some embodiments of the invention the molecule drugs in amounts less than theoretical maximum kongugiruut with the antibody in the reaction of conjugation. The antibody may contain, for example, lysine residues, which do not react with the intermediate connection "drug-linker or linker reagent, as discussed below. Usually antibodies do not contain a large number of free and reactive thiol groups of cysteine, which may be associated with a molecule drugs, and indeed, most of thiol groups of cysteine residues in antibodies are present in the form disulfide bridges. In some embodiments of the invention, the antibody can be recovered under the action of a reducing agent, such as dithiothreitol (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 reaction under denaturing conditions with the formation of reactive nucleotide sequence that is multidisciplinary groups, such as lysine or cysteine.

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

It should be noted that when the intermediate connection "drug-linker or linker reagent, and then with a reagent, such as a molecule drugs, reacts 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 can be calculated for the mixture using a sandwich ELISA analysis using an antibody which is specific for antibodies and drugs. Individual molecules of the ADC in this mixture can be identified using mass spectroscopy and separated by HPLC, for example, hydrophobic chromatography (see, for example, 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, March 2004). In some embodiments of the invention a homogeneous ADC with loading one of the medicinal product can be isolated from the mixture to conjugation by electrophoresis or chromatography.

e. Some ways of getting immunoconjugates

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

Nucleophilic groups present on the antibodies include, but are not limited to: (i) N-terminal amino group, (ii) amino side chain, for example, lysine, (iii) thiol group on the side chain, for example, cysteine, and (iv) hydroxyl or amino sugars, where the aforementioned antibody is glycosylated. Amino groups, thiol groups and hydroxyl groups are nucleophilic and can undergo reactions interact with the formation of covalent bonds with electrophilic groups on linker molecules or linker reagents including: (i) active esters such as NHS-esters, HOBt-ester, halogenfree and acid halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group. Some antibodies are recovered megamachine disulfides, i.e. cysteine bridges. For conjugation with linker reagents antibodies can be made reactive by treatment with 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 of the nucleophile. The antibodies can be entered additional nucleii the performance communications group by modification of lysine residues, for example, through a reaction between lysine with a 2-aminothiophenol (reagent trout)that will lead to transformation of the amine in the thiol. Reactive thiol groups can be incorporated into the antibody by introducing one, two, three, four or more cysteine residues (for example, obtaining variants of antibodies that contain one or more non-natural cysteine amino acid residues).

The conjugates of the antibody-drug” according to the invention can be also obtained by a reaction between the electrophilic group of the antibody, such as a carbonyl group of the aldehyde or ketone, with the 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, hydrazine carboxylate, and originated. In one embodiment of the invention the antibody is modified so that it includes an electrophilic group capable of reacting with nucleophilic substituents on the linker reagent or drug. In another embodiment of the invention sugar glycosylated antibodies can be oxidized, for example, controllable periodic destruction oxidants with the formation of aldehyde or ketone groups that can react with the amino group of the linker is of agentov or molecules of the drug. Received minovia group Chippewa base can form a stable relationship, or they can be recovered, for example, borohydride the reagents to form stable amine linkages. In one embodiment of the invention, the reaction of interaction 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, antibodies of the invention containing N-terminal serine or threonine 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 drug or linker by a nucleophile.

Nucleophilic groups on the molecule drugs include, but are not limited to, amino groups, tirinya, hydroxyl, hydrazide, Joksimovi, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group capable of reacting with the formation of covalent bonds with electrophilic groups on linker molecules and linker reagents including: (i) active complex is Fira, such as NHS-esters, HOBt-ester, halogenfree and acid halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group.

In short, consider the 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, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (for example, supplied by 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-coupling 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 dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (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, the immunotoxin ricin can be obtained, as described in the publication Vitetta et al. Science, 238:1098 (1987).14C-labeled 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is representative chelat forming agent for conjugation of the radionuclide to the antibody. Cm. WO 94/11026.

Alternative hybrid protein containing the antibody and cytotoxic agent may be obtained, for example, recombinant methods or by peptide synthesis. Recombinant DNA molecule may contain a region encoding the antibody and the cytotoxic portion of the conjugate, or adjacent to each other or separated by a region that encodes a linker peptide which does not adversely affect the desired properties of the conjugate.

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

Representative immunoconjugate - conjugates "thio-antibody-drug"

a. Recip is tion constructed 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 antibodies according to the invention, receive a variety of methods that include, but are not limited to, isolation from a natural source (in the case of natural variants of amino acid sequences, the retrieval of the drug by using site-directed (or oligonucleotide-mediated) mutagenesis (Carter (1985) et al. Nucleic 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 cluster mutagenesis (Wells et al. (1985) Gene 34:315-323) previously obtained DNA encoding the polypeptide. Protocols, kits and reagents for performing mutagenesis are commercially available, for example, such as set for the site-directed mutagenesis QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations also 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; and Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500). Variants of recombinant antibodies can also be SK is strayaway by modification of restriction fragments, or by using extension PCR with overlapping, conducted using synthetic oligonucleotides. Mutagenic primers encode the replacement(s) cysteine codons. Standard methods of mutagenesis can be applied for producing DNA encoding such mutant antibodies constructed on the basis of 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 display (McCafferty et al., (1990) Nature 348:552-553) can be used for producing human anti-CD79b antibodies and fragments of antibodies in vitro from sets of genes variable domain of the immunoglobulin (V) from maimonidean donors. In accordance with this method, the genes of the domain V antibody clone with preservation of the reading frame in major or minor gene protein coat of filamentous phage, such as M13 or fd, and present on the surface ragovoy particles as functional antibody fragments. Because filamentous particle contains a copy of the single-stranded DNA genome of the phage, the selection made on the basis of the functional properties of antibodies, also allows you to select the gene encoding the antibody possessing 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 565332; US 5573905; US 5567610; US 5229275).

Anti-CD79b antibodies can be chemically synthesized using known method Oligopeptide synthesis, or they can be obtained and purified recombinant 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 in an automated method. Automated solid-phase synthesis method may be implemented, for example, using t-BOC - or Fmoc - protected amino acids on the peptide synthesizer, Applied Biosystems (Foster City, CA) according to manufacturer's instructions. Different parts of the anti-CD79b antibody or CD79b polypeptide can be obtained by the method of chemical synthesis and combined using chemical or enzymatic synthesis with producing the desired anti-CD79b antibody or CD79b polypeptide.

To obtain fragments of antibodies, various techniques have been developed. 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 can be produced directly by recombinant cell host. Fab, Fv and scFv-fragmentati-CD79b antibodies can be expressed in E. coli and secretariats from E. coli, which facilitates the production of these fragments in large quantities. Antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternative Fab'-SH fragments can be directly isolated from E. coli and chemically bonded with the formation of F(ab')2fragments (Carter et al., Bio/Technology 10:163-167 (1992)), or they can be selected directly from a culture of the recombinant host cells. Anti-CD79b antibody can be a single-chain Fv fragment (scFv) (WO 93/16185; US 5571894; US 5587458). Fragment of the anti-CD79b antibodies can also be a "linear antibody" (US 5641870). Such fragments, linear antibodies can be a monospecific or bespecifically.

The following describes mainly the production of anti-CD79b antibodies by culturing cells transformed or transfected with a vector containing a nucleic acid encoding an anti-CD79b antibody. DNA encoding anti-CD79b antibodies, can be obtained from a cDNA library isolated from tissue, which, obviously, contains mRNA anti-CD79b antibodies and expresses it on detektiruya level. In line with this, the DNA of the 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 can also be obtained from genome the library or the application of known methods of synthesis (for example, automated nucleic acid synthesis).

Methods of designing, selecting and obtaining preparations according to the invention allow to obtain designed on the basis of cysteine anti-CD79b antibodies that react with an electrophilic functional group. These methods also allow you to get a connection-conjugates of antibodies, such as connection-conjugates of the antibody-drug" (ADC), with molecules of drugs in certain designed selective sites. Reactive cysteine residues on the surface antibodies allow specific conjugation of the molecule drugs by reacting with thiol groups, such as maleimido or halogenoacetyl. The nucleophilic reactivity of thiol functional groups of the Cys residue with maleimide group approximately 1000 times higher than the reactivity of any other functional groups of the amino acids in the protein, such as amino group of lysine residues or N-terminal amino group. Toolspecific functional group in iodization and maleimide reagents may react with the amine 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 estimated using the standard is on analysis of Ellman. Immunoglobulin M is an example of the disulfide-bound pentamera, and immunoglobulin G is an example of a protein with an internal disulfide bridges associated with the subunits. In proteins, such as this protein to produce reactive free thiol is necessary to restore the disulfide bonds under the action of the reagent, such as dithiothreitol (DTT) or selenium (Singh et al. (2002) Anal. Biochem. 304:147-156). This procedure can lead to the loss of tertiary structure of the antibody and the binding specificity to the antigen.

Analysis PHESELECTOR (phage ELISA for screening of reactive thiols) allows the detection of reactive cysteine groups in antibodies in the format phage ELISA, which facilitates the design of antibody-based cysteine (Junutula, J.R. et al. (2008) J. Immunol Methods 332:41 to 52; WO 2006/034488; US 2007/0092940). On the surface of the hole causing 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. Mutant proteins presented on the phage can be skanirovaniya fast, reliable and highly effective method. This same method can be obtained from libraries constructed on the basis of cysteine antibodies, which can be subjected to selective binding to identify including the texts of the relevant reactive sites free Cys from randomized phage libraries of proteins, antibodies or other proteins. This method involves carrying out the reaction of cysteine mutant proteins presented on the phage, affinity reagent or reporter group, which also interacts with the thiol.

Analysis PHESELECTOR allows the screening of reactive thiol groups on the antibody. As example is the identification of options AS by this method. To identify a larger number of options thio-Fab with reactive thiol groups can be carried out efficiently search for a full-sized molecules Fab. To identify and quantify the accessibility of solvent to the amino acid residues in the polypeptide was used for this parameter, as the relative accessibility of the surface. The availability of the surface can be expressed as the surface area (A2), which can be contacted with the solvent molecule, for example, with water. The space occupied by water, expressed approximately as the sphere radius of 1.4 Å. In the package of crystallographic software SSR that are either free or require a license payment (Company for the development of the program CCP4, Daresbury Laboratory, Warrington, WA44AD, United Kingdom, Fax: (+44) 1925 603825, or on the Internet: www.ccp4.ac.uk/dist/html/INDEX.html), uses algorithms to calculate the surface accessibility of each amino acid protein with known x-ray crystal is ograficheskie coordinates ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Acta. Cryst. D50:760-763). Two representative software modules with which teach calculate the surface distance are the 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 you to determine the availability of surface protein to the solvent, as the center point of the sphere probe (representing a solvent molecule), when this point of "trying out" van der Vaal's surface protein. The program AREAIMOL allows you to calculate the surface area accessible to solvent, by the generation of surface points on the large sphere around each atom (the distance from the center of the atom is equal to the sum of the radii of the atom and probe) and eliminating those points that are in ekvivalentnykh areas associated with the neighboring atoms. The program AREAIMOL allows to determine the solvent-accessible area of atoms in a PDB file with coordinates and systematize the available area of the remainder of the circuit and of the whole molecule. Square (or difference of squares), 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 different elements.

The program AREAIMOL and allow SURFACE to achieve absolute availability, i.e. the number of angstroms (Å) in the Quad the ATA. The relative accessibility of the surface is calculated in comparison with the availability of standard peptide with the specific amino acids in the polypeptide. Standard peptide is a Tripeptide Gly-X-Gly, where x is the interest amino acid, and this standard peptide must have “extended” conformation, i.e. conformation, such as the beta-chains. This extended conformation maximizes the availability of Agricultural residue Calculated the available area is divided into the available area for the standard peptide in the Tripeptide Gly-X-Gly and receive private, which represents the relative accessibility. The percentage of availability is a relative availability multiplied by 100. Another representative algorithm to calculate the distance of the surface based on SOLV-module program xsae (Broger, C, F. Hoffman-LaRoche, Basel), which allows us to calculate the relative accessibility of amino acid residue for the water sector, based on x-ray coordinates of the given polypeptide. The relative accessibility of the surface for each amino acid in the antibody can be calculated using the data on its crystal structure (Eigenbrot et al. (1993) J MoI Biol. 229:969-995).

DNA encoding constructed on the basis of cysteine antibodies, can be easily isolated and sequenced in accordance with the standard and procedures (for example, using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a source of such DNA. After separation of the DNA may be placed into expression vectors, which are then transferout in cell host, such as E. coli cells, simian COS cells, cells of the Chinese hamster ovary (Cho) or other cells of the host mammal, such as myeloma cells (U.S. patent 5807715; application to U.S. patent 2005/0048572 and 2004/0229310), which usually do not produce protein antibodies, resulting in these recombinant cells masters synthesized monoclonal antibodies.

After the design and selection constructed 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, for example, in 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 the cells of the Chinese hamster ovary (Cho); and (ii) purification by standard methods of protein purification (Lowman et al. (1991) J. Biol. Chem. 266(17):10982-10988).

Thiol groups are designed Cys react with electrophilic linker reagent and with the intermediate compounds "drug-linker" with the formation of the conjugates are "constructed on the basis of cysteine antibody-drug" and the other labeled antibodies, constructed on the basis of cysteine. Residues Cys, which are constructed on the basis of cysteine antibodies in the parent antibody, and which mate to form mesapotamia and interchain disulfide bonds, do not have any reactive thiol groups (unless they are treated with reducing agent) and do not react with electrophilic linker reagents or intermediate compounds “drug-linker”. Just entered the Cys residue may remain unpaired and can respond, that is, to form a conjugate with electrophilic linker reagent or intermediate connection "drug-linker, such as"drug-maleimid". Representative intermediate compounds "drug-linker" is the MC-MMAE, MC-MMAF, MC-vc-PAB-MMAE and MC-vc-PAB-MMAF. Structural provisions designed 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 of 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 with the numbering scheme according to Kabat (bottom row), where the inserts are marked a,b,c. Using the numbering system of Kabat, actual is inana amino acid sequence may contain fewer or more number of amino acids due to a shortening of, or insertion into FR or CDR of the variable domain. Sites constructed on the basis of cysteine variants of the heavy chain identify with application schema sequential numbering and the numbering scheme of Kabat.

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

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

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

Constructed 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 in the heavy or light chains, or a constant or variable domains. Antibody fragments, such as Fab, can be constructed by replacing one or more amino acid fragment of the antibody has one or more cysteine amino acids, with the 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 which incorporates both:

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

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

where indicated, 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 obtaining constructed 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 antibodies; and

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

Stage (b) method of obtaining constructed on the basis of cysteine antibodies can include expression constructed on the basis of cysteine antibodies on viral particle selected from a phage or fahmideh particles.

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

(i) reaction of interaction designed on the basis of cysteine antibodies with affinity reagent that reacts with a thiol, to obtain the affine labeled with konstruirovanie on the basis of cysteine antibodies; and

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

In another embodiment, the present invention relates to a method of screening is designed based on the 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 constructed on the basis of cysteine antibodies;

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

(C) measuring the level of binding affinity labeled 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 cyst is in 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 can include expression 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 designed on the basis of cysteine antibodies with affinity reagent that reacts with a thiol, to obtain the affine labeled constructed on the basis of cysteine antibodies; and

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

b. Design options 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 the full-size recombinant and parental monoclonal anti-CD79b antibodies or to position 205 of the light chain (according to Kabat numbering) (equivalent to position 209 light chain, sequential numbering) in the full-size recombinant and parental monoclonal anti-CD79b antibodies methods of introducing cysteine described in nastoyasheva.

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

Received the following constructed on the basis of cysteine antibodies containing cysteine at position 205 of the light chain (according to Kabat numbering): (a) thio-MA79b-LC(V205C) with a sequence of the heavy chain (SEQ ID NO: 234), and a sequence of light chain (SEQ ID NO: 235), figure 27, and (b) thio-anti-cynoCD79b(ch10D10)-LC(V205C) with a sequence of the heavy chain (SEQ ID NO: 299) and with the sequence of the light chain (SEQ ID NO: 300), figure 49.

These are designed on the basis of cysteine monoclonal antibodies were expressed in cells Cho (ovary of kitayskaya) by the time the fermentation medium, containing 1 mm cysteine.

In one embodiment of the invention is humanized designed on the basis of cysteine anti-CD79b antibodies 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 on the basis of cysteine variants of anti-CD79b antibodies MA79b numbered in accordance with a sequential numbering system numbering according to Kabat and EU numbering system
SequenceSequential numberingNumbering according to KabatEuropean numbering systemSEQ ID NO:
EVQLCESGGGV5CV5C251
LRLSCCASGYTA23CA23C252
MNSLRCEDTAVA88CA84C 253
TLVTVCSASTKS116CS112C254
VTVSSCSTKGPA118CA114CA118C255
VSSASCKGPSVT120CT116CT120C256
WYVDGCEVHNAV282CV278CV282C257
KGFYPCDIAVES375CS371CS375C258
PPVLDCDGSFFS400CS396CS400C259

In one embodiment of the invention chimeric constructed on the basis of cysteine anti-CD79b antibodies 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).

264
Table 3
Comparison of sequences of the heavy chains were constructed on the basis of cysteine variants of anti-CD79b antibodies chMA79b numbered in accordance with a sequential numbering system numbering according to Kabat and EU numbering system
SequenceSequential numberingNumbering according to KabatEuropean numbering systemSEQ ID NO:
EVQLCQSGAEQ5CQ5C260
VKISCCATGYTK23CK23C261
LSSLTCEDSAVS88CS84C262
TSVTVCSASTKS116CS112C263
VTVSSCSTKGPA118CA114CA118C
VSSASCKGPSVT120CT116CT120C265
WYVDGCEVHNAV282CV278CV282C266
KGFYPCDIAVES375CS371CS375C267
PPVLDCDGSFFS400CS396CS400C268

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 chains were constructed on the basis of cysteine variants of anti-CD79b antibodies, anti-cynoCD79b(ch10D10), numbered in accordance with a sequential numbering system numbering according to Kabat and EU numbering system
SequenceSequential numberingNumbering according to KabatEuropean numbering systemSEQ ID NO:
EVQLCESGPGQ5CQ5C269
LSLTCCVTGYST23CT23C270
LNSVTCEDTATS88CS84C271
TTLTVCSASTKS111CS112C272
LTVSSCSTKGPA113CA114CA118C273
VSSASCKGPSVT115CT116CT120C274
WYVDGCEVHNAV282CV278C V282C275
KGFYPCDIAVES370CS371CS375C276
PPVLDCDGSFFS395CS396CS400C277

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

Table 5
Comparison of sequences of the light chain of humanized designed on the basis of cysteine variants of anti-CD79b antibodies MA79b numbered in accordance with a sequential numbering system and numbering according to Kabat
SequenceSequential numberingNumbering according to KabatSEQ ID NO:
SLSASCGDRVTV15CV15C278
EIKRTCAAPSV V114CV110C279
TVAAPCVFIFPS118CS114C280
FIFPPCDEQLKS125CS121C281
DEQLKCGTASVS131CS127C282
VTEQDCKDSTYS172CS168C283
GLSSPCTKSFNV209CV205C284

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

Table 6
Comparison of sequences of the light chain of chimeric constructed on the basis of cysteine variants of anti-CD79b antibodies MA79b numbered in accordance with a sequential numbering system and num is the situation according to the Kabat
SequenceSequential numberingNumbering according to KabatSEQ 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), containing the one or more of the following sequences of the 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 constructed on the basis of cysteine variants of anti-CD79b antibodies, anti-cynoCD79b(ch10D10), numbered in accordance with a sequential numbering system and numbering according to Kabat
SequenceSequential numberingNumbering according to KabatSEQ ID NO:
SLAVSCGQRATL15CL15C292
EIKRTCAAPSVV114CV110C293
TVAAPCVFIFPS118CS114C294
FIFPPCDEQLKS125CS121C295
DEQLKCGTASVS131CS127C296
VTEQDCKDSTYS172CS168C 297
GLSSPCTKSFNV209CV205C298

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

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

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

This method can be used for the conjugation of other reactive thiol reagents, in which the reactive group is, for example, maleimide, todatetime, pyridyldithio or other reactive thiol partner conjugation (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). A reagent that reacts with the thiol may be a molecule drugs, fluorophores, such as a fluorescent dye such as fluorescein or rhodamine, chelate forming agent for visualization or a radioactive metal used in therapy, piptadenia or nematodirinae tag, or label detected, or an agent that modifies clearance, such as various isomers of polyethylene glycol, a peptide that communicates with the third component, or another carbohydrate or popilnya agent.

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

Constructed on the basis of cysteine anti-CD79b antibodies and their conjugates can be used as a therapeutic and/or diagnostic tools. The present invention also relates to methods of prevention, treatment, therapy or mitigate one or more symptoms associated with b-cell-mediated disorder. In particular, the present invention also relates to methods of prevention, treatment, therapy or mitigate one or more symptoms associated with a cell proliferative disorder such as cancer, such as lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex. The present invention also relates to methods of diagnosing CD79b-mediated disorder or a predisposition to develop such disorders, as well as to methods of identifying antibodies and antigenspecific fragments of antibodies, which are mainly associated with b-cell-and solirovanie the CD79b polypeptide.

In another embodiment, the present invention relates to the use of constructed on the basis of cysteine anti-CD79b antibodies for the preparation of a medicinal product for the treatment of 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 the compound-conjugate antibody-drug"containing constructed on the basis of cysteine anti-CD79b antibody (Ab) and the molecule auristatin drugs (D)where the specified constructed on the basis of cysteine antibody is associated with D linker molecule (L) through one or more free cysteine amino acids, where the aforementioned compound 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 antibodies one or more free cysteine amino acids.

In another aspect of the present invention classifies the I to the composition, containing a mixture of compounds of "antibody-drug 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 figures 24-28 and 48-49 options presented conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" (ADC), where the molecule auristatin drugs attached to the introduced cysteine group in the light chain (LC-ADC) or heavy chain (HC-ADC).

Possible advantages conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" are advanced security (higher therapeutic index); improved pharmacokinetic parameters; save miaocheng disulfide bonds of the antibody, which can stabilize the conjugate and maintain its conformation conducive to an active binding; the possibility of identifying sites of conjugation of the medicinal product; and the possibility of obtaining conjugates "constructed on the basis of cysteine antibody-drug" in 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" or "the script" means a chemical group, containing covalent bond or a chain of atoms that covalently bind the antibody to the molecule drugs. In various embodiments of the invention the linker is marked L. “Linker” (L) is a bifunctional or multifunctional molecule that can be used to associate 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. the Conjugates of the antibody-drug” (ADC) are usually obtained by using a linker having a reactive functional group for binding with drug and antibody. The thiol of the cysteine introduced into the antibody (Ab)can form a bond with an electrophilic functional group of the linker reagent molecule drug or an intermediate connection "drug-linker".

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

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

Constructed on the basis of cysteine antibody is subjected to the reaction of interaction 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 publication of Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773, and in accordance with the Protocol described in example 6.

The linker may consist of one or more linker components. Representative of the linker components are 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 ("fairs are forthcoming-Siab), ethylenoxy-CH2CH2O - one or n is how many duplicate items ("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: A represents the extension component covalently linked to a cysteine thiol of the antibody (Ab);

and is 0 or 1;

each W independently represents an amino acid,

w independently represents an integer from 0 to 12,

Y means spacer elements component covalently linked to a molecule drugs; and

y is 0, 1 or 2.

Extending the component

Extension component (-A-), if present, are able to bind the antibody with the amino (-W-). In this case, the antibody (Ab) has functionaluy group that can form a bond with the functional group of the extension component. Suitable functional groups that may be present on the antibody and which can be either natural or chemically synthesized, are, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, anomeric hydroxyl group of a carbohydrate and carboxyl. In one aspect of the invention the functional groups of antibodies are sulfhydryl or amino. Sulfhydryl groups can be generated by recovery of the intramolecular disulfide bond ant the body. Alternative sulfhydryl groups can be formed through the interaction of the amino groups of the lysine molecules are antibodies 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. Representative lengthening 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 bivalent radical selected from -(CH2)rWith3-C8carbocycle, -O-(CH2)rarylene, (CH2)rarylene, Allen-(CH2)r, (CH2)r-(C3-C8-carbazolyl)- (C3-C8-carbazolyl)-(CH2)r-With3-C8-heterocyclyl, (CH2)r-(C3-C8-heterocyclyl), (C3-C8-heterocyclyl)-(CH2)r-, -(CH2)rC(O)NRb(CH2)r, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-, -(CH2)rC(O)NRb(CH2CH2O)r, -(CH2)rC(O)NRb(CH2CH2O)r-CH2, -(CH2CH2O)rC(O)NRb(CH2CH2the) 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 represents an integer of 1-10.

Afilename are divalent aromatic hydrocarbon radicals with 6 to 20 carbon atoms obtained by removing two hydrogen atoms from the original 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 a ring system in which one or more atoms on the ring represent a heteroatom such as nitrogen atom, oxygen and sulfur. Heterocyclic radical comprises 1-20 carbon atoms and 1-3 heteroatoms selected from N, O, P and S. the Heterocycle may be a monocycle having 3 to 7 atoms in the ring (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P and S), or Bicycle with 7-10 atoms on the ring (4-9 carbon atoms and 1-3 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 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 audience who AI "The Chemistry 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, tetrahydropyranyl (piperidyl), thiazolyl, tetrahydrothiophene, oxidized grey tetrahydrothiophene, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianeptine, indolyl, indolinyl, 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-detainer, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine 2N-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, honokalani, hintline, cinnoline, pteridine, 4Ah-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, furutani, phenoxazines, isopropanol, bromanil, imidazolidinyl, imidazolines, pyrazolidine, pyrazoline, PIP is rasini, indolinyl, isoindolyl, hinokitiol, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazole, oxindoles, benzoxazolyl and satanail.

Carbocyclic groups include saturated or unsaturated ring having from 3 to 7 carbon atoms as a monocycle, or from 7 to 12 carbon atoms as Bicycle. 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 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 all of the representative embodiments, the ADC of formula I, such as compound II-VI, even if they are not specifically from 1 to 4 molecules of the drug are associated with the antibody (p=1-4), depending on the number of introduced cysteine residues.

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

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

Another representative of the extension component is a component of the formula II, where R17represents -(CH2CH2O)r-CH2and r is 2:

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

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

In another embodiment of the invention the specified extension component associated with constructed on the basis of cysteine anti-CD79b antibody via a disulfide bond between the sulfur atom of the cysteine antibodies and the sulfur atom of the extension component. A representative of the extension component of this variant of the invention shown in the formula IV, where R17, Ab-, -W-, -Y-, -D, w and y are defined above.

Even the bottom variant of the invention, the reactive group of the extension component contains a reactive thiol functional group, which can form a bond with the free cysteine thiol of the antibody. Examples of the reactive thiol functional groups include, but are not limited to, maleimide, α-halogenoacetyl, activated esters, such as succinimide, 4-nitrophenolate esters, pentafluorophenyl esters, tetraterpene esters, anhydrides, acid chlorides, sulphonylchloride, isocyanates and isothiocyanates. A representative of the extension components of this variant of the invention shown in formulas Va and Vb, where R17, Ab-, -W-, -Y-, -D, w and y are defined above.

In another embodiment of the invention the specified linker may be a linker dendritic type for covalent binding of more than one molecule drugs with the antibody through a branching, multifunctional linker molecule (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 drug to antibody, i.e. when loading, which corresponds to the efficiency of the ADC. Thus, if constructed on the basis of cysteine antibody has only one reactive thiol group of cysteine, the set of molecules of the drug can be covalently linked by dendritic whether the Kera.

Amino acid component

The linker may contain amino acid residues. Amino acid component (-Ww-), if present, binds to the antibody (Ab) molecule drugs (D) constructed on the basis of the cysteine conjugate the antibody-drug" (ADC) according to the invention.

-Ww - is a dipeptide, Tripeptide, tetrapeptide, pentapeptidnogo, Hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptides, undecapeptide or dodecapeptide component. Amino acid residues that make up 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 denotes 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)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH 2, -(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 that is attached to R19is chiral. Each carbon atom is attached to R19independently present in the (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 - are 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 that form the linker component, are the natural amino acid residues, and small amino acids, and unnatural amino acid analogs, such as citrulline.

Amino acid component can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, with the release of a molecule drugs (D)to the I, in one embodiment of the invention, after the release undergoes protonation in vivo with the formation of a 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.

Spacer elements component

Spacer elements component (-Y-), if present (y=1 or 2), binds amino acid component (-Ww-c molecule drugs (D), if such amino acid component is present (w=1-12). Alternatively, if the amino acid component is absent, the spacer elements specified component connects the extension component molecule drugs. If there are no amino acid component and an extension component (w, y=0), the specified component spacer elements also binds the molecule drugs with the antibody molecule. Spacer elements components are components of two General types: smolinerwien and nesmolkayuschee. Nesamierinamais spacer elements component is a component, where part of the spacer elements of the component or all of this component remain associated with the molecule drugs after removal, and in particular, fermentation the th removal of the amino acid component from conjugate the antibody-drug” or conjugate “drug-linker”. If the ADC containing a glycine-glycine spacer elements component or a glycine spacer elements 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 from Ab-Aa-Ww. In one embodiment of the invention independent hydrolysis reaction occurs in the target cells and leads to the breakdown of communication in the molecule glycine-drug with the release of the drug.

In another embodiment of the invention,- Yy - is a p-aminobenzeneboronic component (RAV), fenelonov part of which is substituted Qm, where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), halogen, nitro or cyano, and m is an integer from 0 to 4.

Representative variants nesmolkayuschee spacer elements components (-Y-) are-Gly-Gly-; -GIy-; -Ala - Phe-; -Val-Cit-.

In one of its variants the present invention relates to conjugate the drug-linker” or ADC, in which there is no spacer elements component (y=0), or their pharmaceutically acceptable salts or MES.

Alternative ADC containing carolinensis spacer elements component, maybe the release of a molecule D. In one embodiment of the invention, Y represents a group of PAB, which is associated with-Ww - through nitrogen atom of the amino group PAB, and is directly related to the molecule-D via a carbonate, urethane or ester 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 their electronic properties, similar to the RAV group, such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), heterocyclic analogues of RAV (US 2005/0256030), beta-glucuronide (WO 2007/011968) and ortho - or para-aminobenzoate. Can be used the spacers that are subjected to 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 ring bicyclo[2.2.1]- bicyclo[2.2.2]system (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and amides of 2-aminophenylamino acid (Amsberry et al. (1990) J. Org. Chem. 55:5867). Examples carolinensis spacers used in the ADC are amino compounds of medicinal product substituted in the floor of the terms of glycine (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

Representative spacer elements components (-Yy-) is represented by formulas X-XII:

Dendritic linkers

In another embodiment of the invention the linker L may be a linker dendritic type used for covalent binding of more than one molecule drugs with the antibody through a branching, multifunctional linker molecule (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 drug to antibody, i.e. 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, by dendritic linker can be attached to a large number of molecules of the drug. Representative variants branched dendritic linkers are gendarmerie components, such as 2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-Tris(hydroxymethyl)phenol (WO 2004/01993; Szalai 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 spacer elements component is a branched bis(hydroxymethyl)styrene (BHMS), which can be used to call the program and release a variety of medicines, 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 p is 1-4.

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

Other representative variants of compounds of conjugates of the antibody-drug 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 group that 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. Nucleophilic heteroatom of grupperinger may react with an electrophilic group on the antibody and form a covalent bond with a molecule of antibody. Suitable nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and originated. 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 spacer elements extending and amino acid components. Such spacer elements extending and amino acid components may contain reactive functional groups, which by their nature are electrophilic, nucleophilic or free radical. Reactive functional groups include, but are not limited to, carboxy, hydroxyl, para-nitrophenylarsonic, isothiocyanate and leaving group, such as O-mesyl, O-tosyl, -Cl, -Br, -I or maleimide.

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

Linker reagents

Conjugates of the antibody and auristatin can be obtained using various bifunctional linker reagent, 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 dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis-page (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).

The conjugates of the antibody-drug" can be also obtained using the linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-IAB, sulfo-SMCC, 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 of cysteine antibody thiol-containing molecule drugs to the label or linker intermediate connection. In addition maleimide, other functional groups that react with the thiol group of cysteine antibody molecule drug, a label or an intermediate linker 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 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. 7:180-186; in U.S. patent 6214345; WO 02/088172; patent applications U.S. 2003130189 and 2003096743; WO 03/026577; WO 03/043583 and WO 04/032828.

Ugly is managing the components of the formula (IIIa) can be introduced into a linker by reaction between the following linker reagents 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;

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

,

where X represents Br or I.

Extension components mentioned formula can also be introduced into a linker by reaction between the following bifunctional reagent N-end amino acid component:

Representative dipeptide linker reagent valine-citrulline (val-cit or vc), including maleimides extension component and a para-aminobenzeneboronic (PAB) carolinensis the spacer has a structure:

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

Obtaining conjugates constructed based cysteine anti-CD79b antibody-drug"

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

Thiol group of cysteine antibodies are nucleophilic and have the ability to interact with the formation of covalent bonds with electrophilic groups on linker compounds and intermediate compounds “drug-linker”, including (i) the active complex is e esters, such as NHS-esters, HOBt-ester, halogenfree and halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group; and (iv) disulfides, including pyridylsulfonyl, the resulting sulfide metabolism. Nucleophilic groups on the molecule drugs include, but are not limited to, amine, thiol, hydroxyl, hydrazide, Aksinya, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group capable of reacting with the electrophilic groups on linker parts and linker reagents with the formation of covalent bonds.

Constructed on the basis of cysteine antibodies, capable of forming conjugates with linker reagents, can be obtained by treatment with a reducing agent such as DTT (reagent of Cleland, dithiothreitol) or TCEP (Tris hydrochloride(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 miaocheng and intrachain disulfide bonds (example 5). For example, a full-sized constructed on the basis of cysteine monoclonal antibody (thio-Mab), expressed in CHO cells, is subjected to reduction reaction of approximately 50-fold excess of TCEP for 3 hours at 37 the With to restore the disulfide bonds in the addition products of cysteine, which may be formed between the newly introduced cysteine residues and the cysteine present in the culture medium. Restored thio-Mab diluted and loaded onto HiTrap column S in 10 mm sodium acetate, pH 5, and elute PBS containing 0.3 M sodium chloride. Disulfide bonds between cysteine residues present in the parent Mab, restore again using diluted (200 nm) of aqueous copper sulfate (CuSO4) at room temperature over night. Alternatively, after reductive cleavage of cysteine adducts, to restore intrachain disulfide groups constructed on the basis of cysteine antibodies, can be used for effective oxidant, such as dehydroascorbic acid (DHAA). Can also be used and other oxidants, that is, oxidizing agents, and the conditions of oxidation, known to specialists. Also effective is the oxidation in air. This stage soft partial re-oxidation with a high degree of reliability contributes to the formation of interchain disulfides and preservation of thiol groups newly introduced cysteine residues. Then add approximately 10-fold excess of intermediate connection “drug-linker”, for example, MC-vc-PAB-MMAE, mix and leave at the Erno for 1 hour at room temperature to effect the conjugation with the formation of conjugate anti-CD79b antibody-drug”. Then conjugate mixture is subjected to gel filtration, loaded onto HiTrap column S and elute from the column to remove excess intermediate connection “drug-linker, and other impurities.

Figure 23 illustrates a General 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 environment can form disulfide adducts. These cysteine adducts, circled for a representative thio-Mab (left) figure 23 should be restored with getting constructed on the basis of cysteine antibodies that can be used for subsequent conjugation reactions. Cysteine adducts, presumably with different megamachine disulfide bonds, is subjected to reductive decomposition under the action of vosstanovitelya, such as TSAR, to obtain the reduced forms of the antibodies. Megamachine disulfide bond between related cysteine residues are again formed under conditions of partial oxidation, such as copper sulfate, DHAA or atmospheric oxygen. Newly commissioned, designed and nevasa the major cysteine residues become available to react with linker reagents or intermediate compounds “drug-linker”, resulting in the formation of the conjugates of the antibodies according to the invention. Thio-Mab expressed in cell lines mammals form the external Cys-adduct, conjugated with the introduced Cys through education-S-S-bond. Therefore, purified thio-Mab should be subjected to recovery and oxidation as described in example 5, to obtain the reactive thio-Mab. These thio-Mab is used to obtain a conjugate with maleimides containing cytotoxic drugs, fluorophores and other labels.

Immunoliposome

Described here anti-CD79b antibodies can also be obtained in the form of immunoliposome. "Liposome" is a small vesicles composed of lipids of different types of phospholipids and/or surfactant that can be used to deliver the drug to a 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, get methods known in the art, such as methods described in the 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 WO 97/38731, published on 23 October 1997 Liposomes with increased half-life in blood is described in U.S. patent No. 5013556.

p> Specifically used liposomes composed of these lipids as phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-Feh), can be obtained by evaporation from a reverse phase. Liposomes extruded through filters with defined pore size, resulting in a gain liposomes of the desired diameter. Fab'-fragments of the antibodies according to the invention can be conjugated to the liposomes as described in the publication of Martin et al., J. Biol. Chem. 257:286-288 (1982), as a result of the reaction of the disulfide exchange. The liposome may contain, but not necessarily, the 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 bind to CD79b polypeptide described above. If desired, can also be selected and other antibodies with specific biological properties.

The growth-inhibitory effect of anti-CD79b antibodies according to the invention may be assessed by methods known in the art, e.g., using cells which Express the CD79b polypeptide either endogenously or following transfection with the gene CD79b. So, for example, appropriate tumor cell lines and CD79b-transfetsirovannyh cells can be processed monoclonality-CD79b antibody according to the invention at various concentrations for a few days (for example, 2-7 days), and painted with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation rate may be a comparison of absorption3H-thymidine by the cells treated in the presence or in the absence of anti-CD79b antibodies according to the invention. After treatment, cells are harvested, and the radioactivity incorporated into DNA, and count 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 this cell line. Inhibition of growth of tumor cells in vivo can be determined by various methods known in the art. Tumor cell line may be a cell line, sverkhekspressiya CD79b polypeptide. In one embodiment of the invention the anti-CD79b antibody inhibits proliferation 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%, in comparison with untreated tumor cells when the antibody concentration of about 0.5 to 30 μg/ml of the growth Inhibition can be measured at the antibody concentration of about 0.5 to 30 μg/ml or about 0.5 nm to 200 nm kletochnoi culture, where the specified growth inhibition determined in 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 at about 1 μg/ml to 100 mg/kg body weight results in reduction in tumor size or reduction of the proliferation rate of tumor cells over a period of time from about 5 days to 3 months, and preferably for a period of time from about 5 to 30 days after the first injection of antibodies.

For selection of anti-CD79b antibody, which induces cell death, can be estimated loss of membrane integrity as determined, for example, by the absorption of iodide of propecia (PI), Trypanosoma blue or 7AAD compared with the control. The analysis of PI uptake may be carried out 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 (for example, about 10 μg/ml). Cells are incubated for 3 days. After each treatment, the cells are washed and divided into aliquots in test tubes (35 mm, 12×75) with tight-fitting lid (1 ml per tube, 3 tubes per group processing) to remove accumulations of cells. Then in a test tube add PI (10 μg/ml). Samples can be analyzed on the por is accurate cytometer FACSCAN® and using computer programs 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 antibodies that induce cell death.

For screening of antibodies that bind to an epitope on CD79b polypeptide associated with interest antibody can be carried out routine analysis on cross-blocking, such as the analysis described in Antibodies, 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 test antibody with the same site or epitope that binds a known anti-CD79b antibody. Alternative or in addition may be carried out mapping of epitopes with 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 ensure "proper" installation. In a different method, peptides corresponding to different regions of the CD79b polypeptide, can be used in the analysis of competitive binding with the test antibodies or with a test antibody and an antibody that binds with a characterized or known EP the top.

2. Some methods of screening libraries

Anti-CD79b antibodies according to the invention can be obtained using combinatorial libraries to screen for antibodies with the desired activity or desired activities. For example, the specialists, the number of known methods of producing libraries of phage display and screening such libraries for antibodies with the desired binding properties. Such techniques are generally 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 clones synthetic antibodies are selected by screening phage libraries containing the phage, which is different fragments of variable regions of antibodies (Fv)that is attached to the protein shell of the phage. Such phage libraries are panning using affinity chromatography against the desired antigen. Clones expressing Fv-fragments, the ability to communicate with the desired antigen is subjected to adsorption on the antigen and thereby its separation from nesvezhije clones in the library. Then bind the clones elute with antigen, then the number of these clones can be increased by performing additional cycles of adsorption/elution of the antigen. Any of the anti-CD79b antibody according to the invention can be obtained when the label appropriate screening procedures antigen for selection to represent the interest of the phage clone with the subsequent construction of a full-sized clone of the anti-CD79b antibodies using the Fv sequences, derived from the interest of the phage clone, and appropriate sequences of the constant region (Fc), described by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

In some embodiments of the invention antigennegative domain antibodies consist of two variable (V) regions, consisting of approximately 110 amino acids, one of which is located in the light chain (VL)and the other in the heavy chain (VH), and in these variable regions there are three hypervariable loops (HVR) or complementarity-determining region (CDR). Variable domains can be functionally represented on the phage or as single-chain Fv- (scFv)fragments, in which VH and VL are covalently bound to each other through a short flexible peptide, or as Fab fragments, in which each of these variable domains are joined 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 generally called "fagbemi Fv-clones" or "Fv-clones".

The sets of genes VH and VL can be separately cloned using polymerase chain reaction (PCR) and subjected to non-specific recombination in phage libraries, and then they can be analysed on antihistamie clones, ka is described in the publication Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources make it possible to obtain antibodies with high affinity for the immunogen, and it does not require the procedure of constructing a hybrid. An alternative may be cloned set of human antibodies wild-type one source of human antibodies against non-autoimmune antigens and autoantigens wide range, without resorting to any immunization, as described by Griffiths et al., EMBO J 12:725-734 (1993). And finally, the original library can be also synthesized by cloning paranirvana segments V-gene from stem cells, and using PCR primers containing randomised 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 accession to the minor envelope protein pIII, use of filamentous phage. Antibody fragments can be represented 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, for example, as described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or as Fab fragments, in which one circuit connec is on to pIII, and the other is secreted in periplasm bacterial host cell, where the Assembly structure of the envelope protein Fab, which is displayed on the surface of phage by replacing some of envelope proteins of wild-type, for example, as described in the publication Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

Typically, nucleic acids containing genes encoding antibody fragments, derived from immune cells taken from humans or animals. If you want to get a library consisting mainly of clones of anti-CD79b antibodies, the individual subjected to immunization CD79b to produce it humoral response, and then to construct the library, isolated spleen cells and/or circulating b cells that do not belong to the population of peripheral blood lymphocytes (PBL). In a preferred embodiment of the invention a library of gene fragments of human antibodies that are mostly clones of anti-CD79b antibodies, receive by generating a response in the form of production of anti-CD79b antibodies in transgenic mice bearing an array of functional genes of the human immunoglobulin (and not containing a functional system of production of endogenous antibodies), where such CD79b-immunization will lead to the formation of b cells producing human antibodies against CD79b. Generating transgen the x mice, producing a human antibody, as described below.

Additional enrichment of the cell population reactive anti-CD79b antibodies can be achieved by using a suitable screening procedure for the selection of b-cells expressing CD79b-specific membrane-associated antibody, for example, by separation of cells using CD79b-affinity chromatography or adsorption of cells on CD79b, labeled fluorochromes, and subsequent cell sorting with activation of fluorescence (FACS).

Alternative use of spleen cells and/or b cells or other LPK, taken from unimmunized donor provides a better representation of the possible repertoire of antibodies, and also allows you to construct a library of antibodies in animals of any kind (in humans or in other animals), which CD79b is not antigenic. To create libraries, including the design of antibody genes in vitro, the individual take stem cells and release nucleic acid encoding paranirvana segments of antibody genes. Interest of the immune cells can be isolated from animals of different species such as human, mice, rats, hares, wolves, dogs, cats, pigs, cows, horses, birds, etc.

Nucleic acid encoding the gene segments of variable regions of the antibodies (including VH - and VL-segment is s), allot of interest in cells and amplified. In the case of libraries rearranging genes VH and VL, the desired DNA can be obtained by separating the genomic DNA or mRNA from the cells followed by the polymerase chain reaction (PCR) using primers corresponding to the 5'- and 3'-ends rearranging genes VH and VL, are described in the publication Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), and thereby obtain different sets of V-genes for expression. V-genes can be amplified from cDNA and genomic DNA using reverse primers 5'-end of exon encoding the Mature V-domain, and direct primers derived from the J-segment, as described in publications 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 the leader exon, as described in the publication of Jones et al., Biotechnol., 9:88-89 (1991), and direct the primers can be entered in the constant region, as described in the publication Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarities can be obtained degenerate primers described in the publication 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 the ima is working arrangements VH and VL, 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 by the method described in the publication Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). For cloning the amplified DNA into expression vectors, PCR primer can be introduced rare restriction sites as a label at one end, as described by Orlandi et al. (1989), or such introduction can be carried out using PCR amplification using a labeled primer, as described in 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 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); and these cloned segments (including all major conformation of loop H1 and H2) can be used to obtain different sets of VH genes using PCR primers encoding the H3 loop, with different sequences and lengths, as described in the publication Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Can be also obtained sets VH, with all the variety of sequences, centered in a long H3 loop the same length, as described in the publication Barbas et al., Proc. Nal. Acad. Sci. USA, 89:4457-4461 (1992). Were cloned and sequenced the human segments Vκ and Vλ (as reported in the publication Williams & Winter, Eur. J. Immunol., 23:1456-1461 (1993)), and these segments can be used to produce synthetic sets of light chains. Sets the synthesized V genes derived from the folded structure of a number of VH and VL, and L3 and H3 of different lengths, will encode antibodies with significantly varying structure. After amplification of the coding DNA of the gene V gene segments V germline can be rurangirwa in vitro methods described in the publication Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

Sets the fragments of antibodies can be constructed by combining 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 the publication Hogrefe et al., Gene, 128:119-126 (1993), or in vivo by combinatorial infection, for example, a 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 the double-stranded nature of Fab fragments to eliminate restrictions on the size of the library and associated with a transformation efficiency of E. coli. The original sets of VH and VL clone separately, one in fahmida, and the other in the phage vector. Then two libraries unite by infitsirovanie the bacteria, containing fahmida, phage, so that each cell contained various combinations, and the size of the library was limited by the number of cells present (about 1012clones). Both vectors contain the signals of recombination in vivo, resulting in the genes of the VH and VL undergo recombination to form one replicon together and packaged into phage virions. These huge libraries represent a large number of different antibodies with high affinity (Kd-1about 10-8M).

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

Antibodies produced by the source libraries (natural or synthetic)may have a low affinity (Kd-1about 106 to 10 7M-1), but the affinity maturation can also be simulated in vitro by constructing and re-selection of secondary libraries, as described in the publication Winter et al. (1994), see above. For example, mutations can be introduced randomly in vitro using 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, the affinity maturation can be accomplished through random mutation of one or more CDRs, for example, by PCR performed using primers carrying randomised sequence, covering the interest CDR in selected Fv-clones, and by screening clones with higher affinity. In the application WO 9607754 (published on 14 March 1996) described a method for inducing mutagenesis in a hypervariable region of the light chain of immunoglobulin with obtaining a library of light chain genes. Another effective method is the recombination of domains VH or VL, selected using phage display, with sets of natural variants of the V-domain, isolated from maimonidean donors, and screening for higher affinity, carried out in several rounds of the permutation circuits, as described in the publication Marks et al., Biotechnol., 10:779-783 (1992). This is method allows to produce antibodies and antibody fragments with affiniscape about 10 -9M or less.

Screening of the libraries can be performed using various methods known in the art. For example, CD79b can be used to cover holes adsorption tablets or expressed on the cells of the hosts attached to the adsorption tablets, or it can be used for cell sorting, or anywhereman with Biotin for capture on streptavidin coated, or it can be used in any other method of penning libraries phage display.

Samples of phage libraries are subjected to contact with immobilized CD79b under conditions suitable for binding at least part of the phage particles with the adsorbent. Usually to simulate physiological conditions, choose the appropriate pH, ionic strength, temperature, etc. Phages associated with the solid phase, washed, and then elute the acid, for example, as described in the publication Barbas et al., Proc. Natl. Acad. Sci USA, 88:7978-7982 (1991), or alkali, for example, as described in the publication Marks et al., J. Mol. Biol., 222:581-597 (1991), or by competitive binding with CD79b antigen, for example, in accordance with the procedure similar to that 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, about Gasunie 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 fragments of antibodies simultaneously to contact with the antigen on the same phage. Antibodies with fast kinetics of dissociation and low affinity binding) may be fixed due to the short-term leaching, the use of multivalent phage performance and high density coating antigen on the solid phase. This high density not only helps to stabilize the phage through 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 binding can be improved by a more prolonged leaching and the use of monovalent phage view, described in the publication of Bass et al., Proteins, 8:309-314 (1990) and in WO 92/09690, and also due to the low density of the coating antigen, as described in the publication Marks et al., Biotechnol., 10:779-783 (1992).

Can be carried out selection between fagbemi antibodies with different affiniscape, even with slightly different affinity to CD79b. However, random mutation selected antibody (e.g., vnesennnaya in the implementation of some of the above methods of affinity maturation), probably Bud is t lead to the formation of many mutants, most of which will be in contact with the antigen, and some of them with 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 excess biotinylated CD79b, but this biotinylated 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 paramagnetic spheres, coated with streptavidin. This "equilibrium capture" enables the selection of antibodies by affinity binding sensitivity, which allows of a large excess of phages with lower affinity to allocate mutant clones having 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, usually expressing CD79b. In one of its variants the present invention relates to anti-CD79b antibodies that block the binding of leagues the NDA with CD79b CD79b, but do not block the binding of the ligand CD79b with a second protein. Fv clones corresponding to such anti-CD79b antibodies can be selected by: (1) selection of clones of anti-CD79b antibody of ragovoy library, as described above, and optional amplification of the selected population of phage clones by culturing such population in a suitable bacterial host; (2) selection CD79b and a second protein against which you want to produce blocking and non-blocking activity, respectively; (3) adsorption of phage clones anti-CD79b antibodies immobilized on CD79b; (4) using an excess of the second protein elution 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 performing 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 readily isolated and sequenced according to standard procedures (e.g. the R, using oligonucleotide primers capable of specifically amplifying the target of interest coding region of the heavy and light chains from hybridoma or ragovoy DNA template). After selecting this DNA can be incorporated into expression vectors, which are then transferout in cell host, such as E. coli cells, simian COS cells, cells of the Chinese hamster ovary (Cho or myeloma cells that other cases do not produce a protein of the immunoglobulin, resulting in these recombinant cells masters synthesized the desired monoclonal antibodies. Discussion recombinant expression of the 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 may 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 full-sized heavy and/or light chains, or fragments thereof. It should be noted that for these purposes can be used constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and such constant area is to be derived from human or animals of any species. In the definition used here, the term "chimeric" and "hybrid" antibody is an Fv clone, derived from the DNA of the variable domain of an animal of the same 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(s) sequence(s) "hybrid" full-size heavy chain and/or light chain. In some embodiments of the invention Fv-clone, derived from the DNA of a human variable domain, attached to the DNA of a human constant region coding education(s) sequence(s) for all full-sized human heavy and/or light chains or fragments.

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

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

Antibodies according to the invention can also be used in ADEPT in the form of antibodies conjugated with a prodrug-activating enzyme which converts a prodrug (e.g., peptidyl chemotherapeutic agent, see WO 81/01145) in the active anti-cancer drug. See, for example, WO 88/07378 and U.S. patent No. 4975278.

The enzyme component immunoconjugate suitable for ADEPT, includes any enzyme capable of acting on a prodrug, namely to turn this prodrug into 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 prodrugs into free drugs; Ukrainian, which can be used to turn sulfidogenic prodrugs into free drugs; sitoindosides, which can be used for converting non-toxic 5-fertilizin protivorechivoe drug, namely, 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which can be used to turn petitcodiac prodrugs into free drugs; D-alanismorissette, which can be used for converting prodrugs that contain D-amino acid substituents; carbohydrate-splitting enzymes, such as β-galactosidase and neuraminidase, which can be used for converting glycosylated prodrugs into free drugs; β-lactamase, which can be used for making medicines, derivatizing β-lactams, into free drugs; and penicillin-amidase, such as penicillin V-amidase or penicillin G-amidase, which can be used for making medicines, derivatizing of nitrogen atoms amine phenoxyacetyl or fenilatsetilenom groups, respectively, into free drugs. Alternative antibodies with enzymatic activity, also known in the art as "abzyme", can be used to convert the prodrugs according to the invention into free active drugs (see, for example, Massey, Nature 328:457-458 (1987)). The conjugates of the antibody-Abim" which may be obtained, as described in this application for delivery of abzyme in the population of tumor cells.

The enzymes according to the invention can be covalently attached to an anti-CD79b antibody by methods known in the art, for example, using heterobifunctional cross-linking reagents discussed above. Alternative hybrid proteins containing at least antigennegative region of the antibodies according to the invention, associated with at least a functionally active portion of an enzyme according to the invention, can be designed by methods of recombinant DNA, known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984).

D. Anti-CD79b antibody

In addition to the described here anti-CD79b antibodies, is also considered getting variants of the anti-CD79b antibody. Options anti-CD79b antibodies can be obtained by introducing appropriate nucleotide modifications in coding 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 modify membrane-zakalivayuschie properties.

The changes described here anti-CD79b antibodies may be made, for example, using any of the methods and guidelines for making konservativnyh and nonconservative substitutions, described, for example, in U.S. patent No. 5364934. These modifications can be substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that leads to a change in amino acid sequence compared to the sequence of a natural antibody or polypeptide. Such a modification is, but not necessarily, replacing at least one amino acid residue of any other amino acid in one or more domains of an anti-CD79b antibody. In order to determine whether you can build, replace, or deleteroute amino acid residue without any negative impact on the desired activity of antibodies can be carried out by comparing the sequence of the anti-CD79b antibodies with a sequence homologous known protein molecules to minimize the number of modifications of the amino acid sequences listed in the significant body of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as replacement of leucine, serine, that is conservative amino acid substitutions. Insertions or deletions may, but not necessarily, be in the range of about 1 to 5 amino acids. Allowable modifications can be determined by systematically introduced the I insertion, 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 at the N-Terminus or at the 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 anti-CD79b antibodies can be obtained by any of various standard methods. The desired peptide fragments can be obtained by the method of chemical synthesis. An alternative method involves the production of antibodies or fragments of the polypeptide by enzymatic degradation, for example, by treating the protein with an enzyme known cleaves proteins at sites defined by particular amino acid residues, or by hydrolysis of DNA suitable restricteduse enzymes, and highlight the desired fragment. Another suitable method involves the selection and amplification of the DNA fragment encoding the desired fragment of the antibody or polypeptide using polymerase chain reaction (PCR). Oligonucleotide is, which define the desired ends of the DNA fragment used in PCR as 5'- and 3'-primers. Preferably the fragments of the anti-CD79b antibodies have at least one biological and/or immunological activity similar to the activity described here native anti-CD79b antibodies.

In specific embodiments of the invention representing the interest of conservative substitutions are presented in table 8 under the heading of "Preferred substitutions". If such substitutions result in a change in biological activity, can be entered more significant replacement, referred to in table 8 representative substitutions", or also described below in the section belonging to the class of amino acids, and then the obtained products can be skanirovaniya.

Table 8
The original remainsRepresentative replacementPreferred 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; AlaAla
His (H)Asn; Gln; Lys; ArgArg
Ile (I)Leu; Val; Met; Ala;
Phe; norleucine
Leu

Thr
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)Thr
Thr (T)SerSer
Trp (W)Tyr; PheTyr
Tyr (Y)Trp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe;
Ala; norleucine
Leu

Substantial changes in function or immunological properties of anti-CD79b antibodies can be accomplished by selecting substitutions that 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 remnants 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) acid 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.

Nonconservative substitutions lead to the replacement of a member of one of these classes with a member of another class. Such replacement can be entered in oblastrenovation replacement, or more preferably in the rest (non-conservative) region.

Such modifications can be made by methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning mutagenesis and PCR mutagenesis. For producing variant DNA anti-CD79b antibodies, cloned DNA may 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 mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or mutagenesis carried out by other methods.

To identify one or more amino acids over the entire sequence can also be carried out 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, obviously, but less likely, changes the conformation of the main chain of this variant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Alanine is also preferred because it represents the most common amino acid. In addition, he shall ACOs is 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 provide adequate amounts of variant, it can be used esoteric amino acid.

To improve stability of the molecule to oxidation and to prevent unwanted cross-links can also be replaced by any cysteine residue not involved in maintaining the "right" confomal anti-CD79b antibodies, and usually this 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) to the anti-CD79b antibody may be added(s) cysteine(s) relationship(s).

Particularly preferred substitution is the replacement of one or more residues of the hypervariable region of the parent antibody (e.g., gumanitarnogo or human antibody). In General, the obtained(s) option(s)chosen one(s) for further development, will be(ut) to contribute to improved biological properties compared with the properties of the parent antibody from which they originate. A standard way of generating such variants with substitutions provides for the affinity maturation using the method of phage view. Several sites hypervirial the school area (for example, 6-7 sites) is subjected to mutation to generate all possible amino acid substitutions at each site. Thus obtained variants of the antibodies can be presented on the particles of filamentous phage in the form of monovalent hybrids with the product of the gene III, Packed in every part of the phage M13. Then the options presented on the phage, sceneroot on their biological activity (e.g. binding affinity of), as described in this application. To identify sites in hypervariable region that are candidates for modification may be performed alanine-scanning mutagenesis, which allows to identify the remains of the hypervariable region, play an important role in binding to the antigen. Alternative or in addition it may be beneficial to analyze a crystal structure of the complex antigen-antibody for the identification of contact sites between antibody and CD79b polypeptide. Such contact residues and neighboring residues are candidates for replacement, carried out in accordance with methods developed here. Upon receipt of such options, the panel of these variants is subjected to screening as described in this application, and antibodies that detect superior properties in one or more relevant assays may be selected for further investigation.

Mo is ecoli nucleic acid, encoding amino acid sequence variants of the anti-CD79b antibodies, receive various methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of a natural amino acid sequence variants) or getting through mediated by the oligonucleotide (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis previously received options or unmodified variant anti-CD79b antibodies.

E. modification of the anti-CD79b antibodies

Covalent modification of anti-CD79b antibodies are included in the scope of the present invention. One type of covalent modification is the reaction of interaction desired amino acid residues of an anti-CD79b antibodies 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 carried out, for example, for cross-linking an anti-CD79b antibody with a water-insoluble matrix carrier or surface used in the method of purification anti-CD79b antibodies, and Vice versa. The most commonly used cross-linking agents are, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, N-hydroxysuccinic ideavery, for example, esters formed 4-azidoaniline acid; homobifunctional imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester), bifunctional maleimides, such as bis-N-maleimido-1,8-octane, and such agents as methyl-3-[(p-azidophenyl)dithio]propionamide.

Other modifications are desametasone glutaminergic and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of 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 the changing nature of the native glycosylation of the antibody or polypeptide. The term "the changing nature of the native glycosylation"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 liquiline residues of chemical and/or enzymatic methods), and/or adding one or more glycosylation sites that are not present in the native sequence anti-CD79b antibody. In addition, this term includes qualitative changes in the glycosylation of the native proteins, leading to changes in the nature and number of attendees carbohydrate groups.

Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked glycosylation means attaching carbohydrate group 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 sequences of recognition for enzymatic joining carbohydrate portion to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide promotes the creation of a potential site of glycosylation. O-linked glycosylation means joining one of the sugars, such as N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate mainly to serine 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 way is this modification of amino acid sequence, as a result of 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 carried out by adding to the sequence of the original anti-CD79b antibodies of one or more serine or treoninove residues or their substitutions in the sequence of the original antibody (for sites of O-linked glycosylation). Amino acid sequence of the anti-CD79b antibody can be, but not necessarily, modified by the modification at the DNA level, particularly by mutation of the DNA encoding the anti-CD79b antibody in pre-selected basis, the result will be generated codons transmitted into the desired amino acids.

Another way of increasing the number of carbohydrate molecules on anti-CD79b antibody is a 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 on September 11, 1987, and in the publication 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, which Slu is at 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 Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate molecules to the polypeptide 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 different molecules of non-protein polymers, such as polyethylene glycol (PEG), polypropyleneglycol or polyoxyalkylene, as described in U.S. patents№№ 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337. The antibody can also be made in the prepared microcapsules, for example, methods koatservatsii or interfacial polymerization (for example, in hydroxymethylcellulose or gelatin microcapsule and polymetylmetacrylate microcapsule, respectively, in systems for the delivery of colloidal medicines (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. This technique is described in the manual Remington''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 anti-CD79b antibody associated with other heterologous floor is a peptide or another amino acid sequence.

In one embodiment of the invention, such a chimeric molecule contains a hybrid anti-CD79b antibody with a polypeptide tag, ensure the availability of the epitope, which can be selectively contacted by the antibody against the tag. Epitope tag is typically located at the amino - or carboxy-end of the anti-CD79b antibody. The presence of such labeled epitope forms of anti-CD79b antibodies can be detected using antibodies against the polypeptide tags. In addition, the presence of the epitope tag enables you to easily clear anti-CD79b antibody by the method of affinity purification using an anti-CD79b antibody against the tag or affine matrix of another type, which is associated with the epitope tag. Various polypeptides tags and their respective antibodies are well known in the art. Examples are polyhistidine tag (poly-his) or the label "poly-histidine-glycine (poly-his-gly); polypeptide tag HA of influenza virus and the antibody against the polypeptide, 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)], c-myc-tag and antibodies against such labels, 8F9, 3C7, 6E10, G4, B7 and 9E10 [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and label glycoprotein D (gD) of herpes simplex virus and antibody against such labels [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other polypeptide tags are Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the peptide epitope KT3 [Martin et al., Science, 255:192-194 (1992)]; peptide epitope α-tubulin [Skinner e al., J. Biol. Chem., 266:15163-15166 (1991)]; and label the peptide protein 10, encoded by the genome of T7" [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

In an alternative embodiment of the invention, the chimeric molecule may contain hybrid "anti-CD79b antibody-an immunoglobulin or a particular region of an immunoglobulin". In the case of a bivalent form of the chimeric molecule (also called "immunoadhesins"), this hybrid can be attached to the Fc-region of IgG molecules. IgG-hybrids preferably include the substitution of at least one variable region of the Ig molecule soluble form (with deletionism or inaktivirovannye transmembrane domain) anti-CD79b antibody. In a particularly preferred variant of the invention, the hybrid immunoglobulin includes the hinge region, CH2and CH3or hinge region, CH1CH2and CH3, IgG1 molecule. Description of the production of hybrid immunoglobulins can also be found in U.S. patent No. 5428130, issued June 27, 1995

F. Receiving anti-CD79b antibodies

The following describes mainly the production of anti-CD79b antibodies by culturing cells transformed or transfected with a vector containing a nucleic acid encoding an anti-CD79b antibody. This is provided that to obtain anti-CD79b antibodies can be applied alternative methods, well-known experts who am. 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 can be carried out manually or automatically. Automated synthesis may be achieved, for example, peptide synthesizer, Applied Biosystems Peptide Synthesizer (Foster City, CA) according to manufacturer's instructions. Different parts of the anti-CD79b antibodies may be chemically synthesized separately and in combination with chemical or enzymatic methods to give the desired anti-CD79b antibodies.

1. The isolation of DNA encoding anti-CD79b antibody

DNA encoding anti-CD79b antibody can be obtained from a cDNA library isolated from tissue, which presumably mRNA anti-CD79b antibodies and expresses such mRNA into 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 can also be obtained from a genomic library or by known application methods of synthesis (e.g., automated nucleic acid synthesis).

Libraries can be skanirovaniya using probes (such as oligonucleotide, consisting of 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 conducted in accordance with standard procedures such as the procedure described in the publication Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method of selection of the gene encoding anti-CD79b antibody is PCR technology [Sambrook et al., see above; 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 length sufficient and sufficiently well-defined to minimize false-positive results. The oligonucleotides preferably have been labelled so that they can be detected after hybridization with 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 labels. The hybridization conditions, including conditions of moderate and high rigidity described in Sambrook et al., see above.

Sequences identified in these methods is chininga libraries, can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private databases of sequences. Identity sequence (amino acids or nucleotides) in certain regions of the molecule or across the full sequence can be determined by methods known in the art and described in this application.

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the derived amino acid sequence, first described in this application, and, if necessary, in accordance with standard procedures primer extension described in Sambrook et al., see supra, to detect precursors and processing of intermediate forms of mRNA, which can not be subjected to reverse transcription into cDNA.

2. The selection and transformation of host cells

Cell owners transferout or transform using the here described expression or cloning vectors for the production of anti-CD79b antibodies, and then cultured in standard culture medium, modified if necessary, for inducion the of the promoters, selection of transformants, or amplifying the genes encoding the desired sequences. Culturing conditions, such as environment, temperature, pH, etc. can be selected by a specialist without undue experimentation. Basically the principles, protocols and practices for maximizing the productivity of cell cultures can be found in the publication of the Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., see above.

Methods for transfection of eukaryotic and transformation of prokaryotic cells, including the introduction of DNA into the cell host for DNA replication either as an extrachromosomal or chromosomal integrator, well known to the average person skilled in the art, for example, such methods are methods that are mediated CaCl2CaPO4and liposomes, methods using polyethylene glycol/DMSO and electroporation. Depending on the host cells transformation carried out by standard methods appropriate for such host cells. In the case of prokaryotes, usually spend processing calcium, and calcium chloride, as described in the manual, Sambrook et al., see above, or electroporation. Infection with Agrobacterium tumefaciens spend for transformation of certain plant cells, as described in the publication Shaw et al., Gene, 23:315 (1983) and in the application WO 89/05859, published June 29, 1989 In case the use of mammalian cells, 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 system cells of the host mammal 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 used and other methods, such as microinjection into the nucleus, electroporation, fusion of bacterial protoplasts with intact cells, or methods using polycation, for example, polybrene, poliarnaia. 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, the 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 use is mioticescimi cells masters are Enterobacteriaceae, such as Escherichia, e.g. 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. licheniformis (e.g., B. licheniformis 41P described in DD 266710, published 12 April 1989), Pseudomonas such as P. aeruginosa, Rhizobia, Vitreoscilla, and Paracoccus Streptomyces. These examples are only illustrative, but not limiting purposes. One of the most preferred hosts or parent host cells is strain W3110, because it is the most common strain of the host for the fermentation of recombinant DNA products. Cell host preferably secrete minimal amounts of proteolytic enzymes. For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 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, with 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 degPompT rbs7ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with deletion mutation deP 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 an E. coli strain having mutant periplasmic protease and described in U.S. patent No. 4946783, issued 7 August 1990, also Suitable are 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 provided only for purposes of illustration and are not limiting. Methods of constructing derivatives of any of the aforementioned bacteria with certain 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 cells of bacteria. For example, cells of the species E. coli, Serratia, or Salmonella can be used as hosts, if for delivery replicon are well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Usually cells are the owners must secrete minimal amounts of proteolytic enzymes, and optionally, in a cell culture can be added protease inhibitors. Alternative suitable methods of cloning in vitro are, for example, PCR or other polymerase reactions of nucleic acids.

Full-size antibody, antibody fragments and hybrid proteins, antibodies can be produc is arranged in bacteria, in particular, if there is no need to glycosylation and effectory functions Fc, for example, if therapeutic antibody conjugated with a cytotoxic agent (e.g., toxin), and immunoconjugate is effective for the destruction of tumor cells. Full-size antibodies have a longer half-life in the bloodstream. Production in E. coli is faster and less expensive method. The 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 field of translation initiation (TIR) and signal sequences for optimizing expression and secretion, and these patents are introduced in the present description by reference. After the expression of the antibody isolated from the cell paste of E. coli as a soluble fraction, and this antibody can be purified, for example, on a column of protein a or G-protein, depending on the isotype. Final cleaning can be carried out in a manner analogous 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 eukaryotically microorganisms, such as filamentous fungi. The most commonly used lower eukaryotic microorganism host is Saccharomyces cerevisiae. Other microorganisms are Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 [1981]; EP 139383, published 2 may 1985); cells of Kluyveromyces hosts (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 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 183070; Sreekrishna et al., J. Basic Environ., 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 (application 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 are, but are not limited to, methylotrophy yeast, for example, the 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 of C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Cells-the owners, the right to expression and glycosylation of anti-CD79b antibodies, can be derived from multicellular organisms. Examples of invertebrate cells include insect cells such as cells of Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potatoes, soybeans, Petunia, tomatoes and tobacco. Were identified various baculovirus strains and variants and corresponding permissive cells are the owners of insects occurring 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, the most interesting 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 the virus SV40 (COS-7, ATCC CRL 1651); cell line human embryo kidney (293 cells or 293, subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); kidney cells baby hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (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); carcinoma cells 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); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065 tumor cells in the mouse mammary gland (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); the cells, MRC 5; FS4 cells and cell line human hepatoma (Hep G2).

Cell owners transform the 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 amplifying the genes encoding the desired sequences.

3. The selection and application of the vector can replicate

For recombinant production of antibodies according to the invention nucleic acid (e.g., cDNA or genomic DNA)encoding such antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody can be readily isolated and sequenced according to standard procedures (for example, using oligonu etigny probes, can specifically bind to genes encoding the heavy and light chain antibodies). For this purpose suitable are many vectors. The choice of vector depends in part on the used host cell. Mostly preferred by the 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 particles or phage. The corresponding nucleic acid sequence may be incorporated into the vector in accordance with various procedures. Basically, DNA is inserted into the corresponding(s) site(s) restrictive endonuclease 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, an enhancer element, a promoter and termination sequence transcription. Construction of suitable vectors containing one or more of these components, carry out 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 represent signals the second sequence, or other polypeptide having a specific cleavage site at the N-Terminus of the Mature protein or polypeptide. In General, the specified signal sequence may be a component of the vector, or such a sequence can be part of the DNA encoding the 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, penitsillinazy, lpp or thermostable enterotoxin II. For secretion in yeast signal sequence may be, for example, a leader sequence of yeast invertase, a leader sequence of alpha-factor (including the leader sequence of the 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, a leader sequence glucoamylase C. albicans (patent EP 362179, published 4 April 1990), or signal sequence, as described in the application WO 90/13646, published November 15, 1990, When the expression in mammalian cells, to direct secretion of the protein can be used signal sequence mammals, such as the signal sequence derived from Secretary the activated polypeptides of the same species or related species, as well as viral secretory leader sequence.

A. Prokaryotic cells-owners

Polynucleotide sequences encoding polypeptide components of the antibodies 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 hybridoma cells. Alternative polynucleotide can be synthesized using the synthesis of nucleotides or PCR methods. After receiving sequences encoding polypeptides, these sequences is inserted into a recombinant vector that can replicate and Express heterologous polynucleotide in prokaryotic hosts. For the purposes of the present invention can be used for many affordable and known vectors. The choice of an appropriate vector depends mainly on the size of nucleic acids, embedded in the specified vector, and from a specific 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 a particular cell of the host in which it is located.

Basically plasmid vectors, with the holding of replicon and regulatory sequences, derived from species compatible with the host-cell, are used together with the same owners. 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, yeast and viruses are well known. The origin of replication derived from plasmid pBR322 and contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and also allows you to easily identify transformed cells, is suitable for most gram-negative bacteria; the origin of the 2μ plasmid is suitable for yeast, and various viral origin (SV40, a virus polyoma, 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 these microbial organism for expression of endogenous proteins, or they can be modified so as to include such promoters. Examples of derivatives of pBR322, used for the expression of specific antibodies, are described in detail in U.S. Pat is NTE U.S. No. 5648237, Carter et al.

In addition, phage vectors containing replicon and regulatory sequences that are compatible with the microorganism host, can be used as transformation vectors in combination with these hosts. For example, bacteriophage such as λGEM.TM.-11, can be used to obtain a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

The expression vector according to the invention may contain two or more pairs of the promoter-cistron encoding each polypeptide components. The promoter is noncoding regulatory sequence, localized above (from 5'-end) from cistron, modulating its expression. Prokaryotic promoters are usually divided into two classes, inducible and constitutive. The inducible promoter is a promoter that initiates the increase in the levels of transcription of cistron under its control, in response to changes in 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 masters, well-known to specialists. The selected promoter may be functionally attached to castronno the DNA encoding the light or heavy chain, through allocation of a promoter of a DNA source through its hydrolysis restricteduse enzymes and embedding the selected promoter sequence in 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 used heterologous promoters, because they, compared to the native promoter for the desired polypeptides, can significantly increase the level of transcription and increase yields expressed gene target.

Promoters recognized by a variety of potential cells masters, well-known to specialists. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the promoter 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, Nucleic 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 Shine-dalgarno sequence (S.D.), functionally joined to DNA encoding the anti-CD79b antibody. However, there may be the also used and other promoters, which are functional in bacteria (for example, other known bacterial or phage promoters). The nucleotide sequences of these promoters have been published, which makes it easier for specialists 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 any required restriction sites.

In one aspect of the invention, each cistron in the recombinant vector contains a component such as a secretory signal sequence that regulates the translocation expressed polypeptide through the membrane. In General, the specified signal sequence may be a component of the vector, or such a sequence can be part of the desired DNA-polypeptide, embedded in this vector. The signal sequence is selected in order to carry out the invention must be a sequence recognized and processed (i.e tsepliaeva signal peptidases) the host-cell. In the case of prokaryotic host cells that do not recognize and do not processorbased signal sequences, which are native to the heterologous polypeptides, this signal sequence replace prokaryotes the th signal sequence, selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penitsillinazy, lpp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and the ISI. In one embodiment of the invention the signal sequences used in both cistronic expression systems are STII signal sequences 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 signal sequences in each cistron. In line with this, the light and heavy chain immunoglobulin expressed and are laying and Assembly with the formation of a functional immunoglobulin in the cytoplasm. Some strains of hosts (for example, trxB-strains of E. coli) have the proper conditions in the cytoplasm, which favor the formation of disulfide bonds and thereby facilitate proper installation and Assembly of subunits expressed protein. Proba & Pluckthun, Gene, 159:203 (1995).

The present invention relates to an expression system in which the quantitative ratio expressed polypeptide components can be modulated to maximize the yield of secreted and appropriately collected antibodies with the according to the invention. Such modulation is carried out, at least partially, through simultaneous modulation of the activity broadcast polypeptide components.

One method of modulating the translational activity are described in U.S. patent 5840523 Simmons et al. This method uses the options field of translation initiation (TIR) cistron. For this TIR can be created a series of variants of the amino acid sequence or nucleic acid sequence with a number of translational activity, which 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 codons can be altered, which can modify the amino acid sequence, although preferred are silent mutations in the nucleotide sequence. Modifications in TIR can be, for example, changes in the number of sequences Shine-Dalgarno or distance between them, as well as changes in the signal sequence. One of the ways to obtain mutant signal sequences is the creation of a "Bank codons" in the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e. such modifications t is Auda silent). This can be achieved by replacing the third nucleotide of each codon, and replacement of certain amino acids such as leucine, serine and arginine, in many first and second positions, which can 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 preferably generated using TIR-activity for each cistron. This limited set gives the opportunity to compare the levels of expression of each circuit, and outputs the desired product antibodies at different combinations of TIR-activity. TIR-activity can be determined by quantitative evaluation Wrona gene expression reporter, as described in detail in U.S. patent No. 5840523 Simmons et al. Based on comparison of the translational activities, may be chosen individual needs TIR, which can be combined in the construction of expression vectors according to the invention.

b. Eukaryotic cells-owners

The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a sequence of termination of transcription.

(1) Component signal follow etelnost

The vectors used in eukaryotic cells-the owners may also contain a signal sequence 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 processed (that is cleaved by a signal peptidase) 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 areas predecessor are ligated with the antibody-encoding DNA with preservation of the reading frame.

(2) the origin of replication

Usually this component as the origin of replication is not necessary for expression vectors mammals. For example, the origin of replication of SV40 is typically used only because it contains the early promoter.

(3) The selective gene

Expression and cloning vectors usually contain selective gene, also known as selective marker. Usually selective genes encode proteins that (a) inform the resistance to antibiotics or other toxins, such as the er, to ampicillin, neomycin, methotrexate, or tetracycline, (b) compensate for the deficit caused by auxotrophies, if necessary, or (C) deliver essential nutrients that do not come from complex environments, for example, the gene encoding D-alanine-racemase for Bacilli.

One example of schema selection is the use of drugs, stopping the growth of the host cell. Cells that were successfully transformed with a heterologous gene produce a protein, indicating resistance to the drug and thereby contributing to the survival of these cells in selective medium. For such dominant selection can be used, for example, drugs such as neomycin, mycofenolate acid and hygromycin.

Examples of suitable selective markers for mammalian cells are markers that can identify cells that are able to incorporate nucleic acid encoding an anti-CD79b antibody, such as DHFR genes, timedancing, metallothionein-I and II, and preferably the genes encoding metallothionein primates, adenosine-deaminase, ornithine-decarboxylase, etc. In the case of wild-type DHFR suitable host cell is a cell line CHO, deficient in DHFR activity (e.g., ATCC CRL-9096), and this cell line was obtained and p is smogen, as described in the publication Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed by selective DHFR gene, first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. Alternative cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or cotransformation DNA sequences encoding the antibody, protein DHFR wild-type and the other is a selective marker such as aminoglycoside-3'-phosphotransferase (ARN), can be selected by culturing cells in a medium containing an agent for sampling conducted using the selective marker such as aminoglycoside antibiotic, such as 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 strain of yeast, which can not 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

Expression and cloning vectors usually contain a promoter functionally attached to th the sequences of nucleic acids, coding anti-CD79b antibody to direct mRNA synthesis. Promoters recognized by a variety of potential cells masters, well-known to experts.

In fact, all the genes of eukaryotes have at-rich region localized around the area of 25-30 nucleotides above the site of transcription initiation. Another such sequence, localized approximately in the area of 70-80 nucleotides above the site of initiation of transcription of many genes, is the area CNCAAT, where N can mean any nucleotide. At the 3'end of most eukaryotic genes is the sequence AATAAA, which can serve as a signal for attaching A poly-a tail to the 3'-end of the coding sequence. All of these sequences can be properly embedded 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, glucokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate-isomerase, 3-phosphoglycerate-mutase, pyruvate is inasa, triazolopyrimidine, phosphoglucomutase and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage lies in their ability to regulate transcription in certain culture conditions, are the promoter region of the genes for 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 the anti-CD79b antibody of the vectors in the cells of the host mammal is regulated, for example, by promoters obtained from the genomes of these viruses as polyoma, the smallpox virus in poultry (application UK 2211504, published 5 July 1989), adenovirus (such as adenovirus 2), the virus bovine papilloma virus sarcoma birds, cytomegalovirus, a retrovirus, hepatitis b virus and most preferably simian virus 40 (SV40); heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter; and the promoters of heat shock proteins, provided that such promoters are compatible with the systems of host cells.

Ranni and late promoters of SV40 virus are usually obtained in the form of a restriction fragment of SV40, which also contains the origin of replication of SV40 virus. Pretani the promoter of the human cytomegalovirus usually obtained in the form of a HindIII restriction fragment that is the System the 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 such system is described in U.S. patent No. 4601978. Expression of cDNA for human β-interferon in mouse cells under the control of the promoter timedancing of the herpes simplex virus is also described in the publication Reyers et al., Nature 297:598-601 (1982). Alternatively promoter can be used rous sarcoma virus, having long terminal repeat.

(5) The enhancer element

Transcription of DNA encoding anti-CD79b antibody, in higher eukaryotes can be increased by embedding in vector enhancer sequence. 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 transcriptio. Currently, there are many enhancer sequences derived from mammalian genes (globin genes, elastase, albumin, α-fetoprotein, and insulin). However, it is usually used enhancer from a eukaryotic virus cells. An example is the enhancer of SV40 virus,localized in the late region of origin of replication (BP 100-270), enhancer early promoter of cytomegalovirus enhancer of polyomavirus localized in the late region of origin of replication, and adenovirus enhancers. Cm. publication Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for activation of eukaryotic promoters. The enhancer may be playserver in the vector in the 5'- or 3'-position with respect to the sequence that encodes an anti-CD79b antibody, but it is preferable that he was in the 5'-position from the promoter.

(6) The termination of transcription

Expression vectors used in eukaryotic cells-the masters (yeast, fungi, insects, plants, animals, humans or in nucleus-containing cells derived from other multicellular organisms)will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are usually located on the side of the 5'-end, and sometimes the 3'-end from the untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in netransliruemoi part of the mRNA that encodes the anti-CD79b antibody. One of the suitable components termination of transcription is the region polyadenylation bovine growth hormone. Cm. description expression of Vectora the WO 94/11026 and in this application.

Other methods, vectors and cells of the hosts that are suitable for adaptation to the synthesis of anti-CD79b antibodies 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); EP 117060 and EP 117058.

4. Culturing host cells

Cell owners used for producing anti-CD79b antibodies according to the invention, can be cultured in various media.

A. Prokaryotic cells-owners

Prokaryotic cells used to produce the polypeptides according to the invention, cultured in the medium known in the art and suitable for culturing a selected host cells. Examples of a suitable environment is the broth of Luria (LB) with the necessary nutritional supplements. In some embodiments of the invention it also contains a tool for selection, chosen design expression vector, which promote the selective growth of prokaryotic cells containing the expression vector. For example, in the environment for culturing cells expressing the gene of resistance to ampicillin, add ampicillin.

In addition to the sources of carbon, nitrogen and inorganic phosphate in the medium may also include any necessary additives in appropriate concentrations, administered separately or in the art with other additives or the 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, applied, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated at appropriate temperatures. For culturing 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 even more preferably at about 30°C. the pH of the medium may vary from about 5 to 9, depending mainly on the host organism. For E. coli, pH is preferably about from 6.8 to 7.4, and more preferably approximately 7,0.

If in the expression vector according to the invention is used inducible promoter, expression of the protein is induced under conditions suitable for activation of this promoter. In one aspect of the invention, for the regulation of transcription of polypeptides, used the PhoA promoter. In line with this, the transformed cell hosts are cultivated in the medium to induce with limited phosphate content. The preferred environment with limited phosphate content is Wednesday .R... (see, for example, Simmons et al., J. Immunol. Methods (2002) 263:133-147). In combination with IP is olzoeva vector design can be used and various other inductors, well-known specialists.

In one embodiment of the invention expressed polypeptides according to the invention are secreted into periplasm host cells and can be isolated from this periplasm. The selection of protein, mainly carried out by disruptive microorganism, usually by methods such as osmotic shock, sonication or lysis. After disruptive cells, cellular debris or whole cells can be removed by centrifugation or filtration. These proteins can be further purified, for example, by using affinity chromatography on a resin. Alternative proteins can be transported into the cell medium and separated from her. Cells can be removed from the culture, and the culture supernatant may be subjected to filtration and kontsentrirovaniyu for further purification the proteins. Expressed polypeptides can then be isolated and identified well-known methods such as polyacrylamide gel electrophoresis (SDS page) and Western blot analysis.

In one aspect of the invention, antibodies are produced in large quantities by fermentation. To obtain recombinant proteins can be carried out in various large-scale fermentation routine in the culture with water. Large-scale fermentation is carried out in a fermenter with a capacity of men is our least 1000 liters, and preferably from about 1000 to 100,000 liters. Such fermenters equipped with a paddle stirrer for uniform distribution of oxygen and minerals, and in particular, glucose (the preferred source of carbon/energy). The term “laboratory fermentation”, in General terms, means the fermentation in a fermenter with a volume of not more than about 100 liters, and this amount may vary from about 1 liter to 100 liters.

In the fermentation process inducing the expression of proteins is usually initiated after the cells are cultured in appropriate conditions, achieve the desired density, for example, OD550approximately 180-220, i.e. early stationary phase. In combination with the used vector design can be used and various other inductors, known in the art and described above. Before induction, the cells can be cultured within a smaller period of time. Cells usually induce approximately 12-50 hours, although the time of induction can be increased or decreased.

To increase product yield and to improve the quality of the polypeptides according to the invention can be modified in various fermentation conditions. So, for example, to ensure the “correct” Assembly and installation secreted polypeptide antibodies can be used to complement the global vectors, in which overexpression of 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 cotransformation prokaryotic host cells. It was demonstrated that protein 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. Environ. 39:199-210.

To minimize proteolysis of the expressed heterologous protein (and in particular, proteins that are proteoliticeski sensitive), in the present invention can be used in some strains are the owners of, deficient in proteolytic enzymes. For example, strains of host cells can be modified for the introduction of genetic(a) 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 protease strains of E. coli are available and are described, for example, Joly et al. (1998), see above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:pp.63-72 (1996).

In one the m variant of the invention, strains of E. coli deficient in proteolytic enzymes and transformed 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 Hams F10 (Sigma), minimal maintenance medium ((MEM), (Sigma), RPMI-1640 (Sigma) and modified by Dulbecco Wednesday Needle (DMEM), Sigma). In addition, as a culture medium for culturing the host cells can 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), U.S. patent No. 4767704, 4657866, 4927762, 4560655 or 5122469; WO 90103430; 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 adenosine and thymidine), antibiotics (such as medicine gentamicinTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar doses), and glucose or an equivalent energy source. May is to be also included any other necessary additives in appropriate concentrations, well-known experts in this field. Culturing conditions, such as temperature, pH and the like, similar to that previously used for the expression of the selected host cells, and well known to the average expert in the field.

5. Detection of amplification/gene expression

Amplification and/or expression may be measured in a sample directly, for example, by 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 antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes, DNA-RNA or DNA duplexes protein. The antibodies in turn may be labeled 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 bound to the duplex.

An alternative expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and analysis of cell culture or physiological liquid is TEI for direct and quantitative assessment of the expression of the gene product. Antibodies suitable for immunohistochemical staining and/or analysis of samples of body fluids can be monoclonal or polyclonal and can be obtained from any mammal. Basically can be obtained antibodies against native sequence CD79b polypeptide or against a synthetic peptide derived from these DNA sequences, or against exogenous sequence attached to DNA CD79b and 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 cells-owners. If antibodies are membrane-bound, they can be separated from the membrane using a suitable solution of detergent (such as Triton X-100) or by enzymatic cleavage. Cells used for the expression of anti-CD79b antibodies can be subjected disruptive various physical or chemical methods, such as the conduction cycle of freezing and thawing, sonication, mechanical disruptive or the use of agents for lysis of the cells.

It may be desirable purification of anti-CD79b antibodies from recombinant cell proteins or polypeptides. Suitable purification methods include the following methods: fracc is onirovanie on ion-exchange column, the ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing, electrophoresis in LTO-SDS page, precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; purification on columns with protein a-separate to remove contaminants such as IgG; and purification on columns, forming chelate complexes with metal binding to the labeled epitope forms of anti-CD79b antibody. Can be applied various methods of protein purification, 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). The selection stage(s) of cleaning depends on the nature of the method of production and specifically produced anti-CD79b antibodies.

Using techniques of recombinant DNA antibody can be produced inside the cells, or it can be directly secreted into the medium. If in the first stage, the antibody is produced inside the cells, cell debris or cell owners or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. In the publication of Carter et al., Bio/Technology 10:163-167 (1992) described the procedure for the selection of antibodies that are secreted into periplasmatic space of E. coli. Briefly, cells is Chou paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the environment, usually first focus supernatant 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 breeding accidentally introduced impurity 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 cleaning 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 the antibody. Protein a can be used for purification of an antibody containing 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 recommended G-protein (Guss et al., EMBO J. 5:15671575 (1986)). As the matrices to the th bound affinity ligand, the most commonly used imaging systems, but may be used and other matrices. Mechanically stable matrices such as glass with adjustable pore size or poly(Stradivari)benzene, provide faster flow and reduce processing time than can be achieved with the use of agarose. If the antibody contains a domain WithN3, cleaning it can be used resin Bakerbond ABXTM (J.T. Baker, Phillipsburg, N.J.). Depending on the 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 with poliasparaginovaya acid), chromatofocusing, electrophoresis in LTO-PAG and precipitation with ammonium sulfate.

After conducting any(s) advance(s) phase(s) of purification of a mixture containing an antibody and impurities, may be subjected to hydrophobic chromatography at low pH using an eluting buffer at pH about 2.5 to 4.5, and preferably at a low concentration of salt (e.g., about 0-0 .25 M).

G. Pharmaceutical composition

The conjugates of the antibody-lekarz the governmental agent" (ADC) according to the invention can be introduced in any way, suitable treatment for this condition. The ADC typically administered parenterally, that is by infusion, podhorna, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

In one embodiment of the invention, for the treatment of cancer, the conjugate antibody-drug" administered by intravenous infusion. Dose, administered by infusion, is in the range from about 1 μg/m2to about 10000 g/m2per dose and is usually injected once a week, and only can be entered one, two, three or four doses. Alternative this dose is about 1 mg/m2up to 1000 mg/m2approximately 1 µg/m2up to 800 mg/m2approximately 1 µg/m2up to 600 mg/m2approximately 1 µg/m2to 400 g/m2approximately 10 g/m2up to 500 mg/m2approximately 10 g/m2up to 300 g/m2approximately 10 g/m2up to 200 g/m2and from about 1 μg/m2up to 200 g/m2. To eliminate or mitigate symptoms of the disease, the dose can be entered once a day, once a week, several times a week but not every day; several times a month, but not every day; several times a month but not every week; once a month or occasionally. The introduction can be carried out within any of the specified interval is the time, until then, until you reached the tumor or reduce the symptoms of lymphoma and leukemia under treatment. The introduction can be continued after remission or mitigate symptoms of the disease, if during such continuous introduction remission or mitigation of symptoms still continue.

The present invention also relates to a method of treatment of autoimmune disease, where the method includes the administration to a patient suffering from an autoimmune disease, a therapeutically effective amount of the conjugate "humanitariannet MA79b antibody-drug" in accordance with any of the previous options. In preferred embodiments of the invention the antibody is administered intravenously or subcutaneously. The specified conjugate antibody-drug" injected dose of approximately 1 mg/m2up to 100 mg/m2per dose, and in a particular embodiment of the invention is from 1 mg/m2and up to about 500 g/m2. To eliminate or mitigate symptoms of the disease, the dose can be entered once a day, once a week, several times a week but not every day; several times a month, but not every day; several times a month but not every week; once a month or occasionally. The introduction can be carried out during any and the specified time-frames, until then, until you achieve the elimination or weakening of the symptoms of the autoimmune disease being treated. The introduction can be continued after remission or mitigate symptoms of the disease, if during such continuous introduction of such remission or mitigation of symptoms is still ongoing.

The present invention also relates to a method for treatment of b-cell disorder, comprising the administration to a patient suffering from a b-cell disorder, such as b-cell-proliferative disorder (including, but not limited to, lymphoma and leukemia), or an autoimmune disease, a therapeutically effective amount gumanitarnogo MA79b antibody according to any of the previous versions, where the aforementioned antibody is not conjugated with a cytotoxic molecule or detectable molecule. The specified antibody is typically administered at 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 at least one conjugate anti-CD79b antibody-drug according to the invention. In some embodiments of the invention the pharmaceutical preparation contains (1) ntitle according to the invention and/or its immunoconjugate, and (2) pharmaceutically acceptable carrier. In some embodiments of the invention the pharmaceutical composition comprises (1) the antibody according to the invention and/or its immunoconjugate, and optionally (2) at least one additional therapeutic agent. Additional therapies include, but are not limited to, the tools described below. The ADC typically administered parenterally, that is by injection, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

Therapeutic compositions containing the anti-CD79b antibody or immunoconjugate with CD79b used in accordance with the present invention are convenient for storing forms by mixing the antibody or immunoconjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), with liofilizovannyh drugs or aqueous solutions. Acceptable carriers, excipients or stabilizers used in the doses and concentrations must be non-toxic to recipients, and they are buffers such as acetate, Tris, phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chlorite is hexadecane; the benzalkonium chloride; chloride benzathine; phenol alcohol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (having 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, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; hepatoblastoma agents, such as EDTA; agents, giving toychest, 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 tween®, pluronics® or polyethylene glycol (PEG). The pharmaceutical composition used for administration in vivo, typically must be sterile. This may be readily accomplished by filtration through sterile filtration membranes.

Described here, the composition may also contain several active compounds required for specific indications, and preferably with the additional activity, not have a negative influence on each other. For example, in addition to anti-CD79b antibodies, one composition may be desirable to include additional antibodies, for example, "second" anti-CD79b antibody that binds to a different epitope specific cancer. Alternative or additionally, this composition may also contain chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, antihormonal agent, a cardioprotective agent. Such molecules may be present in combination in amounts that are effective for use in the desired order.

The active ingredients may also be enclosed in a microcapsule obtained, for example, methods koatservatsii or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin microcapsule and polymetylmetacrylate microcapsule, respectively, in systems for the delivery of colloidal medicines (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. This technique is described in the user manual (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

Can be obtained drugs extended release. Suitable examples of sustained release formulations release are semi-permeable matrix of t is erdich hydrophobic polymers, containing the immunoglobulin according to the invention, where these matrices have the form of finished articles, e.g. films, or microcapsules. Examples of the prolonged release matrix include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or polyvinyl alcohol)), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-biodegradable copolymer of ethylene-vinyl acetate, degradable copolymers of lactic acid-glycolic acid such as LUPRON DEPOT® (microspheres for injection, consisting of a copolymer of lactic acid-glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxipropionic acid. Polymers such as a copolymer of ethylene and vinyl acetate and a copolymer of lactic acid-glycolic acid capable of 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 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. To stabilize, depending on the specific mechanism may be a rational strategy. For example, if it was found that the mechanisms of the DG aggregation is the formation of intermolecular S-S-links through thio-disulfide interchange, the stabilization can be achieved by modifying sulfhydryl residues, lyophilization of the acid solutions, the regulation of moisture content, using appropriate additives, and obtain specific compositions based on polymer matrix.

The antibody can be obtained in any suitable form for delivery to the desired cell/tissue. For example, antibodies can be prepared in the form of immunoliposome. "Liposome" is a small vesicles composed of lipids of different types of phospholipids and/or surfactant that can be used to deliver the drug to a 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, get methods known in the art, such as methods described in the 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 WO 97/38731, published on 23 October 1997 Liposomes with increased half-life in blood is described in U.S. patent No. 5013556.

Specifically used liposomes composed of these lipids as phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-Feh), can be obtained by the method of issue of the air traffic management reverse phase. Liposomes extruded through filters with defined pore size, resulting in a gain liposomes of the desired diameter. Fab'-fragments of the antibodies according to the invention can be conjugated to the liposomes as described in the publication of Martin et al., J. Biol. Chem. 257:286-288 (1982), as a result of the reaction of the disulfide exchange. The liposome may contain, but not necessarily, the 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 may be readily accomplished by filtration through sterile filtration membranes.

H. Treatment of anti-CD79b antibodies

To determine the expression of CD79b if your cancer can be conducted by various detection assays. In one embodiment of the invention overexpression of CD79b polypeptide can be assessed using immunohistochemical analysis (IHC). Paraffin sections of tissue obtained by biopsy of the tumor, may be subjected to IHC analysis and evaluated by the intensity of staining CD79b protein in accordance with the following criteria:

Grade 0 - no staining or observed staining of the membranes at less than 10% of tumor cells.

A score of 1+ - very faint/barely perceptible membrane staining, detective more than 10% of tumor cells is K. These cells are colored only in part of their membrane.

Score 2+ - weak or moderate staining of the entire membrane, observed in more than 10% of tumor cells.

Rated 3+ moderate or strong staining of the entire membrane, observed in more than 10% of tumor cells.

Tumors were analyzed for overexpression of CD79b and received a score of 0 or 1+, can be characterized as a tumor, which is not observed overexpression of CD79b, and tumors with estimates of 2+ or 3+ can be characterized as a tumor, sverkhekspressiya CD79b.

Alternative or additional analyses of FISH, such as the INFORM® (developed by Ventana Co., Arizona) or PATHVISION® (Vysis, Illinois)may be carried out on formalin fixed and paraffin tumor tissue to determine the level of overexpression CD79b (if present) in the tumor.

Overexpression or amplification CD79b can be estimated using detection analysis in vivo, for example, by introducing a molecule (such as an antibody), which is associated with a detectable molecule; tagging detectable label (e.g. a radioactive isotope or a fluorescent label) and external scanning the patient for localization of the label.

As described above, the anti-CD79b antibody according to the invention, in addition to therapeutic applications, can be is used in other various purposes. Anti-CD79b antibodies according to the invention can be used to determine the stage of development of a cancer expressing CD79b polypeptide (for example, when radioactive visualization). These antibodies can also be used for cleaning or thus CD79b polypeptide from cells, for detection and quantification of CD79b polypeptide in vitro, e.g., in ELISA analysis or Western blot analysis, in order destruction and elimination CD79b-expressing cells from a population of mixed cells as in the stage of purification of other cells.

Currently, depending on the stage of cancer, cancer treatment includes the following therapy alone or in combination with each other, namely surgery to remove cancerous tissue, radiation therapy and chemotherapy. The treatment with anti-CD79b antibody may be particularly desirable for elderly patients who can not tolerate the toxicity and side effects of chemotherapy, and in metastases when radiation therapy has ogranichennoi application. Antineoplastic anti-CD79b antibodies according to the invention can be used to suppress growth CD79b-expressing cancer after initial diagnosis of the disease or during relapse. In therapeutic use anti-CD79b antibody can be used for the Vano 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. The treatment with anti-CD79b antibody can be carried out in combination with other forms of conventional therapy, either simultaneously with the standard therapy, either before or after the holding of such therapy. In the treatment of cancer, particularly in patients with high risk of developing this disease, are such chemotherapeutic agents 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 introduced anti-CD79b antibody in combination with the introduction of one or more of the above chemotherapeutic agents. In particular, it is also combined therapy, carried out together with the introduction of paclitaxel and its modified derivatives (see, for example, EP0600517). Anti-CD79b antibody is administered with a therapeutically effective dose of a chemotherapeutic drug. In another embodiment of the invention, an anti-CD79b antibody is administered in combination with chemotherapy to enhance the activity and efficacy of chemotherapeutic agents, such as paclitaxel. In SCC, the adjustment guide for physicians (PDR) doses of tools, which were used to treat various cancers. Regimens and doses of the aforementioned chemotherapeutic drugs that are therapeutically effective, depend on the particular cancer being treated, the degree of development of the disease and other factors that are known to the person skilled in the art and may be determined by the treating physician.

In one specific variants of the invention, the patient is given a conjugate containing anti-CD79b antibody conjugated with a cytotoxic agent. Preferably immunoconjugate associated with protein CD79b, internalized in the cell, resulting in increased therapeutic efficacy immunoconjugate in respect of destruction of cancer cells with which they are associated. In a preferred embodiment of the invention, the cytotoxic agent is targeted to a nucleic acid of cancer cells or inhibits its action. Examples of such cytotoxic agents described above, and such means are maytansinoid, calicheamicin, ribonuclease and DNA endonuclease.

Anti-CD79b antibodies or their conjugates with a toxin is administered to a person in accordance with known methods, such as intravenous administration, e.g., in the form of a shock doses or by continuous infusion over ODA is divided by time period, as well as intramuscularly, intraperitoneally, inside the cerebrospinal fluid, subcutaneous, intra-articular, inside the synovial fluid, intrathecally, orally, topically or by inhalation. It is preferable to intravenous or subcutaneous administration of the antibody.

Other courses of therapy may be combined with the introduction of the anti-CD79b antibody. The combined introduction includes co-administration of certain drugs, or a single pharmaceutical preparation and their gradual introduction in any order, preferably in the period of time for which both (or all) of the active agent at the same time show their biological activity. While it is preferable that such a combined therapy gave a synergistic therapeutic effect.

It may also be desirable for combined injection of anti-CD79b antibody or antibody together with the introduction of antibodies against another tumor antigen associated with the particular cancer.

In another embodiment of the invention, therapeutic methods of treatment according to the invention include the combined introduction of the anti-CD79b antibody (or antibodies) and one or more chemotherapeutic agents or growth-inhibiting means, including joint introduction of a mixture of various chemotherapeutic agents other(their) chemotherapy(their) money (funds) or other(s), therapeutic(their) money (money), which also inhibit tumor growth. Chemotherapeutics are phosphate estramustine, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyacetone taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics. Methods of obtaining and schemes such chemotherapeutic agents may be carried out in accordance with the manufacturer's instructions, or they can be empirically adjusted by a specialist. Methods of obtaining and schemes 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, antiestrogenic 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 can be previously subjected antiandrogenna therapy, and then, after the cancer becomes androgen independent, the patient can be introduced anti-CD79b antibody (and optionally other tools described here).

Sometimes it may also be desirable joint introduction to the patient means DL the prevention of cardiovascular diseases (to prevent or reduce the dysfunction of the myocardium, associated with this therapy) or one or more cytokines. In addition to the above therapeutic regimens, the patient may be subjected to surgical removal of cancer cells, and/or it can be subjected to radiation therapy (e.g., external radiation or therapy with radioactive labelled agent, such as an antibody)that were conducted before, during, or after treatment with antibody. Suitable dosages for any of the above in conjunction input means are used here dose, and this dose can be lowered due to the combined (synergistic) effect of said means and anti-CD79b antibodies.

The 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 a particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiological factor causing this disorder, the area in which directed this tool, a way of introduction, 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 bore alkemi modern means, used for the prevention or treatment considered disorders. The effective amount of such other funds depends on the number of antibodies according to the invention, is present in this composition, the type of disorder or the method of its treatment and other factors discussed above. These other drugs are usually injected at the same dose and the same methods which were used previously, or the dose is about 1-99% of the previous dose.

For the prevention or treatment of disease, the appropriate dosage and schemes medicines may be selected by the physician in accordance with 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 injected if the indicated antibody for preventive or therapeutic purposes, from the treatment done before, from the patient's medical history and its susceptibility to a given antibody, and from a doctor's appointment. This antibody can 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 pre-installed dose EN is the body, enter the patient is from about 1 μg/kg to 50 mg/kg of body weight (for example, about 0.1-15 mg/kg/dose), regardless of whether one-time or repeated introduction or continuous infusion of this antibody. Scheme dose may include the introduction of an initial loading dose of anti-CD79b antibody of approximately 4 mg/kg, with subsequent weekly introduction maintenance dose of approximately 2 mg/kg anti-CD79b antibody. However, it can be done and other schemes of doses. A typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. Re-introduction for several days or more, depending on the condition, treatment may be as long until you achieve the desired suppression of disease symptoms. Monitoring the effect of such therapy can be easily carried out by standard methods, using analyses and in accordance with the criteria known to the physician or other specialists in this field.

In this application, apart from the introduction of protein-antibody to the patient, is also considered the introduction of antibodies by the method of gene therapy. This introduction of nucleic acid that encodes the antibody contained in the scope of the term "introduction of a therapeutically effective amount of antibodies". See, example is, application WO 96/07321, published March 14, 1996, and related to the use of gene therapy for intracellular production of antibodies.

There are two main ways of introducing the nucleic acid (optionally contained in a vector) into the cells of a patient, 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 the cells of the patient, and then these selected cells are introduced nucleic acid, and the modified cells are administered to the patient either directly or, for example, in the form of capsules in porous membranes which are implanted to the patient (see, for example, U.S. patents 4892538 and 5283187). There are a number of methods of introducing nucleic acids into viable cells. Such methods may vary depending on, enter whether the nucleic acid into the 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 vitro, are methods with the use of liposomes, electroporation, microinjection, merging of cells, the method using DEAE-dextran method precipitation of calcium phosphate, etc. Most commonly used vector for gene delivery ex vivo is a retroviral vector.

Currently predpochtitelnye methods of transfer of nucleic acids in vivo include 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 tagging of genes and gene therapy can be found in the publication of Anderson et al., Science 256:808-813 (1992). Cm. application WO 93/25673 and cited in her work.

In the volume used here, the term "antibody" can include various forms of anti-CD79b antibodies according to the invention. Thus, these antibodies are full-length or intact antibody, fragments of antibodies, antibody with a native sequence or amino acid variants, humanized, chimeric or hybrid antibodies, immunoconjugates and their functional fragments. In a hybrid antibody sequence antibodies attached to a heterologous polypeptide sequence. Antibodies can be modified in the Fc-region of a message of the desired effector functions. As discussed in more detail in these sections on the corresponding Fc-the fields of the "naked" antibody associated with the cell surface can induce cytotoxicity, for example, by antibody-dependent cellular cytotoxicity (ADCC) or recruitment of complement in the case of the complement-dependent cytotoxicant is or by some other mechanisms. Alternatively, if it is desirable to eliminate or reduce effector function, in order to minimize side effects or complications of therapy, it can be used some other Fc-field.

In one embodiment of the invention, the antibody competes for binding or essentially binds to the same epitope bound antibodies according to the invention. Also discusses antibodies having the biological properties of the anti-CD79b antibody 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-mentioned antibodies described in this application.

Anti-CD79b antibodies according to the invention can be used to treat CD79b-expressing cancer or ameliorate one or more symptoms of cancer in a mammal. Such cancers include, but are not limited to, cancer of the hematopoietic system or blood cancer, such as lymphoma, leukemia, myeloma or lymphoma malignant tumors, and cancer of the spleen and cancer of the lymph nodes. More specific examples of such b-cell associated cancers are, for example, vysokokachestvennaya, srednestaticheskaya and nizkozameshhennoj lymphoma (including In-cleoc what's lymphoma, such as, for example, b-cell lymphoma lymphoid tissue of the mucous membrane and non-Hodgkin's lymphoma, lymphoma cells of the cortex of the brain, Burkitt's lymphoma, small cell lymphocytic lymphoma, lymphoma marginal zone, both diffuse lymphoma, follicular lymphoma, jackinsky lymphoma and T-cell lymphoma and leukemia (including secondary leukemia, chronic lymphocytic leukemia, such as In-cell leukemia (CD5+-b cells), 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 metastasis to 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, the specified antibody is effective to destroy or destruction CD79b-expressing tumor cells or inhibit the growth of such tumor cells in vitro or in vivo after binding of the indicated antibody to CD79b polypeptide on the cell. This antibody is of the "naked" anti-CD79b antibody (not conjugated to any agent). "Naked" antibodies that possess the cytotoxic properties or properties, aimed at inhibiting the growth of cells, can also be attached to the cytotoxic agent, which makes them even more effective against the destruction of tumor cells. Cytotoxic properties can be reported anti-CD79b antibody, for example, by conjugation specified antibody with a cytotoxic agent, with the formation described here immunoconjugate. This cytotoxic agent or a 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 containing an anti-CD79b antibody according to the invention and a carrier. For the treatment of cancer compositions can be administered to the patient in need of such treatment, where this 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, these compositions can contain these antibodies in combination with other therapeutic means, 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 Sitel. In one embodiment, antibodies listed drug is a therapeutic preparation containing pharmaceutically acceptable carrier.

In another aspect the present invention relates to the selection of nucleic acids encoding anti-CD79b antibody. This term also encompasses nucleic acids encoding the H chain and L, and in particular, the remains of the hypervariable region; chain nucleic acid encoding the antibody with a native sequence, and variants, modifications and humanized variants of antibodies.

The present invention also relates to methods for treating a cancer expressing CD79b polypeptide, or to a weakening of one or more symptoms of cancer in a mammal, where the method includes the introduction of a therapeutically effective amount of an anti-CD79b antibody to the mammal. The antibody-containing therapeutic compositions can be introduced within a short period of time (a single injection) or for a long period of time, or periodically, depending on the prescription. The present invention also relates to methods of inhibiting growth of cells expressing CD79b polypeptide, and cytolysis of these cells.

The present invention also relates to kits and industrial products containing at least about the but anti-CD79b antibody. Kits containing anti-CD79b antibodies, can be used, for example, for the analysis of cytolysis CD79b cells, as well as for cleaning or thus CD79b polypeptide from cells. For example, for isolation and purification CD79b specified set may contain anti-CD79b antibody-related fields (for example, sivaratnam areas). Can be obtained sets, 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, may be associated with a label, such as fluorescent or radioactive label.

I. Treatment of the conjugate antibody-drug”

It is assumed that the conjugates of the antibody-drug” (ADC) according to the invention can be used to treat various diseases or disorders characterized by, for example, overexpression of tumor antigen. Representative of conditions, or hyperproliferative disorders are benign or malignant tumors; leukemia and lymphoma malignant disease. Other diseases are disorders associated with dysfunction of nerve cells, glial cells, hypothalamus glandular cells, macrophages, epithelial cells, stroma and blastocele, as well as inflammatory is entrusted, angiogenic and immunologic disorders, including autoimmune disorders.

ADC connections that have been identified in animal models and in cell culture assays can be further tested in the higher primates, with tumors, and clinical trials involving human subjects. Clinical trials involving human subjects can be developed in order to determine the effectiveness of 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, relapsing aggressive NHL, recurrent asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 radiotherapy and/or chemotherapy with the use of known chemotherapeutic and/or cytotoxic agents.

In General, the disease or disorder being treated is hyperpro liferative disease this 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 lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

Cancer may include CD79b-expressing cells, which may contact the ADC according to the invention. To determine the levels of expression of CD79b in cancer can be conducted by various diagnostic/prognostic tests. In one embodiment of the invention, the level of overexpression CD79b can be analyzed using IHC. Paraffin sections of tissue taken by biopsy of the tumor, may be subjected to IHC analysis and assessed for degree of staining and the number of tumor cells in accordance with the following evaluation criteria in the intensity of staining CD79b protein.

For the prevention or treatment of disease, the choice of an appropriate dose of the ADC depends on the type subjected to the treatment of the human disease, above, the severity and course of the disease, the type of molecule, administered for preventive or therapeutic purposes, from its previous therapy, the patient's medical history and its susceptibility to a given antibody, as well as from the doctor's appointment. The specified molecule suitably administered to the patient for one course or several courses of treatment. Depending on the type and severity of the disease, the initial dose of the molecule, scheduled for introduction to the patient is about 1 μg/kg 15 μg/kg (for example, 0.1 to 20 mg/kg), and this dose can be entered as a single dose or fractional doses, or it can be entered by continuous infusion. A typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. Representative dose ADC input patient is from about 0.1 to 10 mg/kg of body weight of the patient.

Re-introduction for several days or more, depending on the patient's condition, treatment may be continued until, until you reach the desired attenuation of the symptoms of the disease. A representative scheme of doses includes the introduction of an initial loading dose of approximately 4 mg/kg, and then weekly introduction maintenance dose of anti-ErbB2 antibodies, comprising approximately 2 mg/kg If this could the t to be applied and other schemes of doses. The effect of this therapy can be easily traced with the use of standard methods and analysis.

J. Combination therapy

Conjugate the antibody-drug" (ADC) according to the invention can be combined with a second compound having anti-cancer properties, obtaining a combined pharmaceutical composition, or it can be used together with other treatments in the form of combination therapy. The specified second connection such pharmaceutical combination compositions or combined treatment provides an additional activity of the combination of the ADC, provided that the components of such combination the combination has no negative impact on each other.

The second connection may be a chemotherapeutic agent, cytotoxic agent, cytokine, agent, inhibiting the growth of cells, protivogelmintnoe tool and/or tool for the prevention of cardiovascular diseases. These molecules are usually present in combination in amounts that are effective to achieve this goal. The pharmaceutical composition containing the ADC according to the invention may also contain therapeutically effective amount of a chemotherapeutic drug, such as an inhibitor of the formation of tubulin, inhib the top or topoisomerase 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 tools, chemotherapeutic agents or growth inhibitory agent. In one of such variants chemotherapeutic agent is a tool or combination of tools, such as, for example, cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapy, such as anti-CD20 antibody (e.g., Rituxan®) or anti-VEGF antibody (e.g., Avastin®).

Combination therapy can be carried out by simultaneous or sequential individual courses of treatment. If p is coherent mode of conducting such courses, this combination of medicines may be introduced in two stages or in several stages. Such a combined treatment includes co-administration of certain drugs or their introduction in the form of a single pharmaceutical preparation, and their consistent introduction in any order, preferably in the period of time for 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 anticancer means and one or more chemotherapeutic agents or growth inhibitors, including joint introduction of a mixture of different chemotherapeutic agents. Chemotherapeutics are taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics. Methods of obtaining and schemes such chemotherapeutic agents may be carried out in accordance with the manufacturer's instructions, or they can empirically chosen specialist. Methods of obtaining and schemes 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 means are simultaneously introduced doses used here is, and these doses may be lowered due to the combined (synergistic) effect just identified tools and other chemotherapeutic agents or treatments.

Combination therapy may provide “synergy” and “synergistic action of the active ingredients, i.e. when the effect achieved in the case of joint use of active ingredients is greater than the sum of the effects achieved when the individual use of these compounds. A synergistic effect may be achieved if the active ingredients (1) prepared in a mixture with each other and introduced or delivered simultaneously in a combined uniform dosage forms; (2) delivered by alternate or parallel administration in the form of separate formulations; or (3) delivered in accordance with some other schemes introduction. With the introduction of through alternative therapy, a synergistic effect can be achieved if the specified connection is injected or delivered sequentially, for example, by different injections in separate syringes. In General, while alternative therapies effective dose of each active ingredient is administered sequentially, i.e. one after another and during combination therapy, effective dosages of two or more active ingr is diantou enter together.

K. Industrial products and kits

In another embodiment, the present invention relates to industrial product contains materials that are used for the treatment, prevention and/or diagnosis of CD79b-expressing cancer. This industrial product contains packaging and label pasted on the packaging, or liner, is attached to this package. Suitable packings are, for example, bottles, vials, syringes, etc. Such packaging can be made of various materials such as glass or plastic. This package contains a composition that 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 package for intravenous solution or vessel having a tube, protegeme needle for subcutaneous injection). At least one active agent in the specified composition is an anti-CD79b antibody according to the invention. On the label or in the leaflet enclosed in the packaging must be indicated that the composition is used for the treatment of cancer. The label or the liner in the package may also contain instructions for the introduction of the composition of the antibody to a patient suffering from cancer. Alternative specified industrial product can be the t also include a second package, containing pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose. In addition, this product can 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, for example, for analyses on the cytolysis CD79b-expressing cells, for cleaning or thus CD79b polypeptide from cells. For isolation and purification of the CD79b polypeptide, the kit can contain an anti-CD79b antibody-related fields (for example, sivaratnam areas). Can be obtained sets, which contain the antibodies for detection and quantification of CD79b polypeptide in vitro, e.g., in ELISA analysis or Western blot analysis. The specified set, as industrial product, contains packaging and label pasted on the packaging, or liner, is attached to this package. This package contains a composition including at least one anti-CD79b antibody according to the invention. Can also be included and other containers that contain, for example, diluents, buffers, and control antibodies. Label or vladishev packaging may include a description of the composition, and instructions for use in vitro or detection.

L. application of the CD79b polypeptide

The present invention encompasses methods of screening compounds to identify compounds that mimic the CD79b polypeptide (agonists)or prevent the action of the CD79b polypeptide (antagonists). The screening assays to identify candidate antagonists used as medicines have been developed to identify compounds that bind or form a complex with the CD79b polypeptide encoded by the identified genes here, or, conversely, inhibit the interaction of the encoded polypeptides with other cellular proteins, including, for example, inhibition of the expression of CD79b polypeptide from cells. Such screening assays include assays that are suitable for large-scale screening of chemical libraries, and therefore suitable for identifying small molecules drug candidates.

Tests can be developed in a variety of formats, including analyses on the binding of protein-protein, biochemical screening assays, immunoassays and cell assays, well known to experts.

All analyses antagonists are standard, i.e. they require contacting the drug candidate with a CD79b polypeptide, codere the output nucleic acid, identified here under conditions and for a period of time sufficient for the interaction of these two components.

In the analysis of the binding of the specified interaction is binding, and the complex formed can be isolated from the reaction mixture or detected in the mixture. In a specific embodiment of the invention the CD79b polypeptide encoded identified here gene or drug candidate immobilized on the solid phase, for example, microtiter tablet, via covalent or non-covalent binding. Non-covalent binding is usually carried out by applying onto a hard surface solution CD79b polypeptide and drying. Alternative immobilizovannoi antibody, for example, a monoclonal antibody specific to immobilizovannogo the CD79b polypeptide, may be used to zakalivanie on a solid surface. This analysis is carried out by adding neimmunizirovannah component that can be marked detectable label, mobilisierung component, for example, on the surface with a coating containing the anchored component. If the reaction is carried out completely, unreacted components are removed, e.g. by washing, and then detect complexes anchored on the solid surface. If not originally mobilisierung component carries detektiruya tag the detection of label immobilized on the surface indicates the formation of the complex. If initially neimmunizirovannah component shall not be detektiruya label, the formation of the complex may be detected, for example, using labeled antibodies that specifically bind to the immobilized complex.

If the connection candidate communicates, but not associated with a specific CD79b polypeptide encoded identified here genome, its interaction with the polypeptide can be analyzed by methods well known as methods for detecting interactions, "protein-protein". Such analyses include the use of traditional methods, such as, for example, cross-linking, coimmunoprecipitate and joint cleaning in the gradient or chromatographic columns. In addition, monitoring of the interaction of "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, such 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 the Tr is scriptie. Expression of the yeast system described in the foregoing publications (generally called "two-hybrid system"), has the advantage that it uses a hybrid of the two proteins, one of which is a protein target that is associated with the DNA-binding domain of GAL4, and the other represents an activating protein-candidate associated with the activating domain. Expression of the reporter GAL1-lacZ under the control of a GAL4-activated promoter depends on the recovery of GAL4 activity via interaction "protein-protein". Colonies containing interacting polypeptides detected using a chromogenic substrate for β-galactosidase. Full set (MATCHMAKERTMto identify protein-protein-interactions between two specific proteins, carried out by methods using 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, as well as to determine the exact amino acid residues that play an important role in such interactions.

Compounds that inhibit the interaction of the gene encoding identified here CD79b polypeptide, with other intra - or extracellular component of the mi, can be tested as follows: usually get the 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 compound candidate to inhibit the binding reaction is carried out in the absence and presence of test compounds. In addition, the third reaction mixture can be added placebo, which serves as a positive control. Monitoring binding (educational complex) test connection with intra - or extracellular component present in the mixture, 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 inhibits the interaction of test compounds with its reaction partner.

For analysis antagonists CD79b polypeptide can be added to cells together with the connection, scrinium on a particular activity and the ability of the compounds to inhibit interest activity in the presence of a CD79b polypeptide indicates that the compound is an antagonist of the CD79b polypeptide. Alternative antagonists can be detektirovaniem Association of CD79b polypeptide and a potential antagonist with membrane-bound receptors of the CD79b polypeptide or recombinant receptors under conditions suitable for analysis on competitive inhibition. The CD79b polypeptide can be labeled, for example, a 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, methods of panning ligand and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). If polyadenylated RNA obtained from cells susceptible to CD79b polypeptide, preferably carry out 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 CD79b polypeptide. Transfetsirovannyh cells grown on slides, obrabatyvat labeled a CD79b polypeptide. The CD79b polypeptide can be labeled by various methods, including iodination or inclusion of a site of recognition for site-specific protein kinase. After fixation and incubation slides subjected autoradiographical analysis. Positive pools are identified, and then get subpoly that re transferout iterative method of obtaining subpool and re-screening, in accordance with the ATA which receive one clone, encoding the presumed receptor.

In an alternative method for the identification of receptor labeled CD79b polypeptide can be photouplink associated with the cellular membrane, or can be obtained preparations expressing the receptor molecules. Cross-linked material separated by electrophoresis in SDS page and exhibiting the x-ray film. The labeled complex containing the receptor, can be cut into peptide fragments, and proteins are microsequencing. Amino acid sequence obtained after mikroekonomia, can be used in order to design a set of degenerate oligonucleotide probes for screening cDNA library conducted in order to identify the gene encoding the presumed receptor.

In another analysis antagonists of mammalian cells or a membrane preparation expressing the receptor, can be incubated with labeled CD79b polypeptide in the presence of the connection candidate. Then may be determined by the ability of this compound to enhance or block this interaction.

More specific examples of potential antagonists are oligonucleotide that binds to the hybrid immunoglobulin and CD79b polypeptide, and in particular, antibodies including, but not limited to, polyclonal and monoclonal antibodies and fragments thereof, single-chain antibodies, antiidiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and their fragments. Alternative potential antagonist may be a closely related protein, for example, a mutated form of the CD79b polypeptide that recognizes the receptor but does not exert any action on it, and thereby competitively inhibits the action of the CD79b polypeptide.

Antibodies that specifically bind to the identified here CD79b polypeptide, as well as other molecules identified in the above screening assays can be introduced 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, the preferred are internalizes antibodies. However, to deliver the antibody or antibody fragment in the cell can also be used lipofectin or liposomes. If you are using fragments of antibodies, the preferred is the shortest inhibitory fragment that specifically binds with the binding domain of the protein target. So, for example, based on the sequences of the variable regions of the antibodies can be designed peptide molecules, which preserves the t ability to communicate with the sequence of the protein target. Such peptides can be synthesized by a chemical method and/or can be produced by the methods of recombinant DNA. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993).

This drug may also contain more than one active connection needed to treat a particular disease, and preferably the connection with complementary activities that do not negatively impact each other. Alternative or additionally, this composition may contain an agent that enhances 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 goals.

M. Derivatives of antibodies

Antibodies according to the invention can be further modified so as to include other nonprotein molecules that are known in the art and are readily available. Preferred molecules suitable for derivatization of the antibody, 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-di is Xalan, 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, polyoxyethylene polyols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. In the industrial production should be preferred to Propionaldehyde of polyethylene glycol due to its stability in water. This polymer can be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if you attach more than one polymer, these polymers may be 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 property or function of antibodies, which should be improved, regardless of the specific conditions of the treatment, which will use the specified derived antibodies, etc.

N. a Method of screening

In yet another embodiment, the present invention relates to a method of determining the presence of a CD79b polypeptide in a sample suspected of containing the CD79b polypeptide, where the decree of the config method includes processing the sample with the conjugate of the antibody-drug", which binds to CD79b polypeptide, and determining the level of binding of the specified conjugate the antibody-drug" with CD79b polypeptide in the sample, where the presence of such binding indicates the presence of a CD79b polypeptide in the sample. Such a pattern may, but not necessarily, contain cells (which may be cancer cells), presumably expressing CD79b polypeptide. Conjugate the antibody-drug"used in this way, it may be, but not necessarily, detektirano labeled, attached to a solid carrier or the like

In another embodiment, the present invention relates to a method of diagnosing the presence of a tumor in a mammal, where the method includes (a) contacting the test sample containing tissue cells isolated from a mammal with a conjugate of the antibody-drug", which binds to CD79b polypeptide, and (b) detecting formation of a complex between the conjugate antibody-drug" and CD79b polypeptide in the test sample, where the formation of the complex indicates the presence of a tumor in a mammal. Conjugate the antibody-drug"used in this way, it may be, but not necessarily, detektirano labeled, attached to a solid carrier or the like, and/or the test sample glue is OK tissues can be obtained from the individual with suspected cancer.

IV. Other ways of using anti-CD79b antibodies and immunoconjugates

A. Diagnostic methods and methods of detection

In one aspect of the invention, an anti-CD79b antibodies and immunoconjugates according to the invention can be used to detect the presence of CD79b in a biological sample. Used herein, the term "detection" includes 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 cancerous tissue compared with other tissues, Express CD79b at a higher level, for example, b-cells and/or tissues associated with b-cells.

In one of its aspects the present invention relates to a method for detecting the presence of CD79b in a biological sample. In some embodiments of the invention the method includes contacting a biological sample with an anti-CD79b antibody under conditions conducive to binding of the anti-CD79b antibody to CD79b, and detecting formation of a complex between the anti-CD79b antibody and CD79b.

In one of its aspects the present invention relates to a method for the diagnosis of disorders associated with increased levels of expression of CD79b. In some embodiments, invented what I specified the method comprises contacting the test cells with the anti-CD79b antibody; the level of expression (either quantitative or qualitative) CD79b in the test cell by detecting binding of an anti-CD79b antibody to CD79b; and comparing the level of expression of CD79b in the test cell with the level of expression of CD79b in the control cells (for example, in normal cells, derived from the same cloth, from which we derive the test cells, or in cells compared with normal cells, Express a CD79b), where a higher level of expression of CD79b in test cells, compared to control cells indicates the presence of a disease associated with an increased level of expression of CD79b. In some embodiments of the invention the test cells from the individual with a suspected disorder associated with an increased level of expression of CD79b. In some embodiments of the invention this disorder is a cellular proliferative disease such as cancer or a tumor.

Representative cell-proliferative disorders that can be diagnosed using the antibodies according to the invention are b-cell disorders and/or b-cell-proliferative disorders, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL relapsing basyntan the Yu NHL untreatable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), 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 surface of cells or in a membrane preparation isolated from cells expressing on their surface CD79b. In some embodiments of the invention the method includes contacting cells with an anti-CD79b antibody under conditions conducive to binding of the anti-CD79b antibody to CD79b, and detecting formation of a complex between the anti-CD79b CD79b antibody and the cell surface. Representative analysis for detection of the binding of anti-CD79b antibody to CD79b expressed on the cell surface, is a "FACS"analysis.

To detect the 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 with the antigen are well known in the art, such as Western blot analysis, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich"-Immunocal the SHL, analyses using thus, fluorescent immunoassays, and immunoassays using protein a, and immunohistochemical analyses of tissues.

In some embodiments of the invention the anti-CD79b antibodies are labeled. Labels include, but are not limited to, labels, or molecules that can be detected directly (such as fluorescence chromophore, electronmobility, chemiluminescent, and radioactive labels), as well as molecules such as enzymes or ligands, which detects the indirect method, for example, by enzymatic reaction or molecular interaction. Representative labels include, but are not limited to, radioisotopes32P,14C,125I3H and131I, fluorophores such as chelate complexes of rare earth metals or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, e.g., Firefly luciferase and bacterial luciferase (U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, such as glucose oxidase, galactosidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase attached to the enzyme, which used the hydrogen peroxide for 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 antibodies immobilized on nerastvorim matrix. Immobilization leads to the separation of the anti-CD79b antibody of any CD79b, remaining in solution in a free state. This procedure is usually carried out by insolubilization anti-CD79b antibody before analysis, as is the case for adsorption at water-insoluble matrix or surface (Bennich et al., U.S. patent 3720760), or by covalent binding (e.g., cross-linking with glutaraldehyde), or by insolubilization anti-CD79b antibody after formation of a complex between the anti-CD79b CD79b antibody and, for example, by thus.

Any of the above options the diagnosis or detection may be carried out using immunoconjugate according to the invention instead of the 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, or i vivo, or in vitro, where the method includes treating the 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 less growth or proliferation of cells of 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 In the cell. In some embodiments of the invention the specified cell is a xenograft, for example, illustrated in the description of this application.

In one aspect of the invention, the antibody or immunoconjugate according to the invention are used for treating or preventing b-cell-proliferative disorder. In some embodiments of the invention specified cell-proliferative disorder is associated with an increased level of expression and/or activity of CD79b. For example, in some embodiments of the invention In a 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 a cancer. Examples of b-cell-proliferative disorders, Podwale what's the treatment with antibodies or immunoconjugates according to the invention are, but not limited to, lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

In one of its aspects the present invention relates to methods for treating b-cell-proliferative disorders, comprising the administration to the individual an effective amount of an anti-CD79b antibody or immunoconjugate. In some embodiments of the invention a method of treating b-cell-proliferative disorder includes an introduction 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 disorder includes an introduction to the individual an effective amount of a pharmaceutical preparation containing (1) immunoconjugate, including anti-CD79b antibody and cytotoxic agent; and optionally (2) at least one additional therape the optical means, such as the means described below.

In one aspect of the invention, at least some of the antibodies or immunoconjugates according to the invention can contact CD79b, derived from species that are not human. In accordance with these 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, abacadabra monkeys and macaques of resursov, as well as pigs or mice)with CD79b, which cross-reacts with the antibody 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 a complex of the antibody-antigen" or "immunoconjugate-antigen and thereby 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 immunoconjugate can be used in the methods of linking CD79b an individual suffering from a disorder associated with increased expression level and/or activity of CD79b, g is E. this method includes the introduction of individual antibodies or immunoconjugate to implement binding CD79b the individual. In one embodiment of the invention the 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 a person. Alternative individual can be a mammal expressing CD79b is associated with anti-CD79b antibody. Besides, the individual can be a mammal, which impose CD79b (e.g., by delivery of CD79b or expression of the transgene encoding CD79b).

Anti-CD79b antibody or immunoconjugate can be introduced to a person for therapeutic purposes. In addition, anti-CD79b antibody or immunoconjugate can be administered to the mammal, not a person, which is expressed CD79b, which cross-linked the antibody (e.g., a Primate, pig, rat or mice), where the specified conduct introduction the purpose of the treatment of the animal or for the use of this animal as a model of human disease. In the latter case, such animal models can be used to evaluate therapeutic efficacy of antibodies or immunoconjugates (for example, determination of dose and time of treatment).

therapy antibodies or immunoconjugate according to the invention can be used in the us alone or in combination with other therapeutic compositions. For example, antibodies or immunoconjugate according to the invention can be introduced together with at least one additional therapeutic agent and/or adjuvant. In some embodiments of the invention other therapeutic agent is a cytotoxic agent, chemotherapeutic agent, or growth-inhibitory agent. In one of such variants chemotherapeutic agent is a tool or combination of tools, such as, for example, cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapy, such as anti-CD20 antibody (e.g., Rituxan®) or anti-VEGF antibody (e.g., Avastin®), where this combination therapy can be applied for the treatment of cancer and/or b-cell disorders, such as b-cell-proliferative disorders, including lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, recurrent asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (ES), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

This above combination therapy includes kombinirov is a great introduction (where two or more therapeutic agents are included in the same drug or certain drugs) and 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 an 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 introduced by any suitable means, including parenteral, subcutaneous administration, intraperitoneal administration, intra-lungs introduction and intranasal introduction, and, if it is necessary for local treatment, the introduction into the affected area. Parenteral injections are intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate can be entered by periodic infusion, particularly with declining doses of the antibody or immunoconjugate. The dose may be entered in any suitable way, for example, by injection, such as intravenous or subcutaneous injection, in part, depending on whether this introduction immediately or within a longer period of time.

Antibodies or immunoconjugate according to the invention is prepared, divided into doses and injected according to the following well-known medical practice. The factors considered for the preparation of such compositions, are a particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiological factor causing this disorder, the area in which directed this tool, a way of introduction, the scheme of administration and other factors known to practitioners. The indicated 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 considered disorders. The effective amount of such other funds depends on the number of antibodies according to the invention, is present in this composition, the type of disorder or the method of its treatment and other factors discussed above. These other drugs are usually injected at the same dose and the same methods which were used previously, or the dose is about 1-99% of the previous dose, or such funds may be entered in any dose, by any means, which may be determined empirically or clinical method.

For the prevention or treatment of disease, the appropriate dosage of the antibody or immunoconjugate according to the invention (when the x used alone or in combination with one or more other therapeutic means, such as chemotherapeutic agents) will depend on the type of disease being treated, the type of antibody or immunoconjugate, severity and treatment of the disease, regardless of injected if the indicated antibody or immunoconjugate preventive or therapeutic purposes, from the treatment done before, from the patient's medical history and its susceptibility to a given antibody or immunoconjugate, and from a doctor's appointment. Such 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 the initial preset dose of the antibody or immunoconjugate entered the patient is from about 1 μg/kg to 100 mg/kg (for example, 0.1 to 20 mg/kg/dose), regardless of whether one-time or repeated introduction or continuous infusion of this antibody. One typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. Re-introduction for several days or more, depending on the condition, treatment may be as long until you achieve the desired suppression of disease symptoms. One representative dose of the indicated antibodies or immunoconjugate may be approximately the 0.05 mg/kg to 10 mg/kg Thus, the patient can be entered in one or more doses of around 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination) antibodies or immunoconjugate. Such doses may be introduced intermittently, e.g. every week or every three weeks (e.g. such that the patient received approximately from 2 to 20 or, for example, about 6 doses of antibodies or immunoconjugates). Initially, there may be introduced a higher loading dose, and then can be entered one or more lower doses. A representative scheme of doses includes the introduction of the initial loading dose, then weekly introduction maintenance dose of about 2 mg/kg of antibody. However, there may be primene and other schemes of doses. Monitoring the effect of such therapy can be easily carried out using standard methods and analysis.

C. Analysis on 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 for identifying anti-CD79b antibody or immunoconjugate possessing biological activity. Biological engineering is I activity may include, for example, the ability to inhibit growth or proliferation of cells (e.g., activity, aimed at "the destruction of the 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 immunoconjugate tested for ability to inhibit growth or proliferation of cells in vitro. Assays for inhibition of growth or cell proliferation are well known in the art. Some of the tests on the proliferation of cells, called here the analysis on "cell killing", allow us to determine the cell viability. One of these analyses is the fluorescence analysis of cell viability CellTiter-GloTMwhich is commercially available and was developed by company 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 for automated high-performance screen (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent (reagent CellTiter-Glo®) directly in cultured cells. This leads to the lysis of cells and the production of a fluorescent signal produced by the luciferase reaction. The luminescent signal is proportional to the amount present of the APR, which is directly proportional to the number of viable cells present in the culture. Data can be recorded in a luminometer or imaging device equipped with a camera with a charge-coupled. Fluorescent signal obtained at the output of the device, expressed in relative light units (RLU).

Another analysis on cell proliferation is an analysis of the "MTT", namely colorimetric analysis, which measure the level of oxidation of bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium to formazan under the action of mitochondrial reductase. Like the analysis of the CellTiter-GloTMthis analysis allows to determine the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65:55 to 63, and Zhang et al. (2005) Cancer Res. 65:3877-3882.

In one aspect of the invention, an anti-CD79b antibody test for the ability to induce cell death in vitro. Tests for the induction of cell death is well known to specialists. Some of the options that inventions such analyses allow us to determine for example, loss of membrane integrity, as indicated by the uptake of iodide propecia (PI), Trypanosoma blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD. In a representative analysis of PI uptake cells were cultured in modified according Dulbecco environment Needle (DMEM):environment 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 were seeded in 100×20 mm plates at a density of 3×106cells in a bowl and leave overnight for adhesion. Then the medium is removed and replaced only with fresh medium or medium containing various concentrations of the antibody or immunoconjugate. The cells are then incubated for 3 days. After processing, the monolayers are washed with PBS and separated by trypsinization. After that, cells are centrifuged at 1200 rpm for 5 minutes at 4°C, then the precipitate resuspended in 3 ml of 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 tight-fitting lid (1 ml per tube, 3 tubes per group processing) to remove accumulations of cells. Then in a test tube add PI (10 μg/ml). Samples will be analyzed by a flow cytometer FACSCAN® and using computer programs FACSCONVERT® CellQuest (Becton Dickinson). Thus the m 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, an anti-CD79b antibody or immunoconjugate tested for the ability to induce apoptosis (programmed cell death) in vitro. Representative analysis on antibodies or immunoconjugate, inducing apoptosis, is tested for binding to annexin. In a representative analysis of the binding of annexin cells are cultivated and sown in the Cup, as described in the previous paragraph. Wednesday, remove and replace only with fresh medium or medium containing 0.001 to 10 μg/ml antibody or immunoconjugate. After incubation for three days, the monolayers are washed with PBS and separated by trypsinization. After that, cells are centrifuged, resuspended in Ca2+binding buffer and divided into aliquots in test tubes as described in the previous paragraph. In a test tube add labeled annexin (e.g., annexin V-FITZ) (1 μg/ml). Samples will be analyzed by a flow cytometer FACSCAN™ and using computer programs FACSCONVERT™ CellQuest (BD Biosciences). Thus identify antibodies or immunoconjugate that induce statistically significant levels of binding to annexin compared with the control. Other representative analysis on antibodies or immunoconjugate that induce up the ptosis, is a colorimetric ELISA analysis using DNA histone, which enables to detect minucioso degradation of genomic DNA. Such analysis can be carried out using, for example, ELISA-kit for the detection of cell death (Roche, Palo Alto, CA).

The cells used in any of the above in vitro, are cells or cell lines that normally Express CD79b or which were so designed that they CD79b expressed. Such cells are tumor cells expressing CD79b compared to normal cells derived from the same tissue. Such cells are cell lines (including tumor cell line), which Express CD79b, and cell lines that do not normally Express CD79b, but were transliterowany nucleic acid that encodes a CD79b.

In one aspect of the invention, an anti-CD79b antibody or immunoconjugate tested for ability to inhibit growth or proliferation of cells in vivo. In some embodiments of the invention the anti-CD79b antibody or immunoconjugate tested for the ability to inhibit tumor growth in vivo. For such testing can be used system model in vivo. In a representative system of xenograft human tumor cells injected the animal with a weakened and the immunity, not a man, for example, SCID mice. This animal is administered an antibody or immunoconjugate according to the invention. Then evaluate the ability of antibodies or immunoconjugate to inhibit or reduce tumor growth. In some embodiments, the above system xenograft human tumor cells are tumor cells derived from human. Such cells, suitable for receiving the xenograft models, are cells of the human leukemia cell line human lymphomas, which include, but are not limited to, cells of BJAB-luc (e.g., EBV-negative cell lines lymphoma Burkitt, 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 injected animal immunocompromised, non-human, by subcutaneous injection or transplantation into a suitable site, for example, in the fat body of the breast.

2. Analysis of binding and other tests

In one aspect of the invention, an anti-CD79b antibody test on antigennegative activity. For example, in some embodiments from which retene anti-CD79b antibody test for the ability to bind to CD79b, expressed on the cell surface. Such testing can be performed using FACS analysis.

In one aspect of the invention to identify a monoclonal antibody which competes with murine MA79b antibody, humanized antibody MA79b.v17 and/or humanized antibody MA79b.v28 and/or humanized antibody MA79b.v32 for binding to CD79b, can be conducted analyses on competitive binding. In some embodiments of the invention, such a competing antibody binds to the same epitope (e.g., linear or conformational epitope)that is bound mouse antibody MA79b, humanitariannet antibody MA79bv.17 and/or humanitariannet antibody MA79b.v18 and/or humanitariannet antibody MA79b.v28 and/or humanitariannet antibody MA79b.v32. Representative analyses on competitive binding include, but are not limited to, routine tests, such as tests, described in the publication 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, which binds the antibody, can be found in the publication Morris (1996) “an 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 of them is blocking the binding of the other by 50% or more.

In epresentatives analysis on competitive binding to immobilized CD79b incubated in the solution, contains the first labeled antibody that binds to CD79b (e.g., a murine antibody MA79b, humanitariannet antibody MA79bv.17 and/or humanitariannet antibody MA79b.v18 and/or humanitariannet antibody MA79b.v28 and/or humanitariannet antibody MA79b.v32)and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CD79b. The second antibody may be present in the supernatant of hybridoma. As a control, immobilized CD79b incubated in the solution containing 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 test sample is significantly lower than the number of labels in the sample, it means that the second antibody is competing with the first antibody for binding to CD79b. In some embodiments of the invention immobilizovannyi CD79b is present on the surface of cells or in a membrane preparation obtained from cells expressing on their surface CD79b.

In one aspect of the invention purified anti-CD79b antibodies can be further characterized by a series of EN is Lisov, including, but not limited to, N-terminal sequencing, amino acid analysis, exclusion liquid chromatography high pressure (ghvd), held in adenocarinoma conditions, mass spectrometry, ion exchange chromatography and papain hydrolysis.

In one of its variants the present invention relates to the 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 an important factor is the half-life of antibodies in vivo, and some effector functions (such as complement and ADCC) are optional or undesirable. In some embodiments of the invention determine the activity of the Fc antibodies to guarantee that will be kept only the desired properties. To confirm the reduction/depletion of CDC and/or ADCC activity can be carried out tests for cytotoxicity in vitro and/or in vivo. So, for example, to ensure that this antibody will not bind to FcγR (and therefore probably will not have ADCC activity), but will retain the ability to bind to FcRn can be carried out analyses on binding to Fc receptor (FcR). Primary cells mediating ADCC, NK cells, Express only Fc(RIII, whereas monocytes Express Fc RI, Fc(RII and Fc(RII. The FcR expression on hematopoietic cells systematized in table 3 on page 464 publication Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). The example in vitro analysis to assess ADCC activity of interest molecules described in U.S. patent No. 5500362 or 5821337. Suitable effector cells for such assays are mononuclear cells of peripheral blood (MCPC) and natural killer cells (NK). Alternative or additionally, ADCC activity of interest molecules can be assessed in vivo, e.g., in a animal model such as the model described in the publication Clynes et al. PNAS (USA) 95:652-656 (1998). To confirm that this antibody is unable to communicate with C1q, and therefore it does not have CDC activity can also be tested for binding to C1q. To analyze the activation of complement may be conducted by CDC analysis, for example, the analysis described in the publication Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). Assessment of binding to FcRn, and clearance/time-life in vivo can also be carried out by methods known to experts.

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 this application, in its entirety introduced into the present description by reference.

Commercially available reagents referred to in the examples were used according to manufacturer's instructions, if it is not specifically mentioned. Antibodies used in the examples are commercially available antibodies, 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 the antibodies produced from hybridomas deposited with the ATCC as HB11413 July 20, 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: Getting gumanitarnogo anti-CD79b antibodies

Rooms residues are given by Kabat . In the description of the invention uses one-letter symbols of amino acids. The degeneracy of the DNA code IUB (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 humanitariannet anti-CD79b antibody

Received various humanized anti-CD79b antibody. Sequence domains VL and VH mouse antibodies MA79b (MA79b) (Roswell Park Cancer Institute; Okazaki et al., Blood, 81:84-94 (1993)) were aligned the sequences of domains of human consensus VL Kappa I (huKI) and human consensus VH subgroup III (huIII). To get the HVR-hybrid used acceptor frame the VH region, which differed from the domain of human consensus VH subgroup III in 3 positions: R71A, N73T and L78A . Hypervariable region of mouse MA79b (MA79b) was attached to the frame region of the acceptor human consensus sequences and received a direct HVR-hybrid MA79b (here called "hybrid MA79b" or "MA79b-hybrid" or "MA79b-related "humanized antibody" or "huMA79b-hybrid"). In the VL domain the following field was added 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 area in positions 26-35 (H1), 49-65 (H2) and 93-102 (H3) (figure 8A-B). In the publication by MacCallum et al. (MacCallum et al., J. Mol. Biol., 262:732-745 (1996)analyzed the crystal structure of the complex antigen-antibody", and it was found that the provisions of 49, 93 and 94 of the heavy chain are part of the contact area, and in the case of 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 was assessed by DNA sequencing.

B. Options for hybrids humanitarian the CSOs anti-CD79b antibodies

Options hybrid anti-CD79b antibodies that include a variety of mutants in hypervariable regions MA79b-linked "gumanitarnogo" antibodies, were obtained using phage libraries. Options hybrid anti-CD79b antibodies include mutations in the same position in the HVR (figure 9) or mutations in many of the provisions in the HVR (figure 10).

C. Selection of phage

For selection of phage extracellular domain CD79b (huCD79becd) (2 μg/ml) was immobilized in PBS on 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 microtiter tablet intensively washed with PBS containing 0.05% tween-20 (PBST) to remove unbound phage, and bound phage were suirable by incubation of the wells with 100 mm HCl for 30 minutes. The rigidity of selection can be increased during 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.

Suirvey phage was neutralized in 1M Tris, pH 8, and amplified using cells XL1-Blue and phage-assistant M13/KO7, and then coltivirus is whether over night at 37°C in 2YT, 50 μg/ml of carbenicillin. The titers of phage, lirovannomu with the target-containing wells was compared with the titers of phage isolated from wells that do not contain targets, to assess the level of enrichment.

D. Obtaining Fab and receiving IgG

For expression of Fab protein to measure the affinity between the heavy chain and g3 in the phage vector representation built in stop-codon. The clone was transferred into E. coli cells 34B8 and 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 the microns of benzamidine, 2.4 mm EDTA, and were destroyed in the clear using microfluidizer. Fab was purified using affinity chromatography on G-protein.

For the screening IgG variants were first produced in 293 cells. Vectors encoding the VL and VH (25 µg), transferred in 293 cells using FuGene. 500 μl of FuGene was mixed with 4.5 ml of DMEM medium containing no FBS and incubated at room temperature for 5 minutes. To this mixture was added to each circuit (25 μg) and incubated at room temperature for 20 minutes and then transferred into the flask for transfection overnight at 37°C in 5% CO2. The next day, the medium containing the transfection mixture for, 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, asetem medium were collected at 1000 rpm for 5 minutes and subjected to sterile filtration through a 0,22 µm filter, bind to the protein present in small numbers. After adding 2.5 ml of 0.1% PMSF for each 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 "gumanitarnogo" antibodies, extracellular domain of human CD79b (huCD79becd) expressed in CHO cells alone or in the form Fc-hybrid (huCD79becd-Fc) and were 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 antibodies MA79b (marked on figure 19 as "test peptide") is described in application for U.S. patent No. 11/462336, filed August 3, 2006, the Epitope for MA79b was localized in the extracellular peptide region located at a considerable distance from the transmembrane domain, and was present in the full-size 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)). A truncated form CD79b did not contain a full-sized extracellular Ig-like domain (extracellular Ig-like domain that is not present in splanirovannaya truncated form CD79b presented in a frame on figure 19).

The level of binding of Fab - and IG-options MA79b, MA79b-linked "gumanitarnogo" antibodies or MA79b-related options "gumanitarnogo" antibodies with immobilized huCD79becdor huCD79b-Fc or with a peptide of 16 amino acids, containing the epitope for MA79b, was measured by means of surface plasmon resonance. Measurement of the affinity was carried out by means of surface plasmon resonance using BIAcoreTM-2000. Antigen, huCD79becdor huCD79b-Fc was immobilized (approximately 50-200 SW) in 10 mm sodium acetate, pH of 4.8, on the sensor 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 with MA79b, which was simultaneously used as a standard. In those experiments in which assessed the level of binding to the peptide of 16 amino acids, containing the epitope for MA79b (ARSEDRYRNPKGSACK) (SEQ ID NO: 16), biotinylated peptide was immobilized (approximately 20 SW) on the sensor chip coated with streptavidin. Purified MA79b-attached option "gumanitarnogo" antibodies (Fab or IgG) (2-fold serial dilution of 0.5-1000 nm in PBST) was injected at a flow rate of 30 ál/min, Each sample was analyzed for 4-minutes of the Association and a 10-minute dissociation. After each injection, the chip p is generated using 10 mm glycine, the pH of 1.7.

The magnitude of the response binding assays were corrected by subtracting the values obtained for the control flow cell, the values obtained for flow-through cuvette containing MA79b-attached option "gumanitarnogo" antibodies (Fab or IgG). For kinetic analysis used langourously model 1:1 curve according to konand koff.

F. Analysis of binding (FACS-analysis)

To further determine the level of binding of the Fab-fragment of the MA79b-linked "gumanitarnogo" antibody or its variants assessed the binding of Fab variants and/or IgG cells DoHH-2 using FACS analysis. In addition, using FACS-analysis assessed the level of binding of the MA79b-related options "gumanitarnogo" antibodies with BJAB cells containing the luciferase.

For conducting FACS analysis of Fab variants MA79b-related options "gumanitarnogo" antibodies (MA79b-linked "gumanitarnogo" antibodies (variant IgG used as control), cells DoHH-2 (1×106volume 100 µl) was first incubated in the presence or absence of 1 μg of the original mouse monoclonal anti-CD79b antibody (MA79b) for 30 minutes and then was added 1 μg individual variant Fab (or control antibodies). As a "second" detecting antibodies used PV conjugial the TES mouse antibody against human light chain Ig Kappa (clone G20-193, BD Biosciences, San Diego, CA), as all options are Fab light chain Kappa, and cells DoHH-2 does not Express on their surface a light chain Kappa.

For additional FACS analysis of IgG variants MA79b-related options "gumanitarnogo" antibodies (variant IgG chMA79b used as a control), and 1.0 μg, 0.1 mg or 0.01 μg antibody was titrated per million cells of BJAB containing the luciferase. As a "second" detecting antibodies used Feh-conjugated mouse antibody against human Ig.

G. Determination of affinity (analysis of Scatchard)

To further determine the level of binding of IgG variants with modifications in HVR-L2 and HVR-H3 (huMA79b L2/H3), we analyzed the binding of iodinated IgG variants with BJAB cells expressing human and CD79b CD79b abacadabra monkeys, and then conducted an analysis of Scatchard.

For analysis of Scatchard 0.5 nm of the I125labeled 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-step serial dilution 1:2) in the presence of transtitional cell line BJAB, stably expressing CD79b copacobana monkeys and endogenous human CD79b. After 4-hour incubation at 4°C cells were washed, and the cell precipitate was read on a gamma counter (AVT is automatic gamma counter 1470 WIZARD Automatic Damme Counter; Perkin Elmer, Walthem, MA). All calculations were performed with three replicates, and the calculation was carried out for 10 minutes. To calculate the Kd used the average number of counts per minute (CPM), and this calculation was performed using the New computer program Ligand (Genentech, South San Francisco, CA).

Results and discussion

A. results of the production gumanitarnogo anti-CD79b antibodies

Human acceptor framework region used to obtain gumanitarnogo anti-CD79b antibody contains a domain VL consensus human sequence 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 consensus sequence at positions R71A, N73T and L78A. Sequence domains VL and VH MA79b were aligned with the human sequence Kappa I and subgroup III, where each HVR identified, and then was attached to the human acceptor framework region with getting the HVR-hybrid, which can be presented on the phage as Fab (figures 7 and 8).

The 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 huCD79becdindicated that the binding affinity of with whom were igalas in more than 100-fold (data not shown), and Biacore analysis indicated a more than 50-fold lower (figure 11).

1. Repair CDR

MA79b-related options "gumanitarnogo" antibodies, which possessed the ability to bind to immobilized huCD79becdidentified using the following substitutions in the sequence.

Changes in HVR in VL were available only in libraries, containing substitutions in one position, and such changes is shown in figure 9 (for the mutations L1: Q27K (SEQ ID NO: 17; mutation SPL-2), (mutation L2: L54R (SEQ ID NO: 18), E55K (SEQ ID NO: 19)), and (mutations L3: E93S (SEQ ID NO: 20; mutation SPL-5), E93K (SEQ ID NO: 21)).

Changes in HVR in L2, L3, H1 and H3 in the HVR was available only in libraries, containing substitutions at several positions, and such changes is shown in figure 10 (for mutation 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)), (mutation L3: E93A (SEQ ID NO: 46); E93Q (SEQ ID NO 47); the absence of 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)), (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 (mutations H3: V98I and F100L (SEQ ID NO: 87; mutation 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 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 IgG for posleduyushego the Biacore analysis and Scatchard.

a. Determination of affinity (Biacore analysis)

As shown in figure 11, which shows the Biacore analysis, this method CDR repair allows you to identify the many changes in a separate sequence, which increase the affinity of the MA79b-linked "gumanitarnogo" antibodies. Analysis using surface plasmon resonance showed that, although none of the tested variants with one HVR-replacement had no affinity, similar to the affinity of MA79b, however, the combination of substitutions identified in HVR-L2 and HVR-H3 (MA79b-associated variant gumanitarnogo" antibodies L2/H3; also referred to here huMA79b L2/H3), resulted in the formation of variants (figure 11), with affinity similar to the affinity of antibodies MA79b when binding to immobilized huCD79becdor huCD79becd-Fc or peptide of 16 amino acids, containing the epitope for MA79b, as determined in the Biacore analysis.

Analysis on linking monomer (Fab) and dimer (IgG) MA79b with antigen (huCD79becd-Fc) (figure 11, number 1, cf. columns for Fab and IgG) gave reason to assume that a component which is 100 times greater avidity and present in MA79b may be absent in variants with improved affinity. In particular, MA79b-associated variant "gumanitarnogo" antibodies L2-2 (also denoted here huMA79b L2-2), which detects 5-kr is you can increase the level of Monomeric binding compared to MA79b (figure 11, rows 1 and 3, see columns for Fab), after changing the format huMA79b L2-2 as IgG (figure 11, number 4, cf. columns for Fab to IgG), any apparent increase in affinity was observed. In addition, the original HVR-bound "humanitariannet" MA79b antibody (huMA79b-hybrid) showed loss of binding of such a component with avidity (figure 11, number 2, cf. columns for Fab to IgG). The ability to enhance binding through avidity may be desirable when binding to antigens on the cell surface.

b. Determination of affinity (analysis of Scatchard)

As was identified in the analysis of Scatchard, this method CDR repair allows you to identify multiple substitutions in a separate sequence, which increase the affinity of the MA79b-linked "gumanitarnogo" antibodies. In particular, analyses at the cellular binding showed that the binding affinity of MA79b and MA79b-associated variant "gumanitarnogo" antibodies L2/H3 (huMA79b L2/H3) (obtained in a different format, such as IgG) BJAB cells stably expressing CD79b abacadabra monkeys and endogenous human CD79b, had Kd values to 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 determined in the analysis of Scatchard.

c. Determining the level of binding (FACS-analysis)

As was assessed using FACS analysis, such CDR repair allows you to identify multiple substitutions in a separate sequence, resulting in increased level of binding of the MA79b-linked "gumanitarnogo" antibodies (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 SP and 6 libraries SR in 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 binding of Fab variants with cells DoHH-2 is blocked by pre-incubation with murine anti-CD79b monoclonal antibody (MA79b) (data not shown).

2. Repair frame region

Modification sequence HVR entered in HVR-L2 huMA79b L2/H3 variant, radically differed from the modifications observed in any of the human germ line. It was found that option huMA79b L2/H3, with its conjugation with DM1, effectively inhibits tumor growth in mice with xenograft model in vivo (table 9). Because the analysis of Monomeric binding (Fab) and dimeric binding (IgG) huMA79b L2/H3-variant antigen showed a loss of avidity (figure 11), then the reparation frame region was carried out as described below.

To determine the role of regulations framed residues in the binding of the dimer to the antigen, designed variant with the provisions of the "all residual frame is area", in which potentially important provisions murine residues frame region were included in the MA79b HVR-bound "humanitariannet" antibody (huMA79b-hybrid). Such an option (referred to in figure 12 option that has "all the remains of the frame region")that does not contain any modifications in the HVR, had dimer affinity binding, similar to the affinity of binding of chimeric MA79b antibody (chMA79b) (figure 12), as determined by Biacore analysis and analysis of Scatchard.

IgG variants, including murine skeleton residues in positions 4 and/or 47 (VL) and/or provisions 47, 48, 67, 69, 71, 73, 74, 78 and/or 80 (VH), was received in order to determine a minimal set of provisions in the frame region that is required for high affinity binding to the dimer (figure 12). The remains of the mouse frame 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 area 47 in VL and 75 and 80 in VH does not play an important role, as shown by the analysis of the MA79b-associated variant "gumanitarnogo antibodies" 17 (huMA79b.v17) (figure 12, row labeled 17).

MA79b-associated variant gumanitarnogo antibodies" 18 (huMA79b.v18; figure 12, row labeled 18), which includes the remains of murine frame region at positions 4 in VL and 48, 67, 69, 71, 73 and 78 in VH, and change in HVR-H3 (marked on figure 12 as "H3-10 and described above as mu is the situation H3-10), including V98I, Y99R and F100L, discover an additional 2-fold increase (figure 12, row labeled 28) level of binding to the dimer compared to option 17 (figure 12, row labeled 17).

In order to avoid possible difficulties in obtaining these antibodies, potential customers, forming seasparrow acid (Asp-Gly) HVR-L1 MA79b-related options "gumanitarnogo antibodies were removed by transformation D28 in Glu (glutamic acid) (D28E; see 28; also designated here as "huMA79b.v28"; figure 12, row labeled 28). Also possible are other substitutions can be introduced to give stability VL MA79b-related options "gumanitarnogo antibodies, including replacement D28 at Ser (serine) (D28E; see 32; also called here "huMA79b.v32"; figure 12, row labeled 32).

MA79b-associated variant gumanitarnogo antibodies" 28 (huMA79b.v28; figure 12, row labeled 28), which includes: (1) residues of murine frame region at positions 4 in VL and 48, 67, 69, 71, 73 and 78 in VH, and also includes (2) replacement HVR-H3 (marked on figure 12 as "H3-10 and described above as mutation H3-10), including V98I, Y99R and F100L, and, in addition, includes: (3) replacement HVR-L1 (D28E described above), was characterized using Biacore analysis.

MA79b-associated variant gumanitarnogo antibodies" 32 (huMA79b.v32; figure 12, row labeled 32), to the that includes: (1) residues of murine frame region at positions 4 in VL and 48, 67, 69, 71, 73 and 78 in VH, and also includes (2) replacement HVR-H3 (marked on figure 12 as “H3-10 and described above as mutation H3-10), including V98I, Y99R and F100L, and, in addition, includes: (3) replacement HVR-L1 (D28S described above), was characterized using Biacore analysis.

a. Determination of affinity (Biacore analysis)

As shown in figure 12, illustrating the Biacore analysis, this method reparations frame circuit allows the identification of multiple substitutions in a separate sequence, which increase the binding affinity of the hybrid MA79b-humanitariannet antibody with huCD79becd. Analyses carried out by means of surface plasmon resonance showed that MA79b-associated variant gumanitarnogo antibodies" 28 (huMA79b.v28; with murine residues frame region at positions 4 in VL, 48, 67, 69, 71, 73 and 78 in VH, as well as mutation H3-10 in HVR-H3 (V98I, Y99R and F100L (also described above) and mutation D28E in HVR-L1 (introduced to improve the stability, see above); figure 12, row labeled 28) and MA79b-associated variant "gumanitarnogo antibodies" 32 (huMA79b.v32; with murine residues frame region at positions 4 in VL, 47, 48, 67, 69, 71, 73 and 78 in VH, as well as mutation H3-10 in HVR-H3 (V98I, Y99R and F100L (also described above) and mutation D28S in HVR-L1 (introduced to improve the stability, see above); figure 12, row labeled 32) have affinity binding to immobilized huCD79becdequivalent to affine the ti binding of the chimeric MA79b antibody (chMA79b) with a specified antigen, as determined by Biacore analysis.

b. Determination of affinity (analysis of Scatchard)

As was assessed using analysis of Scatchard similar to Biacore analysis, this method reparations frame circuit allows the identification of multiple substitutions in a separate sequence, which increase the binding affinity of the hybrid MA79b-humanitariannet antibody" (huMA79b-hybrid). Tests for binding to cells showed that the binding affinity of MA79b, MA79b-associated variant "gumanitarnogo" antibodies 28 (huMA79b.v28; see figure 12, row labeled 28) (obtained in a new format as IgG) and MA79b-associated variant "gumanitarnogo" antibody 32 (huMA79b.v32; see figure 12, row labeled 32) BJAB cells stably expressing CD79b abacadabra monkeys and endogenous human CD79b, has the value of Kd, component 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 determined by analysis of Scatchard.

c. Determining the level of binding (FACS-analysis)

As was determined using FACS analysis, this method reparations frame circuit pozvolit to identify a lot of changes in individual sequences, which enhance binding of the hybrid MA79b-humanitariannet antibody" (MA79b-hybrid) BJAB cells containing Lucifer is zu (data not shown). In particular, FACS-analysis of IgG variants MA79b-related options "gumanitarnogo" antibodies (options huMA79b.v28 and huMA79b.v32) BJAB cells containing the luciferase revealed a binding with the specified BJAB cells containing the luciferase (data not shown).

B. Discussion the production of humanized anti-CD79b antibodies

To identify changes in HVR 1-6, which increase the affinity of binding was carried out repair CDR by attaching 6 mouse HVR MA79b (defined as positions 24-34 (L1), 50-56 (L2), and 89-97 (L3), 26-35 (H1), 49-65 (H2) and 93-102 (H3)) to the human consensus sequences of VL Kappa I and VH subgroup III VH (containing A71, T73 and A78). Substitutions in the sequence HVR identified on figure 10 and 11, or a combination of these substitutions lead to the formation of humanized variants MA79b with affinity similar to the affinity of MA79b.

Alternative repair frame field 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 VH) (figure 12; MA79b-associated variant gumanitarnogo" antibodies 17 (huMA79b.v17)). The binding affinity of the variants with substitutions in the framework region of the antibody against the antigen huCD79becdwas also extended by introducing semen in HVR-H3: V98I, Y99R and F100L (figure 12; MA79b-associated variant gumanitarnogo" antibody 18 (huMA79b.v18)). Potential customers education itsprevious acid in HVR-L1 was removed by introducing mutations D28E (figure 12; MA79b-associated variant gumanitarnogo" antibodies 28 (huMA79b.v28)).

Example 2: Obtaining conjugates of anti-CD79b antibody - drug" (ADC)

To analyze the efficiency of IgG variants MA79b-related options "gumanitarnogo" antibodies listed MA79b-related options "gumanitarnogo" antibodies conjugatively with drugs, such as DM1. Options, conjugated with DM1 are variants with substitutions in HVR-L2 and HVR-H3 (huMA79b L2/H3), huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32.

Drugs used to obtain conjugates of 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), which in its entirety are introduced in the present description by reference). The linkers used to obtain the ADC are BMPEO, SPP or SMCC (also called here "MCC") for DM1 and MC or MC-vc-PAB for MMAE and MMAF. In the case of DM1 antibody was attached to a thio group DM1 through ε-aminogroup the dust lysine using the linker reagent SMCC. Alternatively, in the case of DM1, the antibodies were attached to DM1 via 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 reactive 2-pyridyldithio linker to the protein. Under the action of linkers SPP, after the reaction between free sulfhydryl (e.g., DM1), Peregrina group is removed, resulting in DM1 attached recovered through disulfide bonds. DM1, attached through a linker SPP, released in reducing conditions (e.g., cells), and DM1, attached through a linker SMCC, is resistant to cleavage under conditions of recovery. In addition, the ADC SMCC-DM1 induced cellular toxicity if the ADC is internalized and delivered to lysosome with the release of lysine-Nε-DM1, which represents an effective antimitoticescoe agent present in the cells, and when it is released from cells lysine-Nε-DM1 becomes non-toxic (Erickson et al., Cancer Res., 66:4426-4433 (2006)). In the case of MMAE and MMAF antibody were added to MMAE or MMAF through cysteine through maleimidomethyl-valine-citrulline-(vc)-p-aminobenzeneboronic (MC-vc-PAB). In the case of MMAF these antibodies were alternative attached to MMAF pic what edstam cysteine through maleimidomethyl (MC) linker. MC-vc-PAB-linker cleaved by extracellular proteases, such as cathepsin In, and after splitting is released free drug (Doronina et al., Nat. Biotechnol., 21:778-784 (2003)), and the linker MC is resistant to cleavage by intracellular proteases.

The conjugates of the antibody-drug" (ADC) for anti-CD79b antibodies were obtained using SMCC and DM1 in accordance with the procedure described in the application US 2005/0276812. After cleaning, anti-CD79b antibodies spent a buffer exchange with the introduction of a solution containing 50 mm potassium phosphate and 2 mm EDTA, pH 7.0. SMCC (Pierce Biotechnology, Rockford, IL) was dissolved in dematiaceae (DMA) was added to a solution of antibodies to obtain the final molar ratio of SMCC/Ab=10:1. The reaction was carried out for three hours at room temperature with stirring. SMCC-modified antibody was then purified on a desalting column (GE Healthcare HiTrap (G-25), equilibrated in 35 mm sodium citrate with 150 mm NaCl and 2 mm EDTA, pH to 6.0. DM1, dissolved in DMA was added to SMCC-drug antibodies with getting molar ratio DM1 for the antibody 10:1. The reaction was carried out for 4-20 hours at room temperature with stirring. The solution 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 output ant the body was 40-60%. This drug is usually more than 95% was Monomeric, as was estimated by gel-filtration and method for light scattering from the laser. Because the maximum absorption DM1 was observed at 252 nm, the amount of drug associated with the antibody can be determined by measuring the differential absorption at 252 and 280 nm. Usually the ratio of drug to antibody was 3:4.

Described here conjugates "antibody-drug" (ADC) for anti-CD79b antibodies can be obtained using linkers SPP-DM1 in accordance with the procedure described in the application US 2005/0276812. After cleaning, anti-CD79b antibodies spent a buffer exchange with the introduction of a solution containing 50 mm potassium phosphate and 2 mm EDTA, pH 7.0. SPP (Immunogen) was dissolved in DMA was added to the antibody solution to obtain a final molar ratio of SPP/Ab=10:1, with the exact ratio depends on the desired loading of the drug in the antibody. On the basis of a relationship of 10:1, it is possible to obtain the ratio of the drug to the antibody of approximately 3-4. SPP was left for reaction for 3-4 hours at room temperature with stirring. SPP-modified antibody was then purified on a desalting 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 physiologist the ical solution pH 7.4. DM1 was dissolved in DMA and added to SPP-drug antibodies with getting molar ratio DM1 for the antibody 10:1, which gives a 3-4-fold molar excess compared with the SPP-linkers present in the antibody. Reaction with DM1 was carried out for 4-20 hours at room temperature c with stirring. The solution 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 output of the antibodies was 40-60%. This conjugate the antibody-drug" usually more than 95% was Monomeric, as was estimated by gel-filtration and method for light scattering from the laser. The amount of bound drug was determined by measuring the differential absorption at 252 and 280 nm, as described to obtain conjugates SMCC-DM1 (as described above).

Described here are the conjugates of the 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 dithiothreitol (DT). 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 thiol/Ab was assessed by determining the concentration of the recovered antibodies, based on the optical density of the solution of 280 nm, and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI), and determining the optical density 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, was dissolved in acetonitrile and water, and then added to the cooled recovered antibody in PBS. After incubation for 1 hour was added excess maleimide for quenching the reaction, and any thiol groups unreacted antibody was kepirohi. The reaction mixture was concentrated by centrifugal ultrafiltration, and then conjugate the antibody-drug" purified and absoluely by elution through G25 resin in PBS, and then filtered through the 0.2-μm filters under sterile conditions, and frozen for storage.

The conjugates of the antibody-drug" (obtained using the described here anti-CD79b antibody) was diluted with 2 x 10 µg/ml in the analytical environment. The conjugates were joined by cross-linking of SMCC linkers (for p is soedineniya SPP to maytansinoids to DM1 toxin can be used an alternative disulfide linker) (see the application US 2005/0276812 and US 2005/0238649). In addition, the conjugates can be connected by MC-valine-citrulline-(vc)-PAB or MC with derivatives dolastatin 10, toxin-monomethylaniline E (MMAE) or toxin by monomethylaniline F (MMAF) (see application for U.S. patent No. 11/141344, filed may 31, 2005, and application for U.S. patent No. 10/983340, filed November 5, 2004). The negative control consists of the conjugates on the basis of HERCEPTIN® (trastuzumab) (anti-HER2 antibody) (SMCC-DM1 or SPP-DM1 or MC-vc-MMAE or MC-vc-MMAF). A positive control may include free L-DM1 equivalent loading dose of conjugate. Samples before breeding them intensively stirred to ensure a homogeneous mixture.

Anti-CD79b antibodies for conjugation with the drug are chimeric MA79b antibody (chMA79b) and L2/H3 antibodies huMA79b and described here huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32 (see example 1). Other antibodies for conjugation can be any described herein antibodies (see Example 1).

Example 3: Analysis of in vivo at the destruction of tumor cells

A. Xenografts

To analyze the efficiency of IgG variants MA79b-related options "gumanitarnogo" antibodies with substitutions in HVR-L2 and HVR-H3 (huMA79b L2/H3), option huMA79b L2/H3 conjugatively with DM1 and analyzed the influence of conjugated variant on tumors in mice.

In particular, can be proan the lysed ability of antibodies to ensure that the regression of tumors in many models xenografts, cells including RAMOS, BJAB cells (cell line Burkitt lymphoma, which contains the translocation t(2;8)(p112;q24) (IGK-MYC), mutated P53 gene, and 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 overexpression cycline D1 (BCL1); deletions P16INK4B and P16INK4A and is EBV-positive) (Drexler, H.G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001), cells U698M (b-cell line of lymphoblastic lymphosarcoma; (Drexler, H.G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001) and DoHH2 cells (cell line follicular lymphoma containing the translocation responsible for the development of follicular lymphoma, and it is t(14;18)(q32;q21), which leads to overexpression of Bcl-2, variable heavy chain Ig; P16INK4A deletion, and translocation t(8;14)(q24;q32) (IGH-MYC), and is also an 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 "gumanitarnogo antibodies", 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, or cells Granta-519 by injection into the flanks of mice CB17 ICR SCID, and these mice were left up until the average size of puhalovich grafts did not reach 200 mm 2. Day 0 indicates the day on which the tumor size was about 200 mm2and in which was introduced the first or only one dose that you enter during this processing, if it is not specifically provided below. Tumor volume, expressed in mm3measured with a caliper in two dimensions and was calculated by the formula: V=0.5 a×b2where a and b denote the large and small diameters of the tumor, respectively. Data collected for each experimental group were expressed as mean ± srcpath. Groups of 10 mice were administered a single intravenous (i.v.) dose 50-210 µg associated with the antibody medicines/m2mouse (corresponding to 1-4 mg/kg mouse), namely introduced conjugates "option MA79b-associated gumanitarnogo antibodies or control antibody-drug". During the entire experiment, tumors were measured once or twice a week. Body weight of mice was measured once or twice a week during the entire experiment. When tumor volume reached 3000 mm3or when the tumor was discovered life-threatening signs of ulceration, mice were subjected to euthanasia. All protocols for animal experiments were approved by the Institutional Committee for animal care and use (IACUC).

Used by linkers linking the antibody and the toxin are thioester cross-communicating the second linker SMCC, communicating with DM1. Other linkers may be a disulfide linker SPP or thioester cross-linking, the linker SMCC for DM1 or MC or MC-valine-citrulline(vc)-PAB or dipeptide linker reagent (valine-citrulline(vc)), with maleimide component and a para-aminobenzeneboronic (PAB) carolinensis component for monomethylaniline E (MMAE) or monomethylaniline F (MMAF). Used toxins are DM1. Additional toxins can be MMAE or MMAF.

Anti-CD79b antibodies used in this experiment are chimeric MA79b antibody (chMA79b)described in application for U.S. patent No. 11/462336, filed August 3, 2006, and described here MA79b-related options "gumanitarnogo" antibodies (see example 1A). Other antibodies may be commercially available antibodies, including anti-CD79b antibody, and monoclonal antibodies MA79b obtained from hybridomas deposited with the ATCC as HB11413 July 20, 1993

The negative control consists of the conjugates on the basis of anti-HER2 antibodies (HERCEPTIN® (trastuzumab))(SMCC-DM1).

B. Results

1. The xenografts BJAB containing the luciferase

After conducting a 35-day course of treatment with a conjugate of drugs in the doses indicated in table 9, namely MA79b-associated variant gumanitarnogo antibodies L2/H3 (option huMA79b L2/H3) (obtained in the new format, IgG) and Chimera ant is-CD79b antibody (chMA79b), conjugated with DM1 (huMA79b L2/H3-SMCC-DM1 and chMA79b-SMCC-DM1, respectively), revealed the inhibition of tumor growth BJAB containing the luciferase, SCID mice compared with the negative control, namely the antibody HERCEPTIN® (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1). For all ADC and control were administered one dose of the ADC (as shown in table 9) on day 0. In particular, L2/H3-SMCC-DM1 antibody huMA79b (obtained in the new format as IgG) and chMA79b-SMCC-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"
(µ g/m2)
Dose Ab
(mg/kg)
Ratio (Drug/
Ab)
Control anti-HER2-SMCC-DM10/10 0/1010023,3
chMA79b-SMCC-DM13/103/101002,42,9
chMA79b-SMCC-DM11/100/10501,22,9
huMA79b L2/H3-SMCC-DM12/100/101002,92,4
huMA79b L2/H3-SMCC-DM10/100/10501,42,4

2. The xenografts Granta-519

After conducting a 14-day course of treatment with a conjugate of drugs in the doses indicated in table 10, namely MA79b-associated variant gumanitarnogo antibodies, namely version 17, version 18, option 28 and option 32 (huMA79b.v17, huMA79b.v18, huMA79b.v28 and huMA79b.v32, respectively) (obtained in a 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 chMA7b-SMCC-DM1, respectively), it was revealed that the 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 were administered one dose of the ADC (as shown in table 9) on day 0. In particular, antibodies 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).

In addition, 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 the 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"
(µ g/m2)
Dose Ab
(mg/kg)
Ratio (Drug/
Ab)
Control anti-HER2-SMCC-DM10/100/1020843,4
chMA79b-SMCC-DM10/100/1010723,6
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-DM12/10 2/1020243,4
huMA79b.v32-SMCC-DM10/100/1017242,9

Based on the ability of the ADC MA79b-associated humanitariannet antibody significantly inhibit the progression of tumors in the xenograft, we can say that CD79b molecule can serve as an excellent target for the treatment of tumors in mammals, including b-cell associated cancers, such as lymphomas (i.e., non-Hodgkin's lymphoma), leukemia (i.e., chronic lymphocytic leukemia and other cancers of hematopoietic cells. In addition, MA79b-related 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), leukemia (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 of interest is narisovannyj in b-cell line, 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 constitute a compartment similar to the late endosome/lysosome. HLA-DM is a marker for MIIC.

The Ramos cells were incubated for 3 hours at 37°C With 1 µg/ml MA79b-related humanized antibodies and their variants, with FcR block (Miltenyi) and 25 μg/ml Alexa647-transferrin (Molecular Probes) containing no carbonate medium (Gibco) in the presence of 10 μg/ml leupeptin (Roche) and 5 μm of pepstatin (Roche) for inhibiting the decomposition of lysosomes. The cells are then twice washed, fixed with 3% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature, extinguished 50 mm NH4Cl (Sigma) and made permeable using 0.4% saponin/2% FBS/1% BSA for 20 minutes, then incubated with 1 μg/ml antibodies against mouse Cy3 (Jackson Iunoresearch) for 20 minutes. Then the reaction was blocked for 20 minutes mouse IgG (Molecular Probes), and the mixture is then incubated for 30 minutes with signal amplifier Image-iT FX (Molecular Probes). Finally, 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 of MH class II), within 20 minutes, and then fixed with 3% PFA.

Cells resuspendable in 20 µl tapaninaho buffer and left for adhesion on slides, covered with polylysine (Sigma), and then was placed on a cover glass using DAPI-containing VectaShield vector (Vector Laboratories). For immunofluorescence assay MIIC or lysosomes of the cells were fixed, made permeable, and the signal is 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 manufacturer's instructions (Molecular Probes).

In accordance with this colocalization MA79b-related humanized antibodies or their variants with MIIC or lysosomes In cell lines, as assessed by immunofluorescence assay, may indicate that these molecules are excellent tools for the treatment of tumors in mammals, including b-cell associated cancers, such as lymphomas (i.e., non-Hodgkin's lymphoma), leukemia (i.e., chronic lymphocytic leukemia and other cancers of hematopoietic cells.

Example 5: Getting constructed on the basis of cysteine anti-CD79b antibodies

Constructed on the basis of cysteine anti-CD79b antibodies were obtained as described in this application.

DNA encoding the MA79b antibody (light chain, SEQ ID NO: 4 figure 4; and a heavy chain, SEQ ID NO: 5, figure 5)were subjected to mutagenesis methods described herein for modification of the light and heavy chains. DNA encoding the MA79b antibody (heavy chain SEQ ID NO: 5; figure 5), can also be subjected to mutagenesis methods described herein for modification of the Fc region of the heavy chain.

DNA encoding the antibody huMA79b.v17 (heavy chain SEQ ID NO: 304, figure 15)were subjected to mutagenesis methods described herein for modification of 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), can also be subjected to mutagenesis methods described herein for modification of the light chain or the Fc region of the heavy chain.

DNA encoding the antibody huMA79b.v18 (heavy chain SEQ ID NO: 306, figure 16)were subjected to mutagenesis methods described herein for modification of 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), can also be subjected to mutagenesis methods described herein for modification of 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)were subjected to mutagenesis methods described herein for modification of 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), can also be subjected to mutagenesis op the toboggan here methods for modification of 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), can also be subjected to mutagenesis methods described herein for modification of the light chain and heavy chain.

DNA encoding the antibody against CD79b abacadabra monkeys (light chain, SEQ ID NO: 241, figure 45; and a heavy chain, SEQ ID NO: 243, figure 47)were subjected to mutagenesis methods described herein for modification of the light and heavy chains. DNA encoding the antibody against CD79b abacadabra monkeys (heavy chain SEQ ID NO: 243; figure 47), can be subjected to mutagenesis methods described herein for modification of the Fc region of the heavy chain.

To obtain designed on the basis of cysteine anti-CD79b antibodies, DNA encoding light chain were subjected to mutagenesis by replacing valine-cysteine at position 205 according to the Kabat 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 abacadabra monkeys (ch10D10)). DNA encoding a heavy chain were 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 sequential numbering system; room 114 according to Kabat), as shown in figure 48 (heavy chain SEQ ID NO: 244 thio-Mab-is ntitle against CD79b abacadabra monkeys (ch10D10)), in 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 antibodies can 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; Kabat - 396), as shown in table 2-4.

A. Obtaining constructed on the basis of cysteine anti-CD79b antibodies for conjugation by restoring and re-oxidation

Full size is designed based on the cysteine anti-CD79b monoclonal antibodies (ThioMab) expressed in CHO cells and purified by carrying out affinity chromatography on protein A, and then exclusion chromatography. Purified antibodies were diluted in 500 mm sodium borate and 500 mm sodium chloride at about pH of 8.0, and then restored approximately 50-100-fold molar excess of 1 mm TCEP (Tris hydrochloride(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 onto HiTrap column S in 10 mm sodium acetate, pH 5, and suirable PBS containing 0.3 M sodium chloride. Suirvey restored thio-Mab was treated with 2 mm l is hidroascorbic acid (dhAA) at pH 7 for 3 hours or 2 mm aqueous copper sulfate (CuSO 4) 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 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 thiol, by the reaction of interaction with DTNB (Aldrich, Milwaukee, WI), and determining the optical density at 412 nm.

Example 6: Obtaining conjugates "constructed on the basis of cysteine anti-CD79b antibody-drug" by the reaction of conjugation constructed on the basis of cysteine anti-CD79b antibodies with intermediate connections "drug-linker"

After recovery procedures and re-oxylene described in example 5, were constructed on the basis of cysteine anti-CD79b antibody was diluted in PBS buffer (phosphate buffered saline) and cooled on ice. About 1.5 molar equivalents of intermediate compounds "auristatin drug-linker", such as MC-MMAE (maleimidomethyl-monomethylaniline E), MC-MMAF, MC-val-cit-PAB-MMAE or MC-val-cit-PAB-MMAF, containing reactive thiol functional group, such as maleimido, in relation to zisteinom introduced into the antibody, RA is tarali in DMSO, diluted in acetonitrile and water was added to the cooled restored and re-oxidized antibody in PBS. Approximately one hour was added excess maleimide for quenching the reaction and any unreacted thiol group of the antibody was kepirohi. The reaction mixture was concentrated by centrifugal ultrafiltration, and then the conjugate is constructed on the basis of cysteine anti-CD79b antibody-drug" purified and absoluely by elution through G25 resin in PBS, and then filtered through the 0.2-μm filters under sterile conditions, and frozen for storage.

Getting thioMAb-BMPEO-DM1 huMA79b.v18-HC(A118C) was carried out as follows. The free cysteine on huMA79b.v18-HC(A118C) thio-MAb modified bis-maleimido-reagent BM(PEO)3 (Pierce Chemical), resulting in the unreacted maleimido group remained on the surface of the antibody. This was accomplished by dissolving BM(PEO)3 in 50% ethanol/water to achieve a concentration of 10 mm and adding a 10-fold molar excess of BM(PEO)3 in the solution containing thio-MAb huMA79b.v18-HC(A118C) in phosphate buffered saline solution at a concentration of approximately 1.6 mg/ml (10 micromoles), and then left for 1 hour to complete the reaction. Excess BM(PEO)3 was removed by gel filtration (HiTrap column, Pharmacia) in 30 mm citrate, pH 6, in the presence of 150 mm NaCl buffer. About 10 kPa the hydrated excess of DM1, dissolved in dimethylacetamide (DMA), was added to the intermediate connection thio-MAb-BMPEO huMA79b.v18-HC(A118C). For dissolving reagent, namely molecule drugs, may also 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 medicines. Gel-filtration on columns S200 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, control thio-antibody hu-anti-HER2-HC(A118C)-MC-MMAF, control thio-antibody 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 thio-huMA79b.v28-HC(A118C) and BMPEO-DM1;

6. thio-huMA79b.v28-HC(A118C)-MC-val-cit-PAB-MMAE, obtained by conjugation 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 thio-MA79b-HC(A118C) and MC-MMAF; and

11. thio-MA79b-LC(V205C)-MC-MMAF, obtained by conjugation thio-MA79b-LC(V205C) and MC-MMAF.

Example 7: Characterization of the affinity of binding of conjugates "constructed on the basis of cysteine thio-Mab-drug" with the antigen on the cell surface

The binding affinity of the conjugates "thio-huMA79b.v18, thio-huMA79b.v28-drug and conjugates "thio-MA79b-drug" with CD79b expressed on BJAB cells containing the luciferase, was assessed using FACS analysis. In addition, the binding affinity of the conjugates "thio-anti-cynoCD79b(ch10D10) antibody-drug" with CD79b expressed on BJAB cells expressing CD79b abacadabra monkeys were determined using FACS analysis.

Briefly, approximately 1×106/sup> cells in 100 μl were subjected to contacting with varying amounts (1.0 microgram, 0,1 μg or 0.01 μg Ab per million cells of BJAB containing the luciferase, or BJAB cells expressing CD79b abacadabra monkeys (for anti-cynoCD79b thio-Mab) one of the following conjugates of 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(A118C)-MC-MMAF (see figure 32B-32D, respectively). Feh-conjugated mouse antibody against human Ig was used as the second detection antibody (BD Cat#555787).

Anti-CD79b antibody associated with the cell surface were detected using Feh-conjugated mouse antibodies against human Ig. In the graphs presented in figure 29-32, it was shown that binding to the antigen is approximately the same for all the tested conjugates "thio-MAb-drug".

Example 8: Analysis of in vitro to reduce about what operacii cells under the action of the conjugates of 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 the luciferase; figure 41B, Granta-519; figure 41C, WSU-DLCL2). Fluorescent analysis of cell viability CellTiter-Glo® is 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 amount of ATP present, which is an indicator of metabolic activity of cells (Crouch et al., J. Immunol. Metho., 160:81-88 (1993); US 6602677). Analysis of the CellTiter-Glo® was performed in 96-well format, allowing automated high-throughput screening (HTS) (Cree et al., AntiCancer Drugs, 6:398-404 (1995)). The procedure of such a specially designed analysis involves adding a single reagent (reagent CellTiter-Glo®), which is directly added to cells cultured in medium containing serum.

The procedure is conducted in the format of "add-mix-measure"allows the lysis of cells producing a luminescent signal that is proportional to the number p is outstayed APR. The substrate, luciferin beetles, subjected to oxidative decarboxylation under the action of recombinant Firefly luciferase with the subsequent conversion of ATP to AMP and generation of photons. The number of viable cells corresponds to the relative luminescence units (RLU, oil). Data can be recorded in a luminometer or imaging device equipped with a camera with a CCD. Output fluorescent signal is expressed as RLU measured depending on time. % RLU is the percentage of relative luminescence units, normalized to the data for control, i.e., "antibody that is not conjugated with the drug". Alternative photons generated by the luminescent radiation, can be counted in a scintillation counter in the presence of scintillation fluid. The light unit can be further represented as CPS (number of pulses per second).

The efficacy of conjugates "thio-Mab-drug" was determined by analysis on 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. An aliquot of 40 ml of cell culture containing about 3000 BJAB cells, Granta-519 or WSU-DLCL2 in the environment, the room is Ali into each well of the 384-well plate with opaque walls.

2. TDC (conjugate "thio-Mab-drug") (10 μl) was added to the experimental wells with four replications to a final concentration of 10000, 3333, 1111, 370, 123, 41, 13,7, 4,6 or 1.5 ng/ml, and in the control wells were added only medium that does not contain conjugate with drug and incubated for 3 days.

3. The tablets were balanced 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. Tablet incubated at room temperature for 10 minutes to stabilize the luminescent signal.

7. Luminescence was recorded on a chart expressed in %RLU (relative luminescence units, oil). According to the data obtained for cells at 0.51 ng/ml, incubated with medium containing no conjugate with the medicinal product, build a graph.

Wednesday: BJAB cells, Granta-519 and WSU-DLCL2 cultured in medium RPMI1640/10%FBS/2 mm glutamine.

Example 9: Analysis of the inhibition of tumor growth in vivo using conjugates of anti-CD79b thio-Mab-drug"

A. Granta-519 (lymphoma cells of the cortex of the brain)

In a similar study conducted in accordance with the same Protocol analysis xenograft, as described in the ore 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts Granta-519 (lymphoma cells of the cerebral cortex of man) mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 11.

Control Ab was a hu-anti-HER2-MC-MMAF or MA79b-MC-MMAF. 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 dependence of the change in the average tumor volume in dependence on time for xenograft Granta-519 mice (CB17 SCID, which was introduced conjugate TDC heavy chain A118C or a light chain with V205C anti-CD79b antibodies 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 were 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" did not lead to a significant reduction in the% of the NTA of body weight or lose weight during this period of time.

Moreover, in table 11 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 11
Reduction of tumor volume in vivo by introducing a conjugate thio-chMA79b-HC(A118C) or thio-chMA79b-LC(V205C)MMAF mice CB17 SCID with xenografts Granta-519
The injected antibodyPRCRDose MMAF
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool /Ab)
Control hu-anti-HER2-MC-MMAF0/80/84136,84,0
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/90/91916,8 1,85
Control chMA79b-MC-MMAF1/80/81002,33,0
Control chMA79b-MC-MMAF8/91/93006,83,0
Thio-chMA79b-HC(A118C)-MC-MMAF0/80/8632,31,9
Thio-chMA79b-HC(A118C)-MC-MMAF4/90/91906,81,9
Thio-chMA79b-LC(V205C)-MC-MMAF0/80/8602,31.8
Thio-chMA79b-LC(V205C)-MC-MMAF5/94/91806,81.8

B. Xenografts BJAB containing the luciferase (Burkitt lymphoma)

In a similar study, p is optimom in accordance with the same Protocol analysis xenograft, described in example 3 (see above), but with different conjugates of drugs and different doses, evaluated the effectiveness of other conjugates of drugs in xenografts BJAB containing the luciferase (Burkitt lymphoma) mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 12.

The control antibody was a huMA79b.v28 (conjugated with SMCC-DM1). Control thio-MAb HC(A118C) represented antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (conjugated 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) (conjugated with MC-MMAF). The results are presented below in table 12 and figure 34.

Figure 34A shows a graph of the dependence of the change in the average tumor volume in dependence on time for xenografts BJAB containing the luciferase in mice CB17 SCID treated with conjugates "thio-MAb huMA79b.v28-HC(A118C)-drug, 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" resulted in inhibition of tumor growth compared with the growth of the tumor after treatment with conjugates of the 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), use ishemini as a negative control. Another control was 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 were injected dose was determined by the percentage change of body weight during the first 7 days. The results (figure 34B) showed that the introduction of these conjugates "thio-Mab-drug" did not lead to a significant reduction in the percentage of body weight or lose weight during this period of time.

Moreover, table 12 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 12
Reduction of tumor volume in vivo by introducing a conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenografts BJAB containing the luciferase
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Doses of the Ab
(mg/kg)
DAR (Pharmaceutical-owned vehicle /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-MMAE0/100/104621,55
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
the IO-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE 0/1010/105421,87
Thio-control huMA79b.v28-HC(A118C)0/100/10NA2NA

Thio control hu-anti-CD22(10F4v3)-HC(A118C)-MC-MMAF1/104/105921,96

C. Xenografts WSU-DLCL2 (both diffuse lymphoma)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts follicular lymphoma (diffuse both lymphoma) WSU-DLCL2 mice (CB17 SCID. The drug conjugates and doses are presented below in table 13.

The control antibody was a huMA79b.v28 (conjugated with SMCC-DM1). Control thio-MAb HC(A118C) represented antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (conjug is automatic 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) (conjugated with MC-MMAF). The results are presented below in table 13.

Figure 35A shows a graph of the dependence of the change in the average tumor volume in dependence on time for xenografts WSU-DLCL2 (both diffuse lymphoma) mice (CB17 SCID treated with conjugates TDC heavy chain with AS anti-CD79b antibodies in the dosages indicated 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," led to inhibition of tumor growth compared with the growth of the tumor after treatment of the negative controls (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), thio-huMA79b.v28-HC(A118C). Another control was 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 were injected dose was determined by the percentage change of body weight during the first 7 days. The results (figure 35B) showed that the introduction of these conjugates "thio-Mab-drug" did not lead to a significant reduction in the percentage of body weight or lose weight during this period of time.

Moreover, in table 13 indicated the ANO number of mice out of the total number of tested mice detecting 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).

4
Table 13
Reduction of tumor volume in vivo by introducing a conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenografts WSU-DLCL2
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (the Drug produced a great tool /Ab)
Thio control hu-anti-
HER2-HC(A118C)-BMPEO-DM1
0/100/101144to 1.86
Thio control hu-anti-
HER2-HC(A118C)-MC-MMAF
0/100/1011541,9
Thio control hu-anti
HER2-HC(A118C)-MCvcPAB-
MMAE
0/100/109241,55
Control huMA79b.v28-
SMCC-DM1
1/100/1020243,4
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM10/100/1011041,85
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-MMAF1/100/101181,96

Thio-control huMA79b.v28-HC(A118C)0/100/10NA4NA

D. DOHH2 Xenografts (follicular lymphoma)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the ability of conjugates "thio-Mab-drug" reduce the amount of b-cell tumors in mice CB17 SCID model of DOHH2 xenografts. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 14.

The control antibody was a huMA79b.v28 (conjugated with SMCC-DM1). Control thio-MAb HC(A118C) represented antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (conjugated with BMPEO-DM1, MC-MMAF or MCvcPAB-MMAE), thio-Mab thio-huMA79b.v28-HC(A118C) or thio-Mab thio-hu-anti-CD22-HC(A118C) (conjugated with MC-MMAF). The results are presented below in table 14 and figure 36.

In the figure 36A presents a plot of the change in the average tumor volume in dependence on time for xenografts of DOHH2 cells in mice CB7 SCID, treated with conjugates TDC heavy chain A118C" in the dosages indicated 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-drug" in the dosages indicated in table 14, resulted in inhibition of tumor growth compared with the growth of the tumor after treatment with conjugates of the 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 negative control. Another control was 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, in table 14 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 14
Reduction of tumor volume in vivo by introducing a conjugate thio-HuMA79b.v28-HC(A118C)DM1, MMAF and MAE mice CB17 SCID with xenotar what spuntata DOHH2
The injected antibodyPRCRDose MMAF or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool/
Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/90/91144to 1.86
Thio control hu-anti-HER2-HC(A118C)-MC-MMAF0/90/911541,9
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)1/90/9NA4NA
Thio-control anti-CD22-HC(A118C)-MC-MMAF0/90/911841,96

E. Xenografts BJAB cells containing the luciferase (Burkitt lymphoma)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of the antibody-drug" and with various doses evaluated the effectiveness of other conjugates of drugs in xenografts BJAB containing the luciferase (Burkitt lymphoma) mice (CB17 SCID. The drug conjugates among the STV and dose (administered at day 0 for all ADC and control) are presented below in table 15.

The control antibody was a carrier (only buffer (ADC)). Control thio-MAb HC(A118C) represented antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (conjugated 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) (conjugated with MC-MMAF). The results are presented below in table 15.

In the figure 37A presents a plot of the change in the average tumor volume in dependence on time for xenografts BJAB containing the luciferase in mice CB17 SCID treated TDC heavy chain A118C anti-CD79b antibodies in the dosages indicated in table 15. 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 has led to inhibition of tumor growth compared with the growth of the tumor after treatment of 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 was a thio-huMA79b.v28-HC(A118C) and thio-10F4v3-HC(A118C)-MC-MMAF.

Moreover, in table 15 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 = ratio l is drug money for the antibody).

Table 15
Reduction of tumor volume in vivo by introducing a conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenografts BJAB containing the luciferase
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool/
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-MMAF0/100/10291 1,9

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. Xenotransplant ntati Granta-519 (lymphoma cells of the cerebral cortex)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts Granta-519 (lymphoma cells of the cerebral cortex of man) mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 16.

Control thio-MAb HC(A118C) was a thio-MAb thio-hu-anti-HER2-HC(A118C) (conjugated with BMPEO-DM1 or MC-MMAF). The results are presented in table 16 and figure 38.

Figure 38A shows a graph of the dependence of the change in the average tumor volume in dependence on time for xenograft Granta-519 mice (CB17 SCID, which was introduced conjugate TDC heavy chain A118C anti-CD79b antibodies 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-the drug in doses presented in table 16, resulted in inhibition of tumor growth compared to conjugates of drugs used as control.

In addition, in the same study, for each group, which were injected dose was determined by the percentage change of body weight during the first the e 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 lose weight during this period of time.

Table 16 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)). (DAR = the ratio of drug to antibody).

Table 16
Reduction of tumor volume in vivo by introducing a conjugate thio-huMA79b.v28-HC(A118C)-DM1 and MMAF mice CB17 SCID with xenografts Granta-519
The injected antibodyPRCRDose MMAF or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool /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-DM10/80/811041,85
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-MMAF2/81/8 11541,95
Thio-huMA79b.v28-HC(A118C)-MC-MMAF6/82/822981,95
Thio-huMA79b.v28-HC(A118C)-MC-MMAF4/84/8344121,95

G. Xenografts WSU-DLCL2 (both diffuse lymphoma)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts WSU-DLCL2 (both diffuse lymphoma) mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 17.

The control antibody was a carrier (only buffer (ADC)). Control thio-MAb represented antibody thio-Mab thio-hu-anti-HER2-HC(A118C) (conjugated with BMPEO-DM1, MCvcPAB-MMAE or MC-MMAF). The results are presented in table 17 and figure 39.

Figure 39 shows zavisimostei average tumor volume in dependence on time for xenografts WSU-DLCL2 mice (CB17 SCID, treated with conjugates TDC heavy chain with AS anti-CD79b antibodies in the dosages indicated 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 a 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 the growth of the tumor after treatment of 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 the media).

Moreover, in table 17 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 17
Reduction of tumor volume in vivo by introducing a conjugate thio-HuMA79b.v28-HC(A118C)MMAE, MMAF, and DM1 mice CB17 SCID with xenografts WSU-DLCL2
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool /Ab)
control media0/90/9NANANA
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/90/91144to 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-MMAF5/92/911241,9
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM14/90/911041,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE 1/90/9140,51,9
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/90/9271,01,9
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE2/91/9552,01,9

Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE1/97/91104,01,9

N. The xenografts Granta-519 (lymphoma cells of the cerebral cortex)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts Granta-519 (lymphoma cells of the cerebral cortex of man) mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and to the of ntrolle) are presented below in table 18.

Control thio-MAb was a thio-hu-anti-HER2-HC(A118C) (conjugated with BMPEO-DM1) and antibody thio-MAb thio-hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE. The results are presented below in table 18.

Figure 40A shows a graph of the dependence of the change in the average tumor volume in dependence on time for xenograft Granta-519 mice (CB17 SCID, which was introduced conjugate TDC heavy chain A118C anti-CD79b antibodies in the doses 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 a conjugate thio-HuMA79b.v28-HC(A118C)DM1 and MMAE mice CB17 SCID with xenografts Granta-519
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Dose
Ab
(mg/kg)
DAR (Generic-ment tool /Ab)
Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/100/101144to 1.86
Thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE2/101/109241,55
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM13/100/1011041,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/101/10130,51,87
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE1/100/10271,01,87

Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE1/107/10542,01,87
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE0/1010/101084,01,87

I. Xenotransplant BJAB containing CD79b abacadabra monkeys (BJAB-cynoCD79b)

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts cells of BJAB (Burkitt lymphoma), expressing CD79b abacadabra monkeys (BJAB-cynoCD79b), mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 18.

Control Ab was a carrier (only buffer). Control thio-MAb represented antibodies 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 figure 50.

Figure 50 shows a graph of zavisimost and inhibition of tumor growth depending on the time for xenografts (BJAB-cynoCD79b) mice (CB17 SCID, processed TDC heavy chain A118C anti-CD79b antibodies in the dosages indicated 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-cynoCD79b(ch10D10)-HC(A118C)-MC-MMAF led to inhibition of tumor growth compared with the growth of the tumor after treatment of 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 the 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 a conjugate thio anti-cyno CD79b(ch10D10)-HC(A118C) DM1, MMAF or MMAE or Thio-HuMA79b.v28 DM1, MMAF or MMAE mice CB17 SCID with xenografts BJAB-cynoCD79b
The injected antibodyPRCRDose MMAF, MMA is or DM1
(µ g/m2)
Dose Ab
(mg/kg)
DAR (Generic-ment tool /Ab)
control media0/90/9NANANA

Thio control hu-anti-HER2-HC(A118C)-BMPEO-DM10/90/9572to 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-MMAE1/9 2/9271to 1.86
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-MC-MMAF0/91/92811,9
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM13/90/95521,85
Thio-huMA79b.v28-HC(A118C)-MCvcPAB-MMAE2/92/92711,9
Thio-huMA79b.v28-HC(A118C)-MC-MMAF7/91/92811,9

J. Xenotransplant BJAB-cynoCD79b

In a similar study conducted in accordance with the same Protocol analysis xenograft described in example 3 (see above), but with different conjugates of drugs and different doses evaluated the efficacy of conjugates "thio-Mab-drug" in xenografts cells of BJAB (Burkitt lymphoma), expressing CD79b own capadonna monkeys (BJAB-cynoCD79b), mice (CB17 SCID. The drug conjugates and doses (administered at day 0 for all ADC and control) are presented below in table 19.

Control thio-MAb represented antibodies 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 figure 51.

Figure 51 presents a plot of the inhibition of tumor growth depending on the time for xenografts BJAB-cynoCD79b mice (CB17 SCID treated TDC heavy chain A118C anti-CD79b antibodies in the dosages indicated 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 the growth of the tumor after treatment of the negative controls (thio-anti-HER2-HC(A118C)-BMPEO-DM1). Another control was 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 a conjugate thio anti-cyno CD79b(ch10D10)-HC(A118C) DM1 or thio-HuMA79b.v28-HC(A118C)DM1 mice CB17 SCID with xenografts BJAB-cynoCD79b
The injected antibodyPRCRDose MMAF, MMAE or DM1
(µ g/m2)
Dose
Ab
(mg/kg)
DAR (Generic-ment tool /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-DM11/100/1027 11,85
Thio-huMA79b.v28-HC(A118C)-BMPEO-DM10/102/105521,85
Thio-anti-cynoCD79b(ch10D10)-HC(A118C)-BMPEO-DM10/100/102711,8
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 had been presented only to illustrate certain aspects of the invention, and the scope of the present invention may include any constructs that are functionally equivalent to the claimed designs. The claimed material should not be construed as indicating that this material is inadequate for the practical implementation of any aspect of the invention, including the best version of its realization, 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 of various modifications, which, in addition to the illustrated and described in this application, will also be in the scope of the attached claims.

1. Humanitariannet anti-CD79b antibody or antigen-binding fragment that contains:
sequence HVR:
(i) HVR-L1 containing the sequence A1-A15, where A1-A15 is a KASQSVDYDGDSFLN (SEQ ID NO:131);
(j) the HVR-L2, containing the sequence B1-B7, where B1-B7 is a AASNLES (SEQ ID NO:132);
(k) HVR-L3, containing the sequence C1-C9, where C1-C9 is a QQSNEDPLT (SEQ ID NO:133);
(l) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFSSYWIE (SEQ ID NO:134);
(m) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GEILPGGGDTNYNEIFKG (SEQ ID NO:135); and
(n) HVR-H3 containing the sequence F1-F10, where F1-F10 is a TRRVPIRLDY (SEQ ID NO:204).

2. Humanitariannet antibody against CD79b or antigen-binding fragment that contains:
sequence HVR:
(i) HVR-L1, containing SEQ ID NO:194;
(j) the HVR-L2, containing SEQ ID NO:195;
(k) HVR-L3, containing SEQ ID NO:196;
(1) HVR-H1 containing SEQ ID NO:202;
(m) HVR-H2 containing SEQ ID NO:203; and
(n) HVR-H3 containing SEQ D NO:204.

3. The antibody according .l or 2, where at least part of the frame sequence is a human consensus framework sequence.

4. Humanitariannet anti-CD79b antibody according to claim 1 or 2, where the monovalent affinity of the antibody against human CD79b is substantially similar to the affinity of binding of monovalent antibodies mouse that contains a variable sequence of light and heavy chains shown in figures 7A-B (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14).

5. Humanitariannet anti-079b antibody according to claim 1 or 2, where the binding affinity of the indicated antibody in its bivalent form in relation to human CD79b is essentially similar to the affinity of antibodies of the mouse in its bivalent form, containing variable sequences of the light and heavy chains shown in figures 7A-B (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14).

6. Humanitariannet anti-CD79b antibody according to claim 1 or 2, where the specified affinity of the antibody in its bivalent form in relation to human CD79b is 0.4 nm ± 0,04 measured by analysis of Scatchard.

7. Humanitariannet anti-CD79b antibody according to claim 1 or 2, where the specified affinity of the antibody in its bivalent form in relation to human CD79b is 0.2 nm ± 0,02 measured by analysis of Scatchard.

8. The antibody according to claim 4, where the affinity of binding is measured with the aid of the completion Biacore analysis, or radioimmunoassay.

9. The antibody according to claim 1 or 2, containing the consensus sequence of a frame region of human κ chain subgroup I.

10. The antibody according to claim 1 or 2, containing the consensus sequence of a frame region of a human heavy chain subgroup III.

11. Humanitariannet anti-CD79b antibody according to claim 1 or 2, where the specified humanitariannet antibody, at its conjugation with a cytotoxic agent that inhibits the growth of tumor cells.

12. The antibody according to any one of claims 1 or 2, where the specified humanitariannet antibody is monovalent or bivalent.

13. The antibody according to any one of claims 1 or 2, where the specified humanitariannet antibody contains one Fab-region associated with the Fc-region.

14. The antibody according to claim 2, where the specified variable domain comprises a sequence of FR1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 17 (SEQ ID NO:198-201).

15. The antibody according to claim 2 or 14, where the specified antibody contains a sequence SN and/or Fc presented on figure 17 (SEQ ID NO:205 and/or 206).

16. The antibody according to claim 2, where the specified variable domain comprises a sequence of FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 17 (SEQ ID NO:190-193).

17. The antibody according to claim 2 or 16, where the specified antibody contains a sequence CL1 presented on figure 17 (SEQ ID NO:197).

18. The antibody according to claim 2, obtained by the method:
(i) culturing cells that Express arousih antibody containing the variable domain of the heavy chain variable domain and a light chain according to any one of claim 2 or 14-17; and
(j) selection of antibodies from these cultured cells.

19. The antibody according to claim 2, where the aforementioned antibody is monovalent and contains the Fc-region.

20. The antibody according to claim 1 or 2, where the specified antibody comprises the variable domain light chain having the amino acid sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:207.

21. The antibody according to claim 1 or 2, where the specified antibody comprises the variable domain of the heavy chain having the amino acid sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:208.

22. Humanitariannet antibody that binds to CD79b, where the specified antibody contains a variable domain of the heavy chain with the amino acid sequence SEQ ID NO:208 and the variable domain of the light chain with the amino acid sequence SEQ ID NO:207.

23. Polynucleotide encoding the antibody according to claims 1, 2, 14-18, 20, or 21.

24. The expression vector containing polynucleotide on item 23.

25. A host cell containing a vector according to paragraph 24, where the cell produces an antibody according to claims 1-4, 14-18, 20, or 21.

26. The method of obtaining anti-CD79b antibodies, where the method comprises (a) culturing host cells, select the R group, including eukaryotic cells and cells SNO, under conditions suitable for expression of polynucleotide on item 23 encoding the antibody, and (b) isolation of antibody.

27. The antibody according to claims 1, 2, 14-18, 20, or 21, where the specified antibody binds to the epitope in the field of CD79b, since amino acids 29-39 SEQ ID NO:2 or amino acids 1-11 of SEQ ID NO:16.

28. The antibody according to claims 1, 2, 14-18, 20, or 21, where the specified CD79b expressed on the cell surface.

29. The antibody according p, where the specified cell is In the cell.

30. The antibody according to clause 29, where specified In the cell is associated with b-cell-proliferative disorder.

31. The antibody according to item 30, where the specified b-cell-proliferative disorder is a cancer.

32. The antibody according p, where specified In cell-proliferative disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

33. The antibody according to claims 1, 2, 14-18, 20, or 21, where the aforementioned antibody is a monoclonal antibody.

34. The antibody according p, where the specified antibody fragment selected from Fab, Fab'-SH, Fv-, scFv or (Fab') -fragments.

35. The antibody according to claims 1, 2, 14-18, 20, or 21, where the specified antibody binds to the same epitope which binds an antibody selected from antibody containing the variable domain of the heavy chain SEQ ID NO:170 variable domain and the light chain of SEQ ID NO:169, antibody containing the variable domain of the heavy chain SEQ ID NO:189 variable domain and the light chain of SEQ ID NO:188, antibody containing the variable domain of the heavy chain SEQ ID NO:208 and the variable domain of the light chain of SEQ ID NO:207; and antibodies containing the variable domain of the heavy chain SEQ ID NO:227 variable domain and the light chain of SEQ ID NO:226.

36. Immunoconjugate against CD79b containing the antibody according to claims 1, 2, 14-18, 20, or 21, covalently linked to a cytotoxic agent, where the conjugate is an ADC.

37. Immunoconjugate on p, where the specified cytotoxic agent selected from a toxin, a chemotherapeutic molecule drug, an antibiotic, a radioactive isotope and nucleotidase enzyme.

38. Immunoconjugate on clause 37, where the specified immunoconjugate has the formula: Ab-(L-D)p, where:
(a) AB is an antibody according to claims 1, 2, 14-18, 20, or 21;
(b) L is a linker;
(c) D is a molecule drugs.

39. Immunoconjugate in § 38, where L is selected from 6-maleimidomethyl (MS), maleimidomethyl (MP), valine-citrulline (val-cit), Alani is a-phenylalanine (ala-phe), p-aminobenzonitrile (RAV), N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and N-Succinimidyl-(4-iodates)aminobenzoate (fairs are forthcoming-Siab).

40. Immunoconjugate in § 38, where D is selected from auristatin and dolastatin.

41. Pharmaceutical composition for inhibiting growth of cells that Express CD79b, containing an effective amount immunoconjugate on § 38 and a pharmaceutically acceptable carrier.

42. Method of inhibiting growth of cells that expresses CD79b, where the method includes contacting the specified cell with the antibody according to any one of claims 1, 2, 14-18, 20, or 21, thereby inhibiting the growth of specified cells.

43. The method according to § 42, where the specified antibody conjugated with a cytotoxic agent.

44. The method according to § 42, where the specified antibody conjugated to a growth inhibitory agent.

45. The method of treatment of an individual suffering from cancer, with increased expression of CD79b, where the method includes the introduction to the individual an effective amount of the antibody according to any one of claims 1, 2, 14-18, 20, or 21.

46. The method according to item 45, where the specified cancer is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic limfocitarnaya (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

47. The method according to item 45, where the specified antibody conjugated with a cytotoxic agent.

48. The method according to item 45, where the specified antibody conjugated to a growth inhibitory agent.

49. A method of treating a proliferative disorder in an individual with increased expression of CD79b, where the method includes the introduction to the individual an effective amount of the antibody according to any one of claims 1, 2, 14-18, 20, or 21.

50. The method according to § 49, where the specified proliferative disorder is a cancer.

51. The method according to item 50, where the specified cancer is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

52. The method according to § 49, where the specified antibody conjugated with a cytotoxic agent.

53. The method according to § 49, where the specified antibody conjugated to a growth inhibitory agent.

54. Method of inhibiting cell growth, where the growth in this cell at least in part C is dependent on the growth-potentiating actions CD79b, where the method includes contacting the specified cell with an effective amount of the antibody according to any one of claims 1, 2, 14-18, 20, or 21, thereby inhibiting the growth of specified cells.

55. The method according to item 54, where the specified antibody conjugated with a cytotoxic agent.

56. The method according to item 54, where the specified antibody conjugated to a growth inhibitory agent.

57. The method of therapeutic treatment of a tumor in a mammal, where the growth in this tumor is at least partially dependent upon the growth potentiating action CD79b, and where the method includes contacting the specified cell with an effective amount of the antibody according to any one of claims 1, 2, 14-18, 20, or 21.

58. The method according to § 57, where the tumor is associated with lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

59. The method according to § 57, where the specified antibody conjugated with a cytotoxic agent.

60. The method according to § 57, where the specified antibody conjugated to a growth inhibitory agent.

61. A method of inhibiting the proliferation of b cells,comprising treating the cells with immunoconjugates on p in terms conducive to binding immunoconjugate with CD79b.

62. The method according to p, where this proliferation of b-cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

63. The method according to p where In the cell is a xenograft.

64. The method according to p, where treatment is carried out in vitro.

65. The method according to p, where treatment is carried out in vivo.

66. The method of determining the presence of CD79b in a biological sample suspected of containing CD79b, where the method includes the impact on the specified sample with an antibody according to any one of claims 1, 2, 14-18, 20, or 21, and determining the level of binding of the indicated antibody to CD79b in the specified sample, where the specified binding of an antibody to CD79b in the specified sample is indicative of the presence of the indicated protein in the specified pattern.

67. The method according to p, where the biological sample is collected from a patient with suspected b-cell-proliferative disorder.

68. The method according to p, where specified In cell-proliferative disorder is selected from lymphoma, nehodzhkin the Oh lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed asymptomatic NHL is not curable NHL is not curable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the cerebral cortex.

69. The method of binding the antibody according to any one of claims 1, 2, 14-18, 20 or 21 with a cell that expresses CD79b, where the method includes contacting the specified cell with the antibody according to any one of claims 1, 2, 14-18, 20, or 21.

70. The method according to p, where the specified antibody conjugated with a cytotoxic agent.

71. The method according to p, where the specified antibody conjugated to a growth inhibitory agent.

72. Composition for inhibiting growth of cells that Express CD79b, containing an effective amount of the antibody according to any one of claims 1, 2, 14-18, 20 or 21 and a pharmaceutically acceptable carrier.

73. The composition according to item 72, where the composition comprises a carrier.

74. Immunoconjugate in § 38, where L is selected from 6-maleimidomethyl (MS), maleimidomethyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzonitrile (RAV), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC) and N-Succinimidyl (4-iodine-acetyl)aminobenzoate (fairs are forthcoming-Siab).

75. Immunoconjugate on p, where D is selected from auristatin and dolastatin.

76. Immunoconjugate on item 75, where D is a molecule drugs formula DE or DF:

and where each R2and R6represents methyl, each R3and R4represents isopropyl, R5represents H or methyl, R7represents sec-butyl, each R8independently selected from CH3O-Snz, HE and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8alkyl or -(CH2)2-O-(CH2)2-O- (CH2)2-O-CH3; and R18represents a
-C(R8)2-C(R8)2-aryl; and
R is in the range of about 1-8 and
where, optionally, the drug is selected from MMAE and MMAF.

77. Immunoconjugate in § 38, where immunoconjugate has the formula Ab-(L-MMAE)pwhere L is a linker and R is in the range from 2-5, or has the formula Ab-(L-MMAF)pwhere L is a linker and R is in the range from 2-5.

78. Immunoconjugate on p, where L contains val-cit, MS, RABBI or MS-RAV.

79. Immunoconjugate in § 38, where D is maytansinoid.

80. Immunoconjugate on p, where D is selected from DM1, DM3 and DM4.

81. Immunoconjugate on the .80, where p is equal to 2-4 or 3-4.

82. Immunoconjugate formula Ab-MC-vc-PAB-MMAF

where Ab represents the antibody according to any one of claims 1 to 4, 14-18, 20, or 21; MS represents a 6-maleimidomethyl; vc is a valine-citrulline; RAV is a p-aminobenzeneboronic; and p is approximately 1-8.

83. Immunoconjugate formula Ab-MC-vc-PAB-MMAE

where Ab represents the antibody according to any one of claims 1 to 4, 14-18, 20, or 21; MS represents a 6-maleimidomethyl; vc is a valine-citrulline; RAV is a p-aminobenzeneboronic; and p is approximately 1-8.

84. Pharmaceutical composition for inhibiting growth of cells that Express CD79b, containing an effective amount immunoconjugate on any of PP-83 and a pharmaceutically acceptable carrier.

85. Immunoconjugate in § 38, where immunoconjugate selected from the structures:

where Val is a valine and Cit is citrulline.

86. Immunoconjugate on p, where p represents 1, 2, 3 or 4.



 

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