Humanised antibodies against cd79b and immunoconjugates and methods of application

FIELD: medicine.

SUBSTANCE: claimed invention relates to the field of biotechnology. Claimed are versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a determined amino acid sequence. An epitope of the antibody from 11 amino acids is determined by the Biacore method. Disclosed are: an immunoconjugate of the antibody with a medication or means for inhibiting cell growth, where the antibody is bound with means covalently, and versions of the composition, based on an effective quantity of the immunoconjugate or the antibody, used for inhibiting B-cell proliferation; as well as a method of determining CD79b in a sample with the application of the antibody. Described are: an antibody-coding polynucleotide, as well as an expression vector and an isolated cell, containing the vector for obtaining the antibody. Disclosed are versions of applying the antibody or immunoconjugate for obtaining the medication for inhibiting the growth of CD79b-expressing cells for the treatment of an individual, affected with cancer, for the treatment of proliferative disease or for inhibiting B-cell proliferation.

EFFECT: invention provides novel antibodies, which can find further application in the therapy of proliferative CD79b-associated diseases.

91 cl, 8 tbl, 9 ex, 20 dwg

 

CROSS-REFERENCE TO RELATED APPLICATIONS

On the application, non-provisional filed under 37 CFR §1.53(b), priority is claimed under 35 USC §119(e) of provisional application U.S. serial No. 60/950088, filed July 16, 2007, which are incorporated herein as references in full.

Field of the INVENTION

The present invention relates to compositions suitable for the treatment of hematopoietic tumor in mammals and to methods of using these compositions.

Background of the INVENTION

Malignant tumors (cancer) are the second leading cause of death in the U.S., after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). A malignant tumor characterized by an increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate with the formation of a tumor mass, the invasion of these neoplastic tumor cells in the surrounding tissue, and the formation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and distant sites as a result of a process called metastasis. In the malignant state of the cell proliferates under conditions in which normal cells do not grow. A malignant tumor is manifested in many Fort�, characterized by different degrees of invasiveness and aggressiveness.

Malignant tumors that involve the cells formed in the course of hematopoiesis - process by which formed cellular elements of blood, such as lymphocytes, leukocytes, platelets, erythrocytes and natural killer cells, called hematopoietic malignancies. Lymphocytes, which can be in blood and lymphatic tissue and are important for immune response, are divided into two main classes of lymphocytes: B-lymphocytes (B-cells) and T lymphocytes (T-cells), which mediate humoral and cell-mediated immunity, respectively.

B-cells Mature in the bone marrow and leave the marrow by expressing the antigen binding antibody on their cell surface. When a naive B cell first encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into B-memory cells and effector cells called "plasma cells". B-memory cells have longer life span and continue to Express membrane-bound antibody with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody, instead, they are products�irout antibody in the form, which can secretariats. Synthesized antibodies are the main effector molecule for humoral immunity.

Associated with B-cell disorders include, but are not limited to, malignant lymphoma (nahodkinskuju lymphoma, NHL), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL, B-cell leukemia (CD5+ B-lymphocytes). Non-Hodgkin's lymphoma (NHL), a heterogeneous group of malignant tumors, primarily arising from B-lymphocytes, approximately 4% of all newly diagnosed tumors (Jemal, A. et al., CA-Cancer J Clin., 52: 23-47 (2002)). Aggressive NHL is approximately 30-40% of adult NHL (Harris, N. L. et al., Hematol. J., 1:53-66 (2001)) and includes diffuse large B-cell lymphoma (DLBCL), b-cell lymphoma mantle zone (MCL), peripheral T-cell lymphoma and anaplastic large cell lymphoma. Combination chemotherapy is the first line provides a cure less than half of the patients with aggressive NHL, and most patients eventually die from the disease (Fischer, R. I., Semin. Oncol., 27 (suppl 12): 2-8 (2000)).

T-cells Mature in the thymus, which provides the environment for proliferation and differentiation of immature T-cells. During the maturation of T-cells, T-cells undergo rearrangement of genes, resulting in the formation of T-cell receptor, and positive � negative selection, which helps to determine the cell surface phenotype of Mature T-cells. Characteristic cell surface markers of Mature T-cells are CD3 complex T-cell receptor and one of coreceptors, CD4 or CD8.

In an attempt to find effective cellular targets for therapy of malignant tumors, the researchers conducted a search to identify transmembrane or otherwise membrane-associated polypeptides that have been specifically expressed on the surface of one or more particular type(s) of malignant cells in comparison with one or more normal non-cancerous cell(s). Often, such associate with membrane polypeptides in a greater number of them are expressed on the surface of malignant cells compared with the surface of cancerous cells. Identification of such associate with tumor antigenic cell surface polypeptides provided an opportunity for specific targeting of malignant cells for destruction by therapies based on antibodies. In this regard, it is noted that it was found that a treatment using antibodies is highly effective in the treatment of certain malignant tumors. For example, HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco, California) are antibodies that have been successfully and�use to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a recombinant formed from the DNA of a humanized monoclonal antibody that selectively binds to the proto-oncogene of the extracellular domain of the receptor of epidermal growth factor 2 (HER2). Overexpression of the protein HER2 is observed in 25-30% of cases of primary breast cancer. RITUXAN® is obtained by means of genetic engineering of chimeric monoclonal antibody mouse/human directed against the CD20 antigen located on the surface of normal and malignant B-lymphocytes. Both of these antibodies recombinante produced in CHO cells.

In other attempts to figure out an effective cellular targets for therapy of malignant tumors, the researchers conducted a search to identify (1) is not associated with membrane polypeptides that have been specifically produced by one or more particular type(s) of the malignant cells(QA) compared to one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells on the expression level, which significantly exceeds the level of expression of one or more normal non-cancerous cell(s), or (3) polypeptides, the expression of which is essentially limited to roofing�about one (or a very limited number of different) tissue type(s) as malignant, and in the non-cancerous state (e.g., normal prostate tissue and tumor tissue of the prostate gland). Such polypeptides can remain located intracellular or they can secretariats malignant cell. Furthermore, such polypeptides can be expressed is not the malignant cell but, instead, the cells that produce and/or secrete the polypeptide having the effect of stimulation or enhance the growth of malignant cells. Such synthesized polypeptides often represent proteins that provide malignant cells with an advantage in growth compared to normal cells, and include, for example, such polypeptides as angiogenic factors, cell adhesion factors, growth factors, etc. Can be expected that identification of antagonists such membrane-associated polypeptides can serve as effective drugs for the treatment of such malignancies. Moreover, the identification of the peculiarities of expression of such polypeptides may be useful for specific diagnosis of malignant tumors in mammals.

Despite the above advances in therapy of malignant tumors in mammals, there is a significant need for additional drug �Redstar, capable of detecting the presence of a tumor in a mammal, and effective in the inhibition of neoplastic cell growth, respectively. Thus, the present invention is the identification of polypeptides associated with the cell membrane, secreted or intracellular polypeptides whose expression is specifically limited to only one (or a very limited number of different) type(s) of the tissue, hematopoietic tissue, both in malignant and non-malignant condition, and the use of these polypeptides and encoding nucleic acids to generate compositions suitable for medical treatment and/or detection of malignant hematopoietic tumor in mammals.

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

Taking into account the expression of CD79b, is useful for obtaining therapeutic antibodies to the antigen CD79b, which create minimal antigenicity, or not create it, when administered to patients, especially in long-term care. The present invention satisfies this and other necessary. The present invention relates to antibodies against CD79b, which overcome the limits of current therapeutic compositions, as well as offer additional advantages that will become apparent from the detailed description, below.

The use of conjugates of the antibody-drug (ADC), i.e. immunoconjugates, for local delivery of cytotoxic and cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of malignant tumors (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 and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26: 151-172; US 4975278) allows targeted delivery of the group of drugs to tumors, and intracellular accumulation, where systemic administration of these unconjugated drug m�can result in unacceptable levels of toxicity to normal cells, in addition to tumor cells to be eliminated (Baldwin et al. (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Attempts to improve therapeutic index, i.e., maximum efficiency and minimal toxicity ADC, focused on the selectivity of polyclonal (Rowland et al. (1986) Cancer Immunol. Immunother., 21: 183-87) and monoclonal antibodies (mAb) and on the binding properties of drugs and controlled release of drugs (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Group of drugs used in the conjugate antibody-drug, include protein bacterial toxins such as diphtheria toxin, a protein plant toxins such as ricin, small molecule compounds, 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), above). Group of drugs can affect the cytotoxic and cytostatic mechanisms including tubulin binding, DNA binding or inhibition of topoisomerase. Some cytotoxic drug deterge�VA tend to inactivate or reduce activity upon conjugation with large antibodies or protein receptor ligands.

The peptides auristatin, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin (WO 02/088172), kongugiruut as groups of drugs: (i) chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cAC10 which is specific to CD30 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; US 2004/0018194; (iii) an antibody against CD20, such as Rituxan (WO 04/032828) for the treatment of expressing CD20 tumours and immune disorders; (iv) an antibody against EphB2R 2H9 for the treatment of cancer of the colon and rectum (Mao et al. (2004) Cancer Research 64(3):781-788); (v) an antibody against E-selectin (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab (HERCEPTIN®, US 2005/0238649), and (vi) antibodies against CD30 (WO 03/043583). Options auristatin E described in US 5767237 and US 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, presented March 28, 2004. Analogues of auristatin MMAE and MMAF kongugiruut with different antibodies (US 2005/0238649).

Conventional methods of joining, i.e. linking by covalent bonds, the group of the drug with the antibody, as a rule, lead to a heterogeneous mixture of molecules, where a group of drugs is associated with several participants�AMI antibodies. For example, cytotoxic drugs, as a rule, kongugiruut with antibodies using often numerous lysine residues of the antibody with the formation of a heterogeneous mixture of antibody-drug. Depending on the reaction conditions, the heterogeneous mixture usually contains antibodies with a range of associated groups of the drug from 0 to 8 or more. In addition, each subgroup conjugates with specific numerical ratio of groups of the drug to the antibody is potentially heterogeneous mixture, where a group of medicines associated with different portions of the antibody. Analytical and preparative methods may be insufficient to separate and characterizatio types of molecules of the conjugate antibody-drug in the heterogeneous mixture obtained by the reaction of conjugation. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Their reactivity to linker reagents and intermediate compounds, the drug-linker depend on factors such as pH, concentration, salt concentration and co-solvents. Moreover, the multistage process of conjugation can be unplayable due to the difficulty of controlling the reaction conditions and Karak�erinacei reagents and intermediates.

Thiol groups of cysteine are reactive at neutral pH, unlike most amines that are protonated and less nucleophilic at a pH of about 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often found in the oxidized form as linked by disulfide oligomers or possess disulfide groups with internal bridges. Extracellular proteins usually do not have free thiols (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, page 55). Thiol group of cysteine antibodies, as a rule, are more reactive, i.e. more nucleophilic in respect of electrophilic reagents for conjugation than the amino group or the hydroxyl group of the antibody. Residues of cysteine embedded in proteins by means of genetic engineering for the formation of covalent bonds with ligands or for the formation of new intermolecular disulfide bonds (Better et al. (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al. (1994) Bioconjugate Chem. 5:126-132; Greenwood et al. (1994) Therapeutic Immunology 1:247-255; Tu et al. (1999) Proc. Natl. Acad. Sci USA 96:4862-4867; Kanno et al. (2000) J. of Biotechnology, 76:207-214; Chmura et al. (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; US 6248564). However, embedding tylnej groups of cysteine by mutations of different amino acid residues of the protein at amino acid residues cysteine� is potentially problematic in particular, in the case of the unpaired (free Cys) residues or residues that are relatively accessible for the reaction or oxidation. In concentrated solutions of the protein in the periplasmE. coliand in the culture supernatants or in partially or completely purified protein, unpaired Cys residues on the protein surface can form pairs and to oxidize with the formation of intermolecular disulfides, and, thus, dimers or multimeric proteins. The formation of a disulfide dimer makes the new Cys preaction-able to conjugation with a drug, ligand or with a different label. Furthermore, if protein oxidation by forming an intramolecular disulfide bond between the newly designed Cys residue and the existing Cys residue, both thiol groups of Cys are not available for participation and interactions in the active site. Moreover, the protein may be inactive or non-specific due to misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311: 1-9).

Antibodies with the built-in cysteine residues construct as FAB-fragments of antibodies (thio-Fab) and Express as full-size monoclonal IgG antibody (thio-Mab) (Junutula, J. R. et al. (2008) J Immunol Methods 332:41-52; US 2007/0092940, the contents of which are incorporated herein as ssy�OK). Antibodies thio-Fab and thio-Mab kongugiruut through the linkers to the newly introduced tylnej groups of cysteine using reactive towards thiol linker reagents and reagents the drug-linker with obtaining conjugates antibody-drug (ist-ADC).

All references cited herein, including patent applications and publications, are included as references in full.

Summary of the INVENTION

The invention relates to antibodies against CD79b or their functional fragments, and to method of their use for the treatment of hematopoietic tumors.

In one aspect, the invention relates to an antibody which binds, preferably specifically with any of the above or below polypeptides. Optionally, the antibody is a monoclonal antibody, a fragment of the antibody, including Fab, Fab', F(ab')2- and Fv-fragment, ditelo (diabody), single-domain antibody, a chimeric antibody, a humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. The antibodies of the present invention optionally can be konjugierte with a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin, maytansinoid derived d�of lostatin or calicheamicin, antibiotic, a radioactive isotope, nucleotidase enzyme or similar. The antibodies of the present invention optionally can be produced in CHO cells or bacterial cells and preferably they induce cell death, with which they are associated. For the purposes of detection, the antibodies of the present invention can be labeled amenable to the detection of the label to associate with a firm pad or similar.

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

In another aspect, the invention relates to humanitarianlaw and�titulo against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the antibody in the form of an IgG to CD79b) is 2.0 nm.

In one aspect, provided is an antibody that binds to CD79b, where the antibody comprises: (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-A16 is a KSSQSLLDSDGKTYLN (SEQ ID NO:59)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:60)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a WQGTHFPYT (SEQ ID NO:61)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:62)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD (SEQ ID NO:63), and

(vi) HVR-H3 containing the sequence F1-F6, where F1-F10 is a ARNLYL (SEQ ID NO:64).

In one aspect predusmatrivaetsya antibody that binds to CD79b, where the antibody comprises at least one variant HVR, where the variant HVR sequence contains a modification of at least one residue of the sequence represented in SEQ ID NO.: 59, 60, 61, 62, 63 or 64.

In one aspect, the invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 13 (SEQ ID NO:31-33).

<> In one aspect, the invention relates to an antibody containing the variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 13 (SEQ ID NO:23-25).

In one aspect, the invention relates to antibodies against CD79b containing the variable domain of the heavy chain SEQ ID NO:16. In another aspect, the invention relates to antibodies against CD79b containing the variable domain light chain SEQ ID NO:12.

In one aspect, the invention relates to antibodies against CD79b with built in his cysteine residues containing one or more residues of the amino acid cysteine and a sequence selected from SEQ ID NO:91-122. Antibodies against CD79b with built in his cysteine residues may contact the CD79b polypeptide. Antibodies against CD79b with built in his cysteine residues can be obtained by a process comprising replacing one or more amino acid residues of the original antibody against CD79b cysteine.

In one aspect, the invention relates to antibodies against CD79b with built in his cysteine residues, containing one or a few amino acid residues of cysteine, where antibodies against CD79b with built in his cysteine residues binds to a CD79b polypeptide and is obtained by a process comprising replacing one or more amino acid residues of IP�one antibodies against CD79b cysteine, where the original antibody comprises at least one HVR sequence selected from:

(a) HVR-L1 containing the sequence A1-A15, where A1-A16 is a KSSQSLLDSDGKTYLN (SEQ ID NO:59);

(b) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:60);

(c) HVR-L3, containing the sequence C1-C9, where C1-C9 is a WQGTHFPYT (SEQ ID NO:61) or FQGTHFPFT (SEQ ID NO:79);

(d) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:62);

(e) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD(SEQ ID NO:63); and

(f) HVR-H3 containing the sequence F1-F6, where F1-F10 is a ARNLYL (SEQ ID NO:64).

Antibodies against CD79b with built in his cysteine residues may be a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its respective antigenic epitope. The antibodies of the present invention optionally can be konjugierte with a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin or maytansinoid. The antibodies of the present invention optionally can be produced in CHO cells or bacterial cells and preferably� they inhibit the growth or proliferation of the cell, with which they are associated, or induce cell death. For diagnostic purposes, the antibodies of the present invention can be labeled amenable to the detection of the label to associate with the solid substrate, or similar.

In one aspect, the invention relates to methods for producing antibodies according to the invention. For example, the invention relates to a method of producing antibodies against CD79b (which as defined herein includes intact antibody and its fragments), and said method includes the expression in a suitable the host cell a recombinant vector of the invention encoding the indicated antibody (or its fragment), and the allocation of the specified antibodies.

In one aspect, the invention relates to a pharmaceutical composition containing the antibody of the invention or the conjugate antibody-drug according to the invention, and a pharmaceutically acceptable diluent, carrier or excipient.

In one aspect, the invention relates to a product containing container; and a composition located in the container, where the composition contains one or more antibodies against CD79b according to the invention.

In one aspect, the invention relates to a kit containing a first container containing a composition comprising one or more antibodies against CD79b according to the invention; and a second container containing a buffer.

In one aspect, the invention relates to the use of the product according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder.

In one aspect, the invention relates to the use of the kit according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder.

In one aspect, the invention relates to a method of inhibiting growth of a cell that expresses CD79b, wherein said method comprises contacting the specified cell with the antibody of the invention thereby causing an inhibition of growth of the specified cell. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of medical treatment of a mammal having a malignant tumor containing a cell that expresses CD79b, wherein said method includes the introduction of specified MLC�supply a therapeutically effective amount of the antibody according to the invention, thereby effectively carrying out the treatment of the specified mammal. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method for the treatment or prevention of cell proliferative disorders associated with increased expression of CD79b, wherein said method comprises administering to the individual in need of such treatment, an effective amount of an antibody according to the invention, thereby effectively carrying out the treatment or prevention of a specified cell-proliferative disorders. In one embodiment, the implementation of the specified proliferative infringement is a malignant tumor. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of inhibiting cell growth, where the growth of the indicated cells at least partially depends on the effect of CD79b on strengthening growth, and said method comprises contacting cells with an effective amount of an antibody according to the invention, thereby inhibiting the growth of the specified cell. In one embodiment, the implementation, the antibody conjugial�but with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of medical treatment of a tumor in a mammal, where the specified growth of the tumor is at least partially dependent on the effect of CD79b on strengthening growth, and said method comprises contacting cells with an effective amount of an antibody according to the invention, thereby effectively carrying out the treatment of the indicated tumor. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method for treating malignant tumors, comprising administering to the patient a pharmaceutical composition comprising immunoconjugate described in this document acceptable diluent, carrier or excipient.

In one aspect, the invention relates to a method of inhibiting the proliferation of B-cells, including the impact on the cell immunoconjugate containing the antibody according to the invention, under conditions that allow binding immunoconjugate with CD79b.

In one aspect, the invention relates to a method for determining the presence of CD79b in a sample suspected to contain CD79b, wherein said method comprises exposure to the specified obra�EC antibodies according to the invention, and the definition of the specified binding of the antibody to CD79b in a specified sample, where the specified binding of the antibody to CD79b in a specified sample indicates the presence of said protein in a given sample.

In one aspect, the invention relates to a method of diagnosing a cellular proliferative disorders associated with an increase in the number of cells, such as B-cells expressing CD79b, wherein the method comprises contacting the test cells in a biological sample with any of the above antibodies; determining the level of antibody bound to test cells in the sample by detecting binding of an antibody to CD79b; and comparing the level of antibody bound to cells in a control sample, where the level of bound antibody normalized with respect to the number expressing CD79b cells in the tested and control samples, and where a higher level of antibody associated with the test sample compared to a control sample indicates the presence of cell-proliferative disorders associated with cells expressing CD79b.

In one aspect, the invention relates to a method for the detection of soluble CD79b in blood or serum, the method comprises contacting the test sample of blood or serum from the mammal, presumably suffering from B-cell-proliferative �the violation, with the antibody against CD79b according to the invention, and the detection of increasing soluble CD79b in the sample relative to the control sample of blood or serum from a healthy mammal.

In one aspect, the invention relates to a method of binding antibodies according to the invention with a cell that expresses CD79b, wherein said method comprises contacting the specified cell with the antibody of the invention. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

BRIEF description of the DRAWINGS

Figure 1 shows the nucleotide sequence (SEQ ID NO:1) cDNA PRO36249, where SEQ ID NO:1 is a clone designated herein as "DNA225786" (also referred to in this document as "CD79b"). The nucleotide sequence encodes CD79b, and the initiation codon and the stop codon is shown in bold and underlined.

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

Figure 3 presents the nucleotide sequence (SEQ ID NO:3) light chain chimeric 2F2 (ch2F2) IgG1 (2F2 is a monoclonal antibody against mouse CD79b). The nucleotide sequence encodes Les�forge chain ch2F2, presented on figure 4, and the first codon (encoding the first amino acid of SEQ ID NO:4) is indicated in bold and underlined.

Figure 4 presents the amino acid sequence (SEQ ID NO:4), formed of the coding sequence of SEQ ID NO:3, are presented in figure 3. Variable regions are underlined region.

Figure 5 presents the nucleotide sequence (SEQ ID NO:5) of the heavy chain of chimeric 2F2 (ch2F2) IgG1 (2F2 is a monoclonal antibody against mouse CD79b). The nucleotide sequence encodes a heavy chain ch2F2 presented in figure 6, and the first codon (encoding the first amino acid of SEQ ID NO:6) is indicated in bold and underlined.

Figure 6 presents the amino acid sequence (SEQ ID NO:6), formed of the coding sequence of SEQ ID NO:5, as presented in figure 5. Variable regions are underlined sections.

Figure 7 shows the alignment of sequences of the variable regions of light chains for the following: a consensus sequence of light chain Kappa I (labeled as "huKI"; SEQ ID NO:9) with VL-FR1, VL-FR2, VL-FR3, VL-FR4 (SEQ ID nos:65-68, respectively), antibody 2F2 against mouse CD79b (designated as "mu2F2" and also herein referred to as "2F2"; SEQ ID NO:10), "humanized" antibody with Perez�internal consistency 2F2 (denoted as "hu2F2 with a transplanted sequence"; SEQ ID NO:11) and variant 7 "humanized" antibodies with a transplanted sequence 2F2 (denoted as "hu2F2.D7"; SEQ ID NO:12) (containing 71A, 73T and 78A). Position numbered according to Kabat and hypervariable region (HVR), transplanted from 2F2 mouse in the consensus frame region of the variable region light chain Kappa I, boxed.

In the figures 8A-B presents the alignment of the sequences of variable regions of heavy chains for the following: a consensus sequence of heavy chain subgroup III (designated as "humIII"; SEQ ID NO:13) with VH-FR1, VH-FR2, VH-FR3, and VH-FR4 (SEQ ID NO:69-72, respectively), antibody 2F2 against mouse CD79b (designated as "mu2F2" and also herein referred to as "2F2"; SEQ ID NO:14), "humanized" antibody with a transplanted sequence 2F2 (denoted as "hu2F2 with a transplanted sequence; SEQ ID NO:15) (containing 71A, 73T and 78A) and variant 7 "humanized" antibodies with a transplanted sequence 2F2 (denoted as "hu2F2.D7"; SEQ ID NO:16) (containing 71A, 73T and 78A). Position numbered according to Kabat and hypervariable region (HVR), transplanted from mu2F2 in the consensus frame region of the variable region of the heavy chain subgroup III, boxed.

Figure 9 presents the HVR sequence selected option "humanized" antibodies with a transplanted last�governolo 2F2 (SEQ ID NO:18), where version has many changes of amino acids in a separate area of HVR "humanized" antibodies with a transplanted sequence 2F2 (part HVR-L3 (SEQ ID NO:61) is shown in figure 9 as SEQ ID NO:17). Sequence of variable region light chain and the variable region of the heavy chain shown outside of the amino acid changes were identical transplanted sequence 2F2 and not shown. There were no changes in HVR-L1 (SEQ ID NO:59), HVR-L2 (SEQ ID NO:60), HVR-H1 (SEQ ID NO:62); HVR-H2 (SEQ ID NO:63) or HVR-H3 (SEQ ID NO:64) "humanized" antibodies with a transplanted sequence 2F2.

Figure 10 shows Biacore analysis of selected antibodies against CD79b, including antibody ch2F2 (denoted as "ch2F2"), "humanized" antibody with a transplanted sequence 2F2 (denoted as "hu2F2 with a transplanted sequence" and also referred to herein as "2F2-graft sequence"), and option 7 "humanized" antibodies with a transplanted sequence 2F2 (hu2F2.D7) (89F, 96F; SEQ ID NO:18) against the indicated antigens, including the extracellular domain of human CD79b (huCD79becd), the extracellular domain of human CD79b, merged with Fc (huCD79becd-Fc), and the peptide of 16 amino acids containing the epitope for 2F2 (16-dimensional peptide; SEQ ID NO:78; an epitope from amino acids 1-11 of SEQ ID NO:78). Not revealed the binding of labeled in the figure as "NB".

Figure 11A-B (consensus frame region of the variable region of the heavy chain (VH)) and figure 12 (a consensus frame region of the variable region light chain (VL)) presents illustrative sequence acceptor consensus of frame areas of a person for use in practice of the present invention with the following sequence identifiers: (figure 11A-B) consensus frame region VH subgroup I of a person without CDR by Kabat (SEQ ID NO:36), the consensus frame region VH subgroup I of a person without extended hypervariable regions (SEQ ID NO:37-39), the consensus frame region VH subgroup II human without CDR by Kabat (SEQ ID NO:40), the consensus frame region VH subgroup II human without extended hypervariable regions (SEQ ID NO:41-43), the consensus frame region VH subgroup III a man without a CDR according to Kabat (SEQ ID NO:44), the consensus frame region VH subgroup III person without extended hypervariable regions (SEQ ID NO:45-47), frame acceptor VH region of a person without CDR by Kabat (SEQ ID NO:48), acceptor skeleton of the VH region of a person without extended hypervariable regions (SEQ ID NO:49-50), Acceptera frame area 2 VH man without CDR by Kabat (SEQ ID NO:51) and Acceptera frame area 2 VH man without extended hypervariable regions (SEQ ID nos:52-54) and (figure 12) consensus ka�the Cass region VL subgroup I of human Kappa (SEQ ID NO:55), the consensus frame region VL subgroup II Kappa (SEQ ID NO:56), the consensus frame region VL subgroup III Kappa (SEQ ID NO:57) and the consensus frame region VL subgroup IV Kappa (SEQ ID NO:58).

Figure 13A (light chain) and 13B (heavy chain) shows the amino acid sequence of the antibodies of the invention (2F2.D7). In the figures 13A (light chain) and 13B (heavy chain) represented by the amino acid sequence of a frame region (FR), hypervariable region (HVR), first constant domain (CL or CH1) and Fc region (Fc) of one of the embodiments of the antibodies according to the invention (hu2F2.D7) (SEQ ID NO:19-26 (figure 13A) and SEQ ID NO:27-35 (figure 13B)). Presents a full-sized amino acid sequences (variable and constant regions) of the light and heavy chains 2F2.D7 (SEQ ID NO:89 (figure 13A) and 90 (figure 13B), respectively, and the constant domains are underlined. Shows the amino acid sequence of the variable domains (SEQ ID NO:12 (figure 13A for light chain) and SEQ ID NO:16 (figure 13B for the heavy chain)).

Figure 14 shows the alignment of amino acid sequences of human CD79b (SEQ ID NO:2), cynomolgus monkey (cyno) (SEQ ID NO:7) and mouse (SEQ ID NO:8). CD79b of human and cynomolgus macaque have 85% amino acid identity. Specified signal sequence, the test peptide (epitope consisting of 11 amino acids for antibody 2F2,�isandy in example 1; amino acids 1-11 (ARSEDRYRNPK) SEQ ID NO:78), transmembrane (TM) domain and domain immunoreceptor binding tyrosine activation motif (ITAM). The area enclosed in the frame represents a region of CD79b, which is absent in the embodiment of the splicing CD79b (described in example 1).

Figure 15 presents the image of conjugates of antibodies against CD79b with built in his cysteine residues and medicines (ADC), where a group of medicines associated with an integrated group of cysteine: light chain (LC-ADC); the heavy chain (HC-ADC); and Fc region (Fc-ADC).

Figure 16 illustrates the stages: (i) restoration of disulfide adducts of cysteine and miaocheng and vnutrisemejnyh disulfides in the CD79b antibody against it with the built-in cysteine residues (thio-Mab) with reducing agent TCEP (Tris(2-carboxyethyl)phosphine hydrochloride); (ii) partial oxidation, i.e., re-oxidation to convert miaocheng and vnutrisemejnyh disulfides by dhAA (dehydroascorbic acid); and (iii) conjugation re-oxidized antibody with intermediate connection of a drug-linker to form a conjugate antibodies against CD79b with embedded cysteine residues and medicines (ADC).

Figure 17 shows (A) the light chain sequence (SEQ ID NO:86) and (B) sequence of the heavy chain (SEQ ID NO:85) GU�Anisimovna antibodies against CD79b with built in his cysteine residues (thio-hu2F2.D7-HC-A118C), in which alanine at position 118 in EU (regulation of alanine in the sequence 118; Kabat position 114) of the heavy chain is replaced by cysteine. Group of drugs may be associated with an integrated group of cysteine in the heavy chain. Every figure is a modified amino acid represented in bold double underline. Single underlining shows constant region. Variable regions are underlined region. Fc-region is in italics. "Thio" refers to an antibody with a built-in cysteine, and "hu" refers to humanitarianlaw the antibody.

Figure 18 shows (A) the light chain sequence (SEQ ID NO:88), and (B) sequence of the heavy chain (SEQ ID NO:87) antibodies against CD79b with built in his cysteine residues (Thio-hu2F2.D7-LC-V205C) in which valine at position 205 by Kabat (position in the sequence: valine 210) light chain is replaced by cysteine. Amino acid D at position 6 on EU (shaded in the figure) of the heavy chain alternative can be a E. Group of drugs may be associated with an integrated group of cysteine in the heavy chain. Every figure is a modified amino acid is shown in bold double underline. Single underline specified constant region. Variable regions are not DNV�knotia area. Fc-region is in italics. "Thio" refers to an antibody with built in his cysteine residues.

Figure 19 is a graph showing inhibition of tumor growthin vivoin the model with xenotransplantation BJAB-luciferase, which demonstrates that the introduction of antibodies against CD79b ((a) ch2F2-SMCC-DM1, drug load was approximately 3 (table 7), and (b) hu2F2.D7-SMCC-DM1, drug load was approximately 2.3 (table 7)), SCID mice with B-cell human tumors, significantly inhibited tumor growth. Controls included HERCEPTIN® (trastuzumab)-SMCC-DM1 (antibody against HER2-SMCC-DM1). "hu" refers to humanitarianlaw the antibody and "ch" refers to the chimeric antibody.

Figure 20A is a graph showing inhibition of tumor growthin vivoin the model compared to xenograft Granta-519 (b-cell lymphoma mantle zone), which shows that the introduction of antibodies against CD79b, hu2F2.D7-SMCC-DM1, drug load was approximately 2,8 (table 8), SCID mice with B-cell human tumors, significantly inhibited tumor growth. Controls included HERCEPTIN® (trastuzumab)-SMCC-DM1 (antibody against HER2-SMCC-DM1). Figure 20B shows a graph of the percentage change in weight of the mice in the study compared to xenograft Granta-519 (figure 20A and table 8), which shows that there is no snecial�nye mass change during the first 7 days of the study, "hu" refers to humanitarianlaw the antibody.

DETAILED DESCRIPTION of PREFERRED embodiments

The present invention relates to methods, compositions, kits and articles to identify the compositions suitable for the treatment of hematopoietic tumor in mammals and to methods of using these compositions.

This document presents the details of these methods, compositions, kits and articles.

I.General procedures

In the practice of the present inventionuse, unless otherwise indicated, conventional methods of molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, which are known to specialists in this field. Such methods are fully described in the literature, such as, "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 periodic updated edition); "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 purposes of explanation, consider the description will be used the following definitions, and, where necessary, the terms listed in the only also include the plural �number, and Vice versa. In that case, if the below definitions are contrary to any document incorporated herein by reference, should be guided by the definitions presented in this document.

The terms "marker B-cell surface" or "antigen B-cell surface" in this document refer to the antigen, expressed on the surface of B-cells, which can be aimed antagonist, which is associated with it, including, but not limited to, antibodies to antigen B-cell surface or soluble form of the antigen (B-cell surface, is able to carry out the antagonism of ligand binding to natural antigen of B-cells. Examples of markers of B-cell surface markers include the surface of cells 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 (for descriptions, see The Leukocyte Antigen Facts Book, 2ndEdition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other markers of B-cell surface include RP105, FcRH2, CR2 on B-cells, CCR6, RH, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. Of special interest marker of B-cell surface protein is expressed preferentially in B cells compared to other non-b cell tissues of mammals, and it can be expressed as predestin�ICA B-cells, and Mature B-cells.

The term "CD79b", as used herein, refers to any native CD79b any spinal animal, including mammals such as primates (e.g. humans, cynomolgus monkeys (cyno)) and rodents (e.g. mice and rats), unless otherwise stated. CD79b of human rights also refer herein as "PRO36249" (SEQ ID NO:2) and it is encoded by the nucleotide sequence (SEQ ID NO:1), which is also referred to herein as "DNA225786". The term "CD79b" includes "full-size" reprezentirovannoe CD79b, as well as any form of CD79b, which is formed by processing in the cell. The term also encompasses naturally occurring variants CD79b, for example, a variant on splicing, allelic variants and isoforms. CD79b polypeptides described herein, can be isolated from various sources, such as from human tissue or from other sources, or obtained using recombinant or synthetic methods. The term "CD79b polypeptide with native sequence" includes a polypeptide with the same amino acid sequence as the corresponding CD79b polypeptide derived from a natural source. Such CD79b polypeptides with a native sequence can be isolated from natural sources or can be obtained using recombinant or synthetic means the Term "CD79b polypeptide with native sequence" specifically encompasses naturally occurring truncated or synthesized specific form of CD79b polypeptide (e.g., extracellular domain sequence), forms of naturally occurring variants (e.g., alternative spliced forms) and naturally occurring allelic variants of the polypeptide. In some embodiments of the present invention, the CD79b polypeptide with the natural sequence described herein are Mature or full-length polypeptides with a native sequence of,includes a full-sized amino acid sequence represented in the attached drawings. The initiation codon and the stop codon (if specified) are indicated in bold and underlined in the figures. Residues of nucleic acids, denoted as "N" on the accompanying drawings represent any remnants of nucleic acids. However, although the accompanying figures show that the described CD79b polypeptides begin with methionine residues designated herein as amino acid position 1 in the figures, it is likely and possible that as the starting amino acid residue for CD79b polypeptides can be used other methionine residues located either above or below, from amino acid position 1 in the figures.

The term"2F2" is used herein to refer to monoclonal mouse antibodies against CD79b (also referred to herein as "mu2F2" and�and "2F2" mouse) or chimeric antibodies (also referred to herein as "ch2F2").

The terms"mu2F2" or "2F2 mouse" is used herein to refer to monoclonal mouse antibodies against CD79b, where the mouse antibody comprises the variable domain light chain SEQ ID NO:10 (Fig. 7) and the variable domain of the heavy chain SEQ ID NO:14 (Fig. 8A-B). Mu2F2 can be obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006.

The term"ch2F2 or chimeric antibody 2F2" is used herein to refer to chimeric antibodies (as previously described in patent application U.S. Ser. No. 11/462336, filed August 3, 2006), where the chimeric antibody includes a light chain SEQ ID NO:4 (Fig. 4) where the specified light chain variable domain includes SEQ ID NO:10 (Fig. 7) and light chain constant domain of human IgG1. Chimeric antibody further comprises a heavy chain SEQ ID NO:6 (Fig. 6), where the light chain variable domain includes SEQ ID NO:14 (Fig. 8A-B) and a constant domain of the heavy chain of human IgG1.

The terms "transplanted sequence 2F2" or "humanized" antibody with a transplanted sequence 2F2" or "transplanted sequence hu2F2" is used herein to refer specifically transplanted sequence, obtained by grafting the hypervariable regions of the antibodies 2F2 mouse (mu2F2) to acceptor consensus VL Kappa I (huKI) and consensus VH subgroup III (huIII) with R71A, N73T and L78A (Carter et al., roc. Natl Acad. Sci. USA, 89:4285 (1992)) (see example 1A and figure 7 (SEQ ID NO:11) and 8 (SEQ ID NO:15)).

The term "modification" of amino acid residue/position, as used herein, refers to changing the primary amino acid sequence compared to the original amino acid sequence, where the specified change occurs as a result of changing the sequence involving these amino acid residues/positions. For example, typical modifications include replacing the balance (or in the orientation shown) of another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (usually less than 5 or 3) amino acids adjacent to the specified residue/position and the deletion of the specified residue/position. The term "amino acid substitutions", or its variations, refers to the replacement of an existing amino acid residue in a given (source) amino acid sequence wherein amino acid residue. Typically and preferably, the result of this modification is to change at least one type of physical-biochemical activity of the variant polypeptide compared to a polypeptide that contains the original amino acid sequence (or amino acid sequence of the "wild type"). For example, in the case of antibodies, physico-biochemical activity, which ismene�Xia, may constitute a binding affinity, binding capacity and/or binding effect in respect of target molecules.

The term "antibody" is used in the broadest sense and specifically covers, for example, single monoclonal antibodies against CD79b (including agonist, antagonist and neutralizing, full-length or intact monoclonal antibodies), compositions of antibodies against CD79b with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, polyspecific antibodies (for example, bespecifically antibodies, provided that they exhibit the desired biological activity), obtained from at least two intact antibodies, single-chain antibodies against CD79b and fragments of antibodies against CD79b (see below), including Fab, Fab', F(ab')2- and Fv-fragments, diately, single-chain antibodies (sdAb), provided that they exhibit the desired biological or immunological activity. In this document, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably. The antibody may be a human antibody, humanized antibody and/or antibody with ripe affinity.

The term "antibody against CD79b" or "an antibody that binds to CD79b" refers to an antibody that can bind to CD79b with sufficient affinity so that the antibody is�tsya suitable as a diagnostic and/or drugs when targeting CD79b. Preferably, the degree of binding antibodies against CD79b with an unrelated not CD79b protein is less than about 10% of the binding of an antibody to CD79b in the measurement, for example, radioimmunoassay analysis (RIA). In certain embodiments, an antibody that binds to CD79b, has a dissociation constant (Kd) ≤1 µm ≤100 nm, ≤10 nm, ≤1 nm, or ≤0.1 nm. In certain embodiments, antibodies against CD79b binds to the epitope CD79b, which is conservative among CD79b of various kinds.

A selected antibody" is an antibody that is identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that inhibit therapeutic application of antibodies, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the antibody is purified (1) to more than 95% by weight of antibody as determined by the Lowry method, and most preferably to more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using the sequencer with rotating cups, or (3) to homogeneity in SDSPAGE in reducing or Sevostyanova conditions using Kumasi blue or, preferably, staining with silver. The selected antibody includes the antibody in recombinant cells ofin situsince in this case there are at least one component of the natural environment antibodies. However, as a rule, the selected antibody is obtained by at least one stage of cleaning.

The main element of the antibodies of the 4 circuits is heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 main heterotetrameric elements together with an additional polypeptide called J chain, and therefore, it contains 10 antigen binding sites, while the synthesized IgA antibodies can polymerize with the formation of polyvalent systems containing 2-5 main elements of 4-chains together with the J-chain). In the case of IgG, the element of 4-circuits, usually has a weight of 150,000 daltons. Each L-chain is associated with the H-chain by one covalent disulfide bond, while the two H chains are linked together by one or more disulfide bonds, depending on the isotype of the H-chain. Each H - and L-chain has regularly spaced megamachine disulfide bridges. Each H chain has at the N end of the variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four C Hdomain isotypes for μ and ε. Each L chain has at the N end of the variable domain (VL) followed by a constant domain (CLat its other end. VLis aligned with VHand CLis aligned with the first constant domain of the heavy chain (CH1). I believe that the specific amino acid residues form a contact surface between the variable domains of the light chain and heavy chain. The pairing of VHand VLforms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., 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-chain of any vertebrate species can be assigned to one of two clearly different types, called Kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be attributed to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains, called α, δ, ε, γ and μ, respectively. Classes γ and α are further divided into subclasses on the basis of relatively minor differences in the sequence CHand functions, for example, a person expressed the following� subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

"Variable area" or "variable domain" of an antibody refers to the N-terminal domains of the heavy and light chain antibodies. Variable domain of the heavy chain may be designated as "VH". Variable domain light chain can be designated as "VL". These domains are the most variable parts of an antibody and contain the antigen-binding centers.

The term "variable" refers to the fact that the sequence of certain segments of the variable domains differ significantly among antibodies. V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not uniform throughout the area of the variable domains of 110 amino acids. Instead, V-areas consist of relatively invariant sections, called frame regions (FR) of 15-30 amino acids separated by shorter regions of high variability called "hypervariable regions", the length of which varies from 9 to 12 amino acids. The variable domains of native heavy and light chains contain four FR, mainly riminaldi the configuration of the β-layers, connected by three hypervariable regions that form a loop that combines the structure of β-layers, and, in some cases, forming its frequent�. Hypervariable regions in each chain are located together in the vicinity of FR and, with the hypervariable regions of the other chain, involved in the formation of the antigen-binding centre of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not involved directly in binding the antibody to the antigen, but they exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

"Intact" antibody is an antibody that contains the antigen-binding center, and CLand at least the constant domains of the heavy chain, CH1, CH2 and CH3. The constant domains may represent the constant domains of the native sequence (for example, the constant domains of a person with a native sequence), or a variant amino acid sequence. Preferably, the intact antibody has one or more effector functions.

"”Naked” antibody" for purposes of the present description is an antibody that does not anywhereman with cytotoxic or radioactive group label.

"Antibody fragments" contain a portion of an intact antibody, preferably the antigen-binding or variable�th region of intact antibodies. Examples of fragments of antibodies include Fab, Fab', F(ab')2- and Fv-fragments, diately, linear antibodies (see, e.g., U.S. patent No. 5641870, example 2, Zapata et al., Protein Eng., 8(10): 1057-1062 [1995]); single-chain antibody molecules; and polyspecific antibodies formed by fragments of antibodies. In one embodiment, the implementation, the antibody fragment contains the antigen-binding center of the intact antibody and thus retains the ability to bind antigen.

Cleavage of antibodies with papain leads to the formation of two identical antigen-binding fragments, called "Fab"fragments, and a residual "Fc"fragment, whose name reflects its ability to easily crystallize. Fab fragment consists of an entire L chain along with the domain of the variable segment of the H-chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent in relation to antigen binding, i.e., it has one antigen-binding site. Treatment of antibodies with pepsin leads to a single large F(ab')2-the fragment which roughly corresponds to two connected by disulfide Fab fragments having divalent antigen-binding activity, and, in addition, capable of cross-linking antigen. Fab'fragments differ from Fab fragments by the presence of several additional residues n� C-end domain of C H1, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation for Fab' in which the residue(s) of cysteine constant domains have a free thiol group. F(ab')2-fragments of the antibodies were originally produced as pairs of Fab'fragments that have hinge cysteines between them. Also known other chemical binding fragments of antibodies.

Fc-fragment contains the C-terminal parts of both H chains held together by disulfides. Effector functions of antibodies are determined by sequences in the Fc-plot, which is also the part recognized by the Fc receptors (FcR) found on certain types of cells.

"Fv" is the minimum antibody fragment that contains a complete antipersonnel and the antigen-binding center. This fragment consists of a dimer composed of one variable domain of the heavy chain and one variable domain of the light chain associated strong non-covalent bond. In types of single-stranded Fv (scFv), single variable domain of the heavy chain and one variable domain light chain can be covalently linked movable peptide linker so that the light and heavy chains can associate in a "dimeric" structural analogues of similar types of double-stranded Fv. The folding of these two domains �Brazul six hypervariable loops (3 loops each from the H - and L-chains), which provide amino acid residues for antigen binding and provide the binding specificity of the antigen antibody. However, even a single variable domain (or half of Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

"Single-chain Fv" also abbreviated as "sFv" or "scFv", represent fragments of antibodies that contain VHand VL-antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide also optionally contains a polypeptide linker between the VHand VL-domains that provide the possibility of forming in sFv patterns required for binding to the antigen. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term "diately" refers to small antibody fragments with two antigen-binding sites, and these fragments contain the variable domain of the heavy chain (VH) associated with the variable domain light chain (VL) in the same polypeptide chain (VH-VL). Small fragments of antibodies obtained by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between VHand VL-domains, so �provides megamachine, but not vnutriserdechne, the formation of pairs of V-domains, resulting in a bivalent fragment, i.e., fragment having two antigen-binding centers. Diatel can be divalent or bespecifically. Bespecifically of diately represent heterodimer two "intersecting" sFv fragments in which VHand VLdomains of the two antibodies are on different polypeptide chains. Diately described in more detail, for example, in EP 404097; WO93/11161; Hudson et al., (2003) Nat. Med. 9:129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). Triatel (triabody) and tetrathele (tetrabody) are also described in Hudson et al., (2003) Nat. Med. 9:129-134.

As used herein, the term "monoclonal antibody" refers to an antibody from a population essentially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible mutations, for example, naturally occurring mutations that may be presented in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Moreover, in contrast to the preparations of polyclonal antibodies, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition�s to their specificity, the monoclonal antibodies are predominant in that they can be synthesized without admixtures of other antibodies. The definition of "monoclonal" does not imply that the antibody must be obtained in any particular way. For example, monoclonal antibodies intended for use in accordance with the present invention, can be obtained by the hybrid method, first described by Kohler et al.,Nature,256:495 (1975), or they can be obtained by means of recombinant DNA bacterial, eukaryotic cells, and in cells of animals and plants (see, e.g., U.S. patent No. 4816567). "Monoclonal antibodies" may also be derived from phage libraries of antibodies using the methods described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991).

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a plot heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well�e fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567; and Morrison et al.,Proc. Natl. Acad. Sci USA, 81:6851-6855 (1984)). Of interest in the preparation of this submission chimeric antibodies include "primaryservername" antibody containing the antigen-binding sequence of the variable domain of the non-human Primate (e.g. old world monkey, APE etc) and the sequence of the constant domain of human rights.

"Humanized" forms of non-human antibodies (e.g., rodents) are chimeric antibodies that contain minimal sequence derived from the immunoglobulin, non-human. Basically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region species, non-human (donor antibody) such as mouse, rat, rabbit or non-human primates, which possess the desired specificity, affinity and capacity. In some cases, remnants of the frame region (FR) of human immunoglobulin replace the corresponding residues that are not human. Furthermore, humanized antibodies may contain residues that �otsutstvuet in the recipient antibody or in the donor antibody. These modifications are carried out to further improve the parameters of the antibody. Generally, a humanized antibody contains essentially all of at least one, and typically two, variable domains in which all or essentially all of the hypervariable loops correspond to the hypervariable loops of immunoglobulin, non-human, and all or essentially all of the FR-region represent the FR-region from the sequence of human immunoglobulin. Also a humanized antibody optionally comprises at least the plot of the constant domain (Fc) of an immunoglobulin, typically a constant domain of human immunoglobulin. For further details, see Jones et al.,Nature 321:522-525 (1986); Riechmann et al.,Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited in them: 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).

"Thio", when used herein, refers to an antibody with built in his cysteine residues, and "hu", when used herein, refers to humanitarianlaw the antibody.

"Human antibody" is an antibody that contains the amino acid sequence corresponding to the sequence of the antibody, producer�my person and/or obtained using any of the methods for obtaining human antibodies, as described in this document. This definition of human antibodies, in particular, does not include a humanized antibody containing the antigen-binding residues are not human. Antibodies can be obtained using various methods known in this field, including phage display library. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991). To obtain human monoclonal antibodies available methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147(1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Antibodies can be obtained by introducing antigen transgenic animal modified to produce such antibodies in response to antigen load, but having damaged endogenous loci, for example, the immunized xenonian (see, e.g., U.S. patent No. 6075181 and 6150584 regarding XENOMOUSE technologyTM). Also see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies obtained by the technology of B-cell hybridomas.

The terms "hypervariable region", "HVR" or "HV", as used herein, refer to the sections of the variable domain of antibodies, which are hypervariable in sequence and/or form structurally defined loops. Usually antibodies include estigarribia areas: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Use several ways to indicate the hypervariable regions, and they are included in this description. Defining complementarity region (CDR) according to Kabat, is based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the loop of CDR-H1 by Chothia, the numbering using the rules the numbering of Kabat, varies between H32 and H34 depending on the length of the loop (this is due to the fact that the numbering scheme of Kabat places the insertions at H35A and H35B; if neither 35A or 35B, are not present, the loop ends at 32; if it only has 35A, the loop ends at 33; and if there are 35A and 35B, then the loop ends at 34). Hypervariable region AbM represent a compromise between CDR by Kabat and structural loop by Chothia and they are used in the software simulation of antibodies Oxford Molecular's AbM. "Contact" hypervariable sites based on analysis of the available complex crystal structures. The residues from each of these hypervariable sites noted below.

LoopKabat AbMChothiaContact
L1L24-L34L24-L34L24-L34L30-L36
L2L50-L56L50-L56L50-L56L46-L55
L3L89-L97L89-L97L89-L97L89-L96
H1H31-H35BH26-H35BH26-H32...34H30-H35B
(Numbering according to Kabat)
H1H31-H35H26-H35H26-H32H30-H35
(Chothia numbering)
H2H50-H65H50-H58H52-H56H47-H58
H3H95-H102H95-H102H95-H102 H93-H101

Hypervariable sites may include "extended hypervariable sites": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The remains of the variable domain numbered according to Kabat (Kabat et al., above) for each of these definitions.

The “frame” residues or “FR” residues are variable domains that are not related to the remnants of the hypervariable region, as defined in the present description.

The terms "residue numbering variable domain according to Kabat" or "amino acid numbering of provisions in Kabat", and their variants refer to the numbering system used for the variable domains of the heavy chain or variable light chain domains in the aggregate of antibodies from Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the true linear amino acid sequence may contain fewer or additional amino acids, which corresponds to the shortening of FR or CDR of the variable domain or embedding in them. For example, the variable domain of the heavy chain can include one box of interest of the amino acid (residue 52a according to Kabat) after residue 52 of H2 and embedded residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) p�after residue 82 FR of the heavy chain. Kabat numbering for the residues can be defined for specific antibody by alignment at different sections of the homology sequence of the antibody with a "standard" sequence, with numbering according to Kabat.

The numbering system of Kabat usually used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to a residue in a constant region of the heavy chain of immunoglobulin (e.g., the EU index as specified in Kabat et al., above). "The EU index in Kabat" refers to the residue numbering of the human IgG1 antibody EU. Unless herein otherwise stated, numbers indicate residues in the variable domain of antibodies means residue numbering is in accordance with the numbering system of Kabat. Unless herein otherwise stated, numbers indicate residues in the constant domain of antibodies means residue numbering according to the EU numbering system. (see, for example, the provisional application U.S. No. 60/640323, figures for EU numbering).

Antibody, obtained by "affinity maturation" is an antibody with one or more changes in one one or more HVR, which lead to increase affine�and antibodies to the antigen, compared to the original antibody, which does not have this change(s). Preferred antibodies received by "affinity maturation", have a mu or even low picomolar affinity to the antigen target. Antibodies obtained by affinity maturation, prepared by methods known in this field. To Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by way of "shuffling" VH - and VL-domains. Random mutagenesis HVR and/or wireframe residues is described by: 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 that inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antibody antagonist to partially or completely inhibit the biological activity of the antigen.

"Antibody agonist", as used herein, is an antibody which mimics at least one of the functional activities of interest polypeptide.

"Dependent on kind of an antibody, e.g. an antibody of a mammal against human IgE, is an antibody that has a higher binding affinity to the antigen from the sample production� species of mammal, relative affinity of binding to the homologue of the specified antigen from the sample of the second type of mammal. Usually dependent on the type antibody "specific associated" with the human antigen (i.e., has the value of the binding affinity (Kd) of no more than about 1×10-7M, preferably not more than about 1×10-8and most preferably no more than about 1×10-9M) but has a binding affinity with a homologue of the antigen from a second non-human, species of mammal, which is at least about 50-fold, or at least about 500 times, or at least about 1000 times weaker than the affinity of binding to antigen of a human. Dependent on the type of the antibody may be any of various types of antibodies, as disclosed above, but preferably is a humanized antibody or a human antibody.

The term"binding affinity" generally refers to the total strength of all non-covalent interactions between individual binding site of a molecule (e.g. antibody) and its binding partner (e.g. antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects the interaction of the 1:1 relationship between members�ausasia pair (for example, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in this field, including the methods described herein. Discapline antibodies generally bind antigen slowly and tend to easy dissociation, whereas high-affinity antibodies generally bind antigen faster and tend to stay longer in a bound state. To experts there are different ways to measure the affinity of binding, and any of them can be used for the purposes of the present invention. Specific illustrative implementation options described below.

The expression"or better" when used herein, when referring to the binding affinity refers to a stronger binding between the molecule and its binding partner. "Or better" when used herein refers to a stronger binding, characterized by a lower numerical value Kd. For example, in the case of antibodies, affinity to the antigen which is 0.6 nm or better," the affinity of the antibody to the antigen is <0.6 nm, i.e. of 0.59 nm, of 0.58 nm, 0,57 nm, etc., or any value that is less than 0.6 nm.

In one embodiment, the "Kd" or "Kd value" according to this and�attainment measured by analysis of binding of labeled tracer antigen (RIA), carried out with the Fab version of interest antibody and its antigen as described in the following analysis, which measure the affinity of binding of the Fab to the antigen in solution by balancing minimal Fab concentration (125(I)-labeled antigen in the presence of a series of titles unlabeled antigen, with subsequent fixation associated antigen on the tablet, covered with antibody against Fab (Chen, et al., (1999) J. Mol. Biol 293:865-881). To set the conditions for analysis, on microplates for titration (Dynex) is applied during the night of 5 µg/ml fixing antibodies against Fab (Cappel Labs) in 50 mm solution of sodium carbonate (pH 9,6), and then blocked with 2% (wt./about.) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-absorbent tablet (Nunc #269620), 100 PM or 26 PM [125I]-antigen are mixed with serial dilutions of Fab of interest (e.g., in accordance with the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Then Fab of interest were incubated over night; however, the incubation can be continued for a longer period (for example, 65 hours) to ensure that equilibrium is attained. Thereafter, the mixture is transferred to the locking tablet for incubation at room temperature (e.g. within 1 hour). Then R�the target is removed and the plate washed eight times with 0.1% Tween-20 in PBS. After the plates dry, add 150 ál/well of scintillator (MicroScint-20; Packard), and shall count in tablets using gamma counter pulses (Packard) for 10 minutes. The concentration of each Fab, which ensure the binding of less than or equal to 20% of the maximum binding is chosen for use in competitive binding assays. In accordance with another variant of the Kd or Kd value is measured using the analysis method of surface plasmon resonance, such analyses can be used BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with CM5 chips with immobilized them with antigen at ~10 response units (RU). Briefly, biosensor chips from carboxymethylamino dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. After the injection of antigen was injected 1 M ethanolamine to block unreacted groups. For determining the kinetics, two-fold serial dilutions of Fab (0,78 nm to 500 nm) inje�irout in PBS with 0.05% Tween 20 (PBST) at 25°C with a flow rate of approximately 25 μl/min. The Association constant (kon) and the dissociation constant (koff) is calculated using a simple model of binding Langmuir one-to-one (BIAcore Evaluation Software version 3.2) by simultaneously align sensogram Association and dissociation. The equilibrium dissociation constant (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 Association constant of the antibody is greater than 106M-1C-1in the analysis method of surface plasmon resonance mentioned above, then the Association constant can be determined using the method of fluorescence quenching, which 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 20 nm antibodies against the antigen (Fab form) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen in the measurement using a spectrophotometer, such as a spectrophotometer with stop jet (Aviv Instruments) or a spectrophotometer SLM-Aminco 8000 series (Thermo Spectronic) with a stirred cell.

"Binding rate" or "rate of Association" or "connection speed" or "kon"according to this invention can also be determined using this method of surface plasmon resonance described above using a BIAcoreM -2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) as described above.

The expression "essentially similar" or "essentially the same" as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparative antibody), so that the person skilled in the art may consider the difference between two values little or no biological and/or statistical significance within the context of the biological characteristic measured with the specified values (e.g., Kd values). The difference between these two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value for the reference/comparative antibodies.

The expression "significantly reduced" or "significantly different", as used herein, denotes a significant high degree of difference between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparative antibody), so that the person skilled in the art may consider the difference betweenthe two values is statistically significant in the context of the biological characteristic measured with the specified values (e.g., Kd values, the HAMA response). The difference between these two values is preferably more than about 10%, preferably more than about 20%, preferably more than about 30%, preferably more than about 40%, preferably more than approximately 50% as a function of the value for the reference/comparative antibodies.

"Antigen" is a predetermined antigen with which the antibody selectively binds. The antigen target can be a polypeptide, a hydrocarbon, a nucleic acid, a lipid, hapten or other naturally occurring or synthetic compound. Preferably, the antigen is a target is a polypeptide.

"The acceptor of a frame region of a person" for the purposes of the present description is a frame region containing the amino acid sequence of a frame region VL or VH formed of a frame region of a human immunoglobulin or a consensus sequence of frame area of the person. Acceptor of a frame region of a person, "educated" in a frame region of a human immunoglobulin or a consensus sequence of frame area of the person itself may contain their amino acid sequence, or it can �to win a pre-existing changes in amino acid sequences. Where pre-existing amino acid changes, preferably are present not more than 5 and preferably 4 or less, or 3 or less, pre-existing amino acid changes. Where pre-existing amino acid changes in VHpreferably, these changes existed in only three, two or one of the positions 71H, 73H and 78H; for example, amino acid residues at these positions can be 71A, 73T and/or 78A. In one embodiment, the implementation, frame acceptor VL region of a man identical in sequence frame region VL of human immunoglobulin or a consensus sequence of frame area of the person.

"Consensus sequence of frame area of man" is a frame region, which represents the most frequently occurring amino acid residues in the choice of frame sequences of the VL or VH of a human immunoglobulin. Usually the choice of sequences of the VL or VH of a human immunoglobulin is carried out in a subgroup of sequences of variable domains. Usually, the subgroup of sequences is a subgroup according to Kabat. In one embodiment of the implementation, for the VL, the subgroup is a subgroup Kappa I on Kabat. In one �the Ariant implementation for VH, the subgroup is a subgroup III as in Kabat.

"The consensus frame region VH subgroup III includes a consensus sequence formed by amino acid sequences of variable regions of heavy chain subgroup III as in Kabat. In one embodiment, the implementation of the amino acid sequence of the consensus frame region VH subgroup III includes at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:69)-H1-WVRQAPGKGLEWV(SEQ ID NO:70)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:71)-H3-WGQGTLVTVSS(SEQ ID NO:72).

"The consensus frame region VL subgroup I" comprises the consensus sequence formed by amino acid sequences of variable regions of light chain Kappa subgroup I of Kabat. In one embodiment, the implementation, the amino acid sequence of the consensus frame region VL subgroup I includes at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:65)-L1-WYQQKPGKAPKLLIY(SEQ ID NO:66)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:67)-L3-FGQGTKVEIKR (SEQ ID NO:68).

"Unmodified frame region of a human" is a frame region of a person that has the same amino acid sequence as the acceptor of a frame region of a person, for example, it lacks substitutions(substitutions) of an amino acid the amino�islote, no person in the acceptor skeleton of human rights.

"Modified hypervariable region" for the purposes of the present description represents a hypervariable region comprising one or more (for example from one to about 16) amino acid substitution(substitutions).

The term "unmodified hypervariable region" for the purposes of the present description represents a hypervariable region having the same amino acid sequence as the antibody is not human, from which it is obtained, i.e. hypervariable region that lacks one or more amino acid substitutions.

An antibody "which binds" an interest antigen, for example, associated with a tumor polypeptide antigen target, is an antibody that binds the antigen with sufficient affinity so that the antibody is suitable as a drug to target it to the cell or tissue expressing the antigen, and which does not cross react with other proteins to a significant degree. In such embodiments, the degree of binding of the antibody to the protein is"not a target" is less than about 10% of the binding of the antibody with its specific protein target, as determined in the analysis of sorting fluorescence activated cell (FACS) or p�power radioimmune precipitation (RIA). As to the binding of the antibody to the molecule-target, the terms "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or epitope on a particular polypeptide target means binding that is measurable level differs from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, with an excess of unlabeled target. In this case, specific binding indicates that the binding of the labeled target probe competitive inhibited by excess unlabeled target. The terms "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or epitope on a particular polypeptide target as used herein can be illustrated, for example, a molecule having a Kd for the target of at least approximately 10-4M, alternative at least about 10-5M, alternative less� least about 10 -6M, alternative at least about 10-7M, alternatively at least about 10-8M, alternative at least about 10-9M, alternative at least about 10-10M, alternative at least about 10-11M, alternative at least about 10-12M, or more. In one embodiment, the implementation, the term "specific binding" refers to binding, when a molecule binds to a particular polypeptide or epitope on a particular polypeptide without binding on a significant level with any other polypeptide or epitope of the polypeptide.

An antibody that "inhibits the growth of tumor cells expressing CD79b polypeptide" or a "growth inhibitory" antibody is an antibody, which results in measurable growth inhibition of cancer cells expressing or sverkhekspressiya corresponding to a CD79b polypeptide. The CD79b polypeptide may be a transmembrane polypeptide expressed on the surface of malignant cells, or it may be a polypeptide that is produced and secreted by malignant cells. Preferred inhibiting the growth of antibodies against CD79b inhibit the growth of expressing CD79b tumor to�etok more than 20%, preferably from about 20% to about 50%, and more preferably more than 50% (e.g. from about 50% to about 100%) compared with appropriate control, the control typically are tumor cells that are not treated with the test antibody. In one embodiment, the implementation, growth inhibition can be measured when the antibody concentration from about 0.1 to 30 μg/ml or about 0.5 nm to 200 nm in cell culture, where the growth inhibition is determined 1-10 days after exposure of tumor cells to the antibody. The inhibition of growth of tumor cellsin vivocan be defined in various ways, such as the methods described in the Experimental section examples", below. The antibody is growth inhibitoryin vivoif the introduction of antibodies against CD79b in a concentration of from about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or the proliferation of tumor cells within about 5 days to 3 months after the first administration of the antibody, preferably within about 5 to 30 days.

An antibody that "induces apoptosis" is an antibody that induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, crinkling�of cells, the expansion of the endoplasmic reticulum, fragmentation of cells and/or formation of membrane vesicles (called apoptotic bodies). Typically, such a cage is a cage, sverkhekspressiya CD79b polypeptide. Preferably, the cell is a tumor cell, e.g., hematopoietic cell, such as B-cell, T-cell, basophil, eosinophils, neutrophil, monocyte, platelet or erythrocyte. Available different ways of measuring cellular events associated with apoptosis. For example, translocation of phosphatidylserine (PS) can be measured by binding of annexin; DNA fragmentation can be evaluated by DNA ladder; and condensation of the nucleus/chromatin along with DNA fragmentation can be evaluated by the increase in the number hypodiploidy cells. Preferably, the antibody which induces apoptosis, is an antibody that causes the induction of binding of annexin approximately 2 to 50 times, preferably about 5 to 50 times, and most preferably from about 10 to 50 times greater than the binding of annexin untreated cells in the analysis of the binding of annexin.

An antibody that "induces cell death", is an antibody that causes the living cells of the become lifeless. The cell is a cell that expresses CD79b polypeptide and is the cell� of this type, that specifically expresses or sverkhekspressiya CD79b polypeptide. Such a cell can be malignant or normal cell specific cell type. The CD79b polypeptide may be a transmembrane polypeptide expressed on the surface of malignant cells, or is a polypeptide that is produced and secreted by malignant cells. The cell may be a malignant cell, e.g., B-cell or T-cell. Cell deathin vitroyou can determine in the absence of complement and immune effector cells to distinguish it from cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, the analysis of cell death can be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody to induce cell death, we can estimate the loss of membrane integrity, as assessed by the capture of propidium iodide (PI), tripan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD, relative to untreated cells. Preferred inducing cell death antibodies are antibodies that induce seizure PI in the analysis savatar in BT474 cells.

"Effector functions" of antibodies refer to such kinds of biological activity which are peculiar to the Fc region (Fc-region with the native sequence or variant Fc-region amino acid sequence) of an antibody, and vary depending on the isotype of the antibody. Examples of effector functions of antibodies include: C1q binding and complement-dependent cytotoxicity; binding of Fc-receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g. B cell receptor; BCR) and the activation of B-cells.

The term "Fc region" is used herein for the definition of the C-terminal region of the heavy chain of immunoglobulin, including Fc-region with the native sequence and variants Fc-regions. Although the boundaries of the Fc region of the heavy chain of an immunoglobulin may vary, the Fc-region of the heavy chain of human IgG is usually defined as the segment from amino acid residue at position Cys226, or from Pro230, to its C-end. C-terminal lysine (residue 447 in the EU numbering system) of the Fc region may be removed, for example, in the process of obtaining or purification of the antibody, or by constructing recombinant nucleic acid that encodes a heavy chain antibody. Accordingly, the composition of intact antibodies may include aggregate and�Titel, all of which removed the K447 residue, a combination of antibodies, which are not deleted by the K447 residue, and the mixture of antibodies containing a mixture of antibodies containing and not containing the K447 residue.

"Functional Fc region" possesses "effector function" Fc-region with the native sequence. Examples of "effector functions" include C1q binding; CDC; the binding of Fc-receptor; ADCC; phagocytosis; negative regulation of cell surface receptors (e.g., receptor of b-cells; BCR), etc. Such effector functions generally require the Fc-region has been combined with a binding domain (e.g., variable domain antibodies) and can be assessed using various assays as described, for example, in the section "Definitions" herein.

"The Fc-region with the native sequence" includes an amino acid sequence identical to the amino acid sequence Fc region found in nature. Fc-region of a person with a native sequence include the Fc-region of a human IgG1 with a native sequence (allotype not-A and A); Fc-region of human IgG2 with a native sequence; Fc-region of human IgG3 with a native sequence; and the Fc-region of human IgG4 with a native sequence, as well as their naturally occurring variants.

A "variant Fc region" comprises an amino acid consisten�etelnost, which differs from the Fc-region with the native sequence that contains at least one amino acid modification, preferably one or more amino acid substitution(substitutions). Preferably, a variant Fc region comprises at least one amino acid substitution compared to the Fc-region with the native sequence or a Fc-region of the original polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc-region with the native sequence or in the Fc-region of the original polypeptide. Variant Fc region herein preferably has at least about 80% homology to the Fc-region with the native sequence and/or Fc-region of the original polypeptide, and most preferably it has a homology with them at least about 90%, more preferably it has a homology with them at least about 95%.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which synthesized Ig related to Fc-receptors (FcR) found on certain cytotoxic cells (e.g. natural killer (NK) cells, neutrophils and macrophages), C�s specific to these cytotoxic effector cells binding antigen carrier cage-target, and then destroy the target cell by means of cytotoxins. Antibodies are the "weapons" the cytotoxic cells and are absolutely required for such destruction. Basic cells for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is presented in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To evaluate the activity of interest molecules against ADCC analysis can be performed ADCCin vitrosuch as analysis, described in U.S. patent No. 5500362 or 5821337. Suitable for these assays effector cells include peripheral mononuclear blood cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the activity of interest molecules against ADCC is possible to estimatein vivofor example , in animal models, such as described in Clynes et al.PNAS (USA)95:652-656 (1998).

The terms "Fc receptor" or "FcR" refers to a receptor that binds to the Fc-region of antibodies. The preferred FcR is FcR person with a native sequence. Moreover, a preferred FcR is FcR, which binds an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and alternative-spliced forms of these receptors. Recipe�ry FcγRII include 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 in its cytoplasmic domain contains immunoreceptor tyrosine-binding activating motif (ITAM). Inhibiting receptor FcγRIIB in its cytoplasmic domain contains immunoreceptor tyrosine-binding inhibitory motif (ITIM), (see review M. in to Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR is described in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al.,J. Lab. Clin.Med. 126:330-41 (1995). By the term "FcR" in the present description include other FcR, including FcR, which will be revealed in the future. The term also includes the neonatal receptor, 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 FcRnin vivoand the half-life in serum for polypeptides with high affinity binding of human FcRn can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates, which is administered polypeptides with a variant Fc-region. In WO 2000/42072 (Presta) describes variants of antibodies with increased or decreased binding to FcR. Cm. also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

"�factorie human cells" are leukocytes, which Express one or more FcR and perform effector functions. Preferably, the cells Express at least FcγRIII and perform effector function is ADCC. Examples of human leukocytes that carry out ADCC include peripheral mononuclear blood cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; thus are preferred PBMC and NK cells. Effector cells can be isolated from their natural sources, e.g., from the blood.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of the target cells in the presence of complement. Activation of the classical complement cascade begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are associated with them recognizable antigen. To assess complement activation, you can analyze the CDC, for example, as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996). Variants of polypeptides with altered amino acid sequences of the Fc-region and increased or decreased ability to bind C1q described in U.S. patent No. 6194551B1 and WO1999/51642. Cm. also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term "containing the Fc-region of an antibody refers to an antibody that comprises an Fc-region. C-terminal lysine (residue 447 according to the EU numbering system) of the Fc-region� can be removed for example, during purification of the antibody or by recombinant construct of the antibody encoded by the nucleic acid. Thus, the composition comprising containing the Fc-region of an antibody according to the present invention can include an antibody that contains K447, the antibody from which fully K447 removed, or a mixture of antibodies containing and not containing the K447 residue.

The term "extracellular domain of CD79b polypeptide or "ECD" refers to a form of CD79b polypeptide, which essentially does not contain the transmembrane and tsitoplazmaticheskogo domains. Typically, ECD CD79b polypeptide contains less than 1% of such transmembrane and/or cytoplasmically domains, and preferably it contains less than 0.5% of such domains. It is understood that any transmembrane domains identified for CD79b polypeptides of the present invention, identified in accordance with the criteria commonly used in this field to identify the hydrophobic domain of this type. The exact boundaries of a transmembrane domain may vary but most likely not more than about 5 amino acids at either side of the domain, as originally defined in the present document. Thus, optionally the extracellular domain of CD79b polypeptide can contain about 5 or fewer amino acids on either side of the border between the transmembrane d�Mohn/extracellular domain as indicated in the examples or description, and such polypeptides, with or without the associated signal peptide, and encoding nucleic acid provided by the present invention.

The approximate location of the "signal peptides" of the CD79b polypeptide described herein, can be shown in the present description and/or in the respective figures. However, it should be noted that the C-terminal boundary of the signal peptide may vary, but most likely not more than about 5 amino acids at either side of the C-terminal boundary of the signal peptide, as source identified in this document, where the C-terminal boundary of the signal peptide may be determined in accordance with criteria which are commonly used in this area to identify this item type amino acid sequence (see e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Furthermore, it is known that in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely the same, which results in more than one secreted polypeptide. These Mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal faces�s signal peptide, as identified in this document, and encoding the polynucleotides provided by the present invention.

The term"variant of the CD79b polypeptide" means a CD79b polypeptide, preferably an active CD79b polypeptide as described herein having at least about 80% amino acid sequence identity with a full-length CD79b polypeptide with native sequence as described herein, the sequence CD79b polypeptide that lacks the signal peptide, as described herein, an extracellular domain of CD79b polypeptide with a signal peptide, or without him, or any other fragment of a full-sized sequence CD79b polypeptide as described herein (such as fragments that are encoded by nucleic acid, which constitutes only a portion of the complete coding sequence of the full-sized CD79b polypeptide). Such options CD79b polypeptide include, for example, CD79b polypeptides, where added or removed one or more amino acid residues at the N - or C-end full-sized native amino acid sequence. Usually a variant of the CD79b polypeptide has at least about 80% amino acid sequence identity alternatively at least approx�an astounding 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a full-length CD79b polypeptide with native sequence as described herein, the sequence CD79b polypeptide that lacks the signal peptide, as described herein, an extracellular domain of CD79b polypeptide with a signal peptide, or without it, as described herein or any other specifically defined fragment of the full-size sequence CD79b polypeptide, as described herein. Usually versions of the CD79b polypeptide has a length of at least about 10 amino acids, alternative 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. Optional versions of the CD79b polypeptides contain no more than one conservative amino acid replacement compared to the CD79b polypeptide with native sequence, no more alternative 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to the CD79b polypeptide with native sequence.

"Percent (%) identity of amino acid sequences in the peptide sequence or �of polypeptide, i.e. sequences of CD79b polypeptides identified herein is defined as the percentage of amino acid residues in the sequence of the candidate that are identical with amino acid residues in a specific sequence of the peptide or polypeptide, i.e., the sequence CD79b polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the identity of the sequences. Alignment for purposes of determining percent identity of amino acid sequences can be performed by various methods known in this field, for example, using publicly available computer software such as software BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this field can determine appropriate parameters for performing the alignment, including any algorithms needed to achieve maximal alignment over the entire length of the compared sequences. However, for purposes herein, the % value of identity of amino acid sequences derived from a computer program to compare followers�of Inesta ALIGN-2, where is the full source code for the program ALIGN-2 is provided in table 1 below. A computer program to compare sequences ALIGN-2 was prepared at Genentech, Inc. and the source code shown in table 1, below, was presented with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The program ALIGN-2 is publicly available through Genentech, Inc., South San Francisco, California or you can compile from source code is presented in table 1, below. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All parameters of comparison sequences is established by the program ALIGN-2 and do not change.

In cases where ALIGN-2 is used for comparisons of amino acid sequences % identity of amino acid sequences for a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A that has a certain % amino acid sequence identity or contains % amino acid sequence identity to, with, or against a given amino acid sequence B), or calculated as follows:

100 multiplied by the quotient X/Y

where X represents the number of amino acid residues, evaluated as identical matches by the program to align sequences ALIGN-2 in the alignment of this program, A and B, and where Y represents the total number of amino acid residues in B. it is Clear that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % identity of amino acid sequence A to B will not equal the % identity of amino acid sequences B to A.

"Variant of the polynucleotide CD79b" or "variant nucleic acid sequence CD79b" means a nucleic acid molecule which encodes a CD79b polypeptide, preferably an active CD79b polypeptide, as defined herein and which has at least about 80% identity nucleic acid sequence with a nucleic acid sequence that encodes a full-sized sequence CD79b polypeptide with native sequence identified herein, full-sized sequence CD79b polypeptide with native sequence lacking the signal peptide as described herein, an extracellular domain of CD79b polypeptide with a signal peptide, or without, as described herein, or any others�GOI fragment of the full-size sequence CD79b polypeptide, as described herein (such as fragments that are encoded by a nucleic acid that represents only a portion of the complete coding sequence of the full-sized CD79b polypeptide). Typically, such variants of polynucleotides CD79b possess at least about 80% identity nucleic acid sequence, alternative, at least approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity nucleic acid sequence with a nucleic acid sequence that encodes a full-sized sequence CD79b polypeptide with native sequence, as described herein, a full-sized sequence CD79b polypeptide with native sequence lacking the signal peptide as described herein, an extracellular domain of CD79b polypeptide with a signal peptide, or without it, as described herein or any other fragment of a full-sized sequence CD79b polypeptide, as described herein. Options do not include the native nucleotide sequence.

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

"Percentage identity (%) of sequences of nucleic acids" in respect of nucleic acid sequences encoding CD79b identified herein is defined as the percentage of nucleotides in the sequence of the candidate that are identical with the nucleotides in interest with a nucleic acid sequence CD79b, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent identity of nucleic acid sequences can be performed in various ways that are within the special knowledge, for example, using publicly available computer software such as software about�AspectJ BLAST BLAST-2, ALIGN or Megalign (DNASTAR). However, for purposes herein, the % value identity of nucleic acid sequences is derived from a computer program to compare sequences ALIGN-2, where the full source code for the program ALIGN-2 is provided in table 1 below. A computer program to compare sequences ALIGN-2 was prepared at Genentech, Inc. and the source code shown in table 1, below, was presented with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The program ALIGN-2 is publicly available through Genentech, Inc., South San Francisco, California or you can compile from source code is presented in table 1, below. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All parameters of comparison sequences is established by the program ALIGN-2 and do not change.

In cases where ALIGN-2 is used for the comparison of nucleic acid sequences, percent identity of nucleic acid sequences for a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which may alternatively be phrased as a given nucleic acid sequence C, which� has or comprises a certain % identity nucleic acid sequence, with, or against a given nucleic acid sequence D) is calculated as follows:

100 multiplied by the quotient W/Z

where W represents the number of nucleotides, evaluated as identical matches by the program to align sequences ALIGN-2 in the alignment of this program C and D, and where Z represents the total number of nucleotides in D. it is Clear that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % identity nucleic acid sequence C to D will not equal the % identity nucleic acid sequence D relative to C. If not specifically stated otherwise, all values % identity to the nucleic acid sequences used in the present document, obtained, as described in the previous paragraph using the computer program ALIGN-2.

In other embodiments, variants of the polynucleotides CD79b are nucleic acid molecules that encode the CD79b polypeptide and which are capable of gibridizatsiya preferably under stringent conditions of hybridization and washing of nucleotide sequences encoding a full-CD79b polypeptide, as described herein. Versions of the CD79b polypeptide can be a polypeptide, which to�droutsa variants of polynucleotides CD79b.

The term "full-coding region" when used in relation to nucleic acid that encodes a CD79b polypeptide, refers to a sequence of nucleotides that encodes a full-sized CD79b polypeptide of the present invention (which is often symbolized by the initiating codon and stop codon, inclusive, in the accompanying drawings). The term "full-coding region" when used in relation to nucleic acid deposited with the ATCC, refers to the encoding of the CD79b polypeptide part of the cDNA, which is integrated into the vector deposited with the ATCC (which is often symbolized start codon and stop codon, inclusive, in the accompanying drawings (the initiating codon and the stop codon is indicated in bold and underlined in the figures)).

"Isolated", when applied to describe different CD79b polypeptides described herein, means polypeptide that is identified and separated and/or selected from a component of its natural environment. Contaminant components of its natural environment are materials that would normally preclude diagnostic and therapeutic use of the polypeptide, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, such polypeptides Bud�t clear (1) to the extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using the sequencer with rotating cups, or (2) to homogeneity in SDS-PAGE in reducing or Sevostyanova conditions using Kumasi blue or, preferably, silver staining. Such polypeptides selected include related polypeptides in recombinant cells ofin situas in this case, will be missing at least one component of the natural environment of the CD79b polypeptide. However, typically, such polypeptides can be obtained through at least one stage of cleaning.

"Isolated" nucleic acid that encodes a CD79b polypeptide, or nucleic acid that encodes another polypeptide, is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source encoding the polypeptide nucleic acid. The selected nucleic acid molecule that encodes the polypeptide is in a different form or terms than the form and context in which it occurs in nature. Thus, the selected nucleic acid molecule encoding a polypeptide different from the specific molecules well�Lanovoy acid, encodes a polypeptide that exists in natural cells. However, the selected nucleic acid molecule that encodes a polypeptide includes nucleic acid molecules that encode polypeptides that are contained in cells that normally Express the polypeptide where, for example, the nucleic acid molecule is in position on the chromosome that is different from the position on the chromosome in natural cells.

The term "sequence control" refers to DNA sequences necessary for expression of the functionally linked coding sequence in a particular organism, the host. Sequence control, which are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence and the site of binding of ribosomes. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "functionally linked" when it is in functional relationship with another nucleic acid sequence. For example, DNA for proposedvalue or secretory leader sequence functionally linked to DNA polypeptide if it is expressed as propelca, which is involved in secretion of the polypeptide; a promoter or enhancer is functionally linked to the coding �posledovatelnostyu, if it affects the transcription of the sequence; or the binding site of the ribosome is functionally linked to a coding sequence if it is positioned in such a way as to facilitate translation. As a rule, "functionally linked" means that the linked DNA sequences are contiguous, and, in the case of a secretory leader sequences are contiguous and are located in the reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation into the corresponding restriction sites. If such sites do not use synthetic oligonucleotide connectors or linkers in accordance with conventional practice.

"Rigidity" of hybridization reactions is easily determined by a person skilled in the art, and, as a rule, it is an empirical calculation dependent upon probe length, temperature of washing and salt concentration. As a rule, for proper annealing of longer probes require higher temperatures, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in the environment at a temperature below its annealing temperature. The higher is with�Epen desired homology between the probe and hybridizing sequence, the higher is the relative temperature that can be used. As a result, it follows that higher relative temperatures would provide the trend towards more stringent reaction conditions, while lower temperatures reduce the stiffness. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions" or "conditions of high stringency," as defined herein, can be defined as the conditions under which: (1) employ low ionic strength and high temperature for washing, for example, of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) use during hybridization a denaturing agent, such as formamide, for example, 50% (vol./about.) formamide with 0.1% bovine serum albumin/0.1% of Ficoll/0.1% polyvinylpyrrolidone/50 mm sodium phosphate buffer at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) hybridization is carried out overnight in a solution, which uses 50% formamide, 5 x SSC (0,75 M NaCl, of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5 x solution Denhardt, irradiated ultrasonic irradiation DNA salmon sperm (50 µg/ml), 0.1% of SDS and 10% dextran sulfate at 42°C, rinsed for 10 minutes at 42°C in 0.2 x SSC (chloride NAT�Oia/sodium citrate) followed by rinsing with high rigidity for 10 minutes consisting of 0.1 x SSC containing EDTA at 55°C.

"Conditions of moderate stringency" can be defined, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and they include the application of a less stringent solution for washing and hybridization conditions (e.g., temperature, ionic strength and %SDS) than the solution for washing and hybridization conditions described above. An example of moderately stringent conditions is incubation over night at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mm NaCl, 15 mm trinitrate sodium), 50 mm sodium phosphate (pH 7.6), 5 x solution Denhardt, 10% dextran sulfate and 20 mg/ml denatured cleaved DNA salmon sperm, followed by washing the filters in 1 x SSC at about 37-50°C. the Specialist will be clear how to adjust the temperature, ionic strength, etc., if necessary to adapt to factors such as probe length, etc.

The term "labeled epitope", when used in this document, refers to the chimeric polypeptide containing the CD79b polypeptide, or antibody against CD79b, fused with polypeptide-tagged". The polypeptide tag has enough residues to provide epitope against which it is possible to obtain an antibody, but at the same time is short enough not to interfere with the activity of the polypeptide with which it merged. Also polypep�d-mark preferably is completely unique so that the antibody essentially does not enter into cross-react with other epitopes. Suitable polypeptides tags, generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

"Active" or "activity" for the purposes herein refers to form(s) of the CD79b polypeptide which retains the biological or immunological activity of native or naturally occurring CD79b, where "biological" activity refers to a biological function (either inhibitory or stimulatory) provided by a native or naturally occurring CD79b distinct from ability to induce the formation of antibodies against an antigenic epitope possessed by a native or naturally occurring CD79b, and "immunological" activity refers to the ability to induce the formation of antibodies against an antigenic epitope, possessed by a native or naturally occurring CD79b.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or fully blocks, inhibits or neutralizes a biological activity of native CD79b polypeptide. Similarly, the term "agonist" is used in the most shirou�Ohm sense and includes any molecule that mimics a biological activity of native CD79b polypeptide. Suitable molecules agonists and antagonists specifically include antibodies, or antagonists antibodies, agonists, or fragments of these antibodies, fragments or variants amino acid sequence of native CD79b polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods of identifying antagonists or agonists of the CD79b polypeptide may include contacting CD79b polypeptide molecule with a candidate agonist or antagonist and measuring amenable to the detection of changes in one or more biological activities normally associated with a CD79b polypeptide.

"Purified" means that the molecule is present in the sample at a concentration of at least 95% by weight, or at least 98% by weight of the sample in which it is contained.

"Allocated" nucleic acid molecule is a nucleic acid molecule which is separated from at least one other nucleic acid molecule with which it is usually associated, for example, in its natural environment. The selected nucleic acid molecule, in addition, includes a nucleic acid molecule contained in cells that normally Express molecule nucleic� acid, but, a nucleic acid molecule is outside of the chromosome or in the region of the chromosome that differs from its natural location on the chromosome.

The term "vector" as used herein refers to a nucleic acid molecule capable transport another nucleic acid to which it is associated. One type of vector is a "plasmid", which refers to a circular double-stranded loop of DNA that can be ligitamate additional segments of DNA. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be ligitamate in the viral genome. Certain vectors are capable of Autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal vectors mammals). Other vectors (e.g., episomal vectors mammals) can be integrated into the genome of the host cell when introduced into a host cell, and, thus, they are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to herein as a "recombinant expression vectors" (or simply, "recombinant vectors"). As a rule, ex�rezerwuj vectors, used in recombinant DNA methods, often have the form of plasmids. In the present description, the terms "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

The term "polynucleotide" or "nucleic acid" as used herein interchangeably, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. Before and after Assembly of the polymer may occur modification of the nucleotide structure, if it occurs. The sequence of nucleotides may be interrupted dinucleotide components. In addition, the polynucleotide can be modified after polymerization, for example, by conjugation with carrying out the tagging component. Other types of modifications include, for example, "cap", substitution of one or more naturally occurring nucleotide analogue, magnolioideae modification, for example, modifications such as uncharged St�same (for example, methylphosphonate, phosphotriesterase, phosphoamide, carbamates, etc.) and with charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), modification exposed groups, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modification intercalators (e.g., acridine, psoralen, etc.), modification of the chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modification of alkylating compounds, modification with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). In addition, any hydroxyl group normally present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, they can be protected by protective groups, or activated to form additional linkages to additional nucleotides, or they can be anywhereman with solid or semi-solid substrates. 5' and 3'terminal OH can be fosforilirovanii or substituted with amines or organic cejrowski groups of from 1 to 20 carbon atoms. Other hydroxyls can also be converted to standard protecting groups. Polynucleotides can also contain analogous forms of ribose sugars� or deoxyribose, which is mainly known in this field, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-atidarymas, carbocyclic analogues of sugars, alpha-anomeric sugars, epimeria sugars, such as arabinose, xylose or lyxose, sugar pyranose, sugar furanose, sedoheptulose, acyclic analogs and basicosta nucleoside analogues, such as methylribose. One or more fosfolipidnyh ties can be replaced by alternative linker groups. These alternative linker groups include, but are not limited to, variants of implementation, where the phosphate is replaced by P(O)S ("tiat"), P(S)S ("ditial"), "(O)NR2("amidate"), P(O)R, P(O)OR', CO or CH2("Formatul"), where each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C) optionally containing simple ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not necessary that all of the links in the polynucleotide were identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

"Oligonucleotide", as used herein, refers mainly to short, generally single stranded, generally synthetic polynucleotides in length, typically, but not necessarily, less than 200 nucleotides. The terms "oligonucleotide�" and "polynucleotide" are not mutually exclusive. The above description of the polynucleotides is equally and fully applicable to oligonucleotides.

The terms "malignant tumor" and "malignant" refer to the physiological state or describe the physiological condition in mammals that is typically characterized by unregulated growth of cells. Examples of malignant tumors include, but are not limited to, hematopoietic malignant tumors or tumor-related blood malignancies such as lymphoma, leukemia, myeloma or lymphoid malignancies, and malignant tumors of the spleen and malignant tumors of the lymph nodes and carcinoma, blastoma, and sarcoma. More specific examples of malignant tumors include associated with B-cell malignancies, including for example, low-grade, intermediate-and high-grade lymphomas (including B cell lymphomas such as B-cell lymphoma associated with mucosa lymphoid tissue and nehodgkinski lymphoma (NHL), b-cell lymphoma mantle zone, Burkitt lymphoma, malcolmfeijten lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma and Hodgkin lymphoma, and T-cell lymphomas) and leukemias (including secondary leukemia, XP�technical lymphocytic leukemia (CLL), such as B cell leukemia (CD5+ B lymphocytes), myeloid leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia (ALL) and myelodysplasia), and other hematological and/or associated with B-cells or T-cells of a malignant tumor. Also included malignant tumors subsidiary of hematopoietic cells, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes and natural killer cells. Also included malignant B-cell proliferative disorders selected from the following: lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone. Sources of B-cell malignancies include the following: B-cell lymphoma marginal zone is derived from B-memory cells in the marginal zone, follicular lymphoma and diffuse large cell B-lymphoma originate from centrocytes in the light zone of germinal centers, chronic lymphocytic leukemia and IU�coletocna lymphocytic leukemia originate from B1 cells (CD5+), the lymphoma cells of the mantle zone is derived from naïve B-cells in the mantle zone, and Burkitt lymphoma occurs from centroblasts in the dark zone of germinal centers. Tissues, including hematopoietic cells, referred to herein as a "tissue stem cells" include the thymus and bone marrow and peripheral lymphoid tissues such as spleen, lymph nodes, lymphoid tissue associated with mucous membranes, such as those associated with intestinal lymphoid tissue, tonsils, Peyer's patches and the Appendix and lymphoid tissue associated with other mucous membranes, for example, with a lining of the bronchi. Other specific examples of such malignant tumors include 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, Mechanochemistry cancer, cancer of the gastrointestinal tract, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, cancer of the colon and rectum, carcinoma of the endometrium or uterine carcinoma of the salivary gland, kidney cancer, liver cancer, prostate cancer, cancer of the female external genital organs, thyroid cancer, carcinomas� liver, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

"B-cell malignant tumor" in this document includes nehodgkinski lymphoma (NHL), including low-grade/follicular NHL, malcolmfeijten NHL (SL), intermediate-differentiated/follicular NHL, intermediate-differentiated diffuse NHL, low-grade immunoblastic NHL, low-grade lymphoblastic NHL, low-grade small cell NHL with unsplit cores, NHL with a massive defeat cell lymphoma mantle zone that is associated with AIDS lymphoma, and waldenstrom's macroglobulinemia; nehodgkinski lymphoma (NHL), Hodgkin disease with a predominance of lymphocytes (LPHD), malcolmfeijten lymphoma (SLL), chronic lymphocytic leukemia (CLL), indolent NHL including recurrent indolent NHL and refractory to rituximab indolent NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; lymphoma cells, mantle zone; and other hematologic malignant tumors. These malignant tumors can be treated with antibodies directed against markers of B-cell surfaces, such as CD79b. This document provides that�their disease can be treated by administering antibodies, directed against a marker of B-cell surfaces, such as CD79b, and it includes administration of an unconjugated ("naked") antibody or an antibody conjugated with a cytotoxic agent, as described herein. Also in this document it is envisaged that such diseases can be treated with combined therapy, including antibody against CD79b or conjugate antibodies against CD79b with a medicament according to the invention in combination with another antibody or antibody conjugate and medicinal products, other cytotoxic agent, radiation therapy, or other method of treatment carried out simultaneously or sequentially. In the illustrative method of treatment according to the invention, the CD79b antibody against according to the invention is administered in combination with an antibody against CD20, immunoglobulin, or binding the CD20 fragment, either together or sequentially. The antibody against CD20 can be a “naked” antibody or conjugate of the antibody and the drug. In one of the embodiments of combination therapy, the antibody against CD79b is a fully human antibody of the present invention, and the antibody against CD20 is a Rituxan® (rituximab).

As used in the context of the present application, the term "nehodgkinski lymphoma" or "NHL" refers to malignant tumors of the lymphatic�tion system, non Hodgkin lymphomas. Hodgkin's lymphoma can be distinguished from non-Hodgkin's lymphomas, mainly, by the presence of cells, reed-Sternberg lymphomas Hodgkin's lymphoma and the absence of these cells in nehodgkinski lymphomas. Examples nehodgkinski lymphomas, which covers used in the present application the term include any kind of lymphoma, which the person skilled in the art (e.g., an oncologist or pathologist) can be defined as nahodkinskuju lymphoma in accordance with the classification systems known in this field, such as the Revised European-American classification of lymphomas (REAL), as described in Color Atlas of Clinical Hematology, (Third Edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited 2000). See, in particular Fig. 11.57, 11.58 and 11.59. More specific examples include, but are not limited to, recurrent or refractory NHL, borderline low-grade NHL, the NHL stage III/IV, resistant to chemotherapy NHL, lymphoblastic leukemia and/or lymphoma predecessors of B-cells, malcolmfeijten lymphoma, B-cell chronic lymphocytic leukemia and/or prolimfocitarnoj leukemia and/or malcolmfeijten lymphoma, B-cell prolimfocitarnuû lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, B-cell lymphoma marginal zone lymphoma marginal zone of the spleen, extranodal MALT lymphoma of marginal� zone hairy cell leukemia, plasmacytoma and/or plasmacytoma myeloma, low-grade/follicular lymphoma, intermediate-differentiated/follicular NHL, b-cell lymphoma, mantle zone lymphoma of the Central follicle (follicular), intermediate-differentiated diffuse NHL, diffuse large cell B-cell lymphoma, aggressive NHL (including borderline NHL with aggressive and recurrent aggressive NHL), NHL, retidivirovania after autologous stem cell transplant or resistant to it, primary mediastinal large cell B-cell lymphoma, primary effusion lymphoma, low-grade immunoblastic NHL, low-grade lymphoblastic NHL, low-grade small cell NHL with unsplit cores, NHL with a massive defeat, Burkitt's lymphoma, lymphoblastic leukemia and/or lymphoma of large granular precursors of T-cells (peripheral), mycosis fungoides and/or Sezary syndrome, lymphoma of the skin (cutaneous), anaplastic large cell lymphoma, angiocentric lymphoma.

"Violation" is any condition which can be useful in the treatment of substance/molecule or method of the invention. It includes chronic and acute disorders or diseases,�Lucie pathological conditions which predispose the mammal to the disorder. Non-limiting examples of disorders to be treated according to the present description, include malignant conditions, such as malignant and benign tumors; leukemias and lymphoid malignant tumors; neuronal, glial, astroglial, hypothalamic violations and violations of other glands, macrophagal, epithelial, stromal and blastocele disorders; and inflammatory, immunologic and other associated angiogenesis disorders. In addition, disorders include conditions, such as B-cell-proliferative disorders and/or B-cell tumors such as lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

The terms "cell-proliferative violation" and "proliferative violation" refers to disorders associated with abnormal to some extent, of cell proliferation. In one embodiment, the implementation, the cell-proliferative infringement is a malignant tumor.

<> "Tumor", as used herein, refers to any neoplastic cell growth and proliferation, both malignant and benign, and any predislocation and malignant cells and tissues.

"Autoimmune disease" herein is a disease or disorder arising in their own tissues of an individual and directed against them, or a symptom or manifestation or condition that occurs as its consequence. In many of these autoimmune and inflammatory disorders, there may be a number of clinical and laboratory markers, including, but not limited to, hand hypergammaglobulinemia, high levels of autoantibodies, deposition of antigen-antibody in the tissues, the benefits of treatment with corticosteroids or immunosuppressive agents, and aggregates of lymphoid cells in the affected tissues. Not limited to any theory concerning mediated B-cell autoimmune disease, it is believed that B-cells demonstrate a pathogenic effect in autoimmune human diseases using a variety of mechanical cascades, including the production of autoantibodies, formation of immune complexes, activation of dendritic cells and T-cells, synthesis of cytokines, which direct the release of chemokines, and providing focus for ectopic �of olimporte. Each of these cascades may be involved in the pathology of autoimmune diseases in various degrees.

"Autoimmune disease" may be organ-specific disease (i.e., the immune response is directed against specific organ systems such as the endocrine system, hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and liver systems, the renal system, the thyroid, the ears, the neuromuscular system, Central nervous system, etc.) or a systemic disease that can affect many organ systems (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, etc.). Preferably, such diseases include autoimmune rheumatologic disorders (such as rheumatoid arthritis, Sjogren syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid syndrome antibodies, and psoriatic arthritis), autoimmune gastrointestinal disorders and liver disorders (such as inflammatory bowel disease (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac enteropathy), vasculitis (for example, such as ANCA-negative�individual vasculitis and ANCA-associated vasculitis, including vasculitis, Cerca-Strauss, Wegener's granulomatosis and microscopic polyangiitis), autoimmune neurological disorders (such as multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, optic Milosevic, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as glomerulonephritis, goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as psoriasis, urticaria, urticaria, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as thrombocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusion purpura and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune disease hearing (such as a disease of the inner ear and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation, and autoimmune endocrine disorders (such as diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease and autoimmune thyroid disease (e.g., disease graves. and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated in�whines, lupus, multiple sclerosis, Sjogren's syndrome, the disease is graves., IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Specific examples of autoimmune diseases, as defined herein, which in some cases cover the diseases listed above, include, but are not limited to, arthritis (acute and chronic, rheumatoid arthritis including rheumatoid arthritis with juvenile early-stage arthritis, such as rheumatoid synovitis, arthritis gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis induced by collagen type II arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, still's disease, arthritis of the spine, osteoarthritis, chronic progredient arthritis, deforming arthritis, primary chronic polyarthritis, reactive arthritis associated with menopause arthritis that is associated with the depletion of estrogen arthritis 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, �AIRMATIC, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, urticaria, herpetiformis dermatitis, coin-like dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis and atopic dermatitis, x-linked Hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as Spino-optical MS, primary progressive MS (PPMS) and remitting clinical MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis and ataxic sclerosis, optic Milosevic (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease mediated by the immune system of the gastrointestinal tract, inflammation of the gastrointestinal tract, colitis such as ulcerative colitis,colitis ulcerosamicroscopic colitis, collagenosis colitis, polypoid colitis, necrotizing EN�erakala and transmural colitis, and autoimmune inflammatory bowel disease), inflammatory bowel disease, gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including respiratory distress syndrome in adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the choroid, inflammation of the iris, chorioidea, autoimmune hematological violation, graft versus host disease, angioedema such as hereditary angioedema, damage to cranial nerves, for example, in meningitis, herpes pregnancy, pemphigoid pregnant, itching of the scrotum, autoimmune premature ovulation disorder, sudden hearing loss due to an autoimmune condition, IgE mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic encephalitis or encephalitis of the brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, agranulocytosis autoimmune uveitis, glomerulonephritis (GN) with nephrotic syndrome and without it, such as chronic or acute glomerulonephritis such as primary GN, mediated by the immune system GN, membranous GN (membranous nephropathy), ID�Opticheskie membranous GN or idiopathic membranous nephropathy, membrane or membranosa-proliferative GN (MPGN), including type I and type II, and rapidly progressive GN, proliferative nephritis, autoimmune multiple insufficiency endocrine glands, balanitis, including restricted plasma cell balanitis, balanoposthitis, annular erythema, persistent diskhromicheskie erythema, polymorphic erythema, annular granuloma, shiny ringworm, sclerotic and atrophic lichen, simple chronic zoster, subulate zoster, oral lichen planus, lamellar ichthyosis, epidermolitichesky hyperkeratosis, predzakaznoy keratosis, gangrenous pyoderma, allergic conditions and responses, food allergies, allergies to medicines, allergies to insects, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, esteatose eczema, digitations eczema, and vesicular palmoplantar eczema, asthma such asasthma bronchiale, bronchial asthma and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory response, immune reactions against foreign antigens such as fetal blood group A-B-O in the process of pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, impaired leukocyte adhesion, lupus, vklyuchayuschey jade, lupus cerebri, childhood lupus, depositnow lupus, extrarenal lupus, discoid lupus or discoid lupus erythematosus with lupus alopecia, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus, juvenile diabetes mellitus (type I), including pediatric IDDM, adult diabetes (type II), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic violation of large arteries, immune responses associated with acute and delayed hypersensitivity, mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, agranulocytosis, vasculitis (including vasculitis of large vessels, such as polymyalgia rheumatica and giant cell arteritis (Takayasu), vasculitis of medium-caliber vessels, such as Kawasaki's disease and polyarteritis nodosa, immunovaccine, vasculitis in the Central nervous system, cutaneous vasculitis, at hypersensitivity vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis and systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as syndrome Cerca-Strauss (CSS), Wegener's granulomatosis and microscopic polyangiitis)the Viso�tion arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, anemia of Diamond-Blackfan, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), a deficiency of factor VIII, hemophilia A, autoimmune neutropenia, cytopenia, such as pancytopenia, leukopenia, diseases that involve diabetes of leukocytes, inflammatory disorders in the Central nervous system, Alzheimer's disease, Parkinson's disease, the syndrome of multiple organ failure, such as a secondary syndromes septicemia, trauma or hemorrhage, diseases, mediated by complexes of antigen-antibody, a disease associated with antibodies against the glomerular basement membrane, antiphospholipid syndrome, neuritis motor neurons, allergic neuritis, disease/Behcet syndrome, syndrome Castellana, goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson, pemphigoid or pemphigus such as bullous pemphigoid, cicatricial pemphigoid (pemphigoid mucous membranes), skin pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceous, pemphigus mucous membranes and erythematous pemphigus, acquired epidermolysis bullosa, vos�removing the eye, preferably, allergic inflammation of the eye such as allergic conjunctivitis, bullous disease associated with linear IgA, autoimmune inflammation of the conjunctiva, autoimmune polyendocrinopathy, disease or Reiter's syndrome, thermal powerscene due to an autoimmune condition, preeclampsia, immune complex violation, such as immune complex nephritis, mediated by antibodies jade, neuro-inflammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM mediated neuropathy, thrombocytopenia (e.g., such as occurs in patients with myocardial infarction), including thrombotic thrombocytopenic purple (TTP), posttransfusion purple (PTP), heparin-induced thrombocytopenia, and autoimmune or immunopositive or thrombocytopenia, for example, idiopathic thrombocytopenic purple (ITP) including chronic or acute ITP, scleritis such as idiopathic keratoconic, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, or subacute thyroiditis (Hashimoto's thyroiditis) or subacute tiroidite, autoimmune� disease of the thyroid gland, idiopathic hypothyroidism, disease graves., eye disease graves. (ophthalmopathy or associated with thyroid ophthalmopathy), polyglandular syndromes, such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as myasthenic syndrome Lambert-Eaton syndrome or Eaton-Lambert syndrome, muscle stiffness or stiff individual", encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as associated with thymoma myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (regardless of transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA-nephropathy), idiopathic IgA-nephropathy, linear IgA-dermatosis, acute febrile neutrophilic �armatos, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis, pneumocytes syndrome, autoimmune enteropathy, celiac disease, celiac disease, abdominal sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amyotrophic lateral sclerosis (ALS; disease Lou Gehring), coronary artery disease, autoimmune ear disease such as autoimmune disease of the inner ear (AIED), autoimmune hearing loss, polyhedra, such as refractory or recurrent polyhedric, pulmonary alveolar proteins, keratitis, such as syndrome Kogan/nezirroticski interstitial keratitis, bell's palsy, a disease/sweet syndrome, autoimmune red acne associated with shingles pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (e.g., benign a monoclonal gammopathy and a monoclonal gammopathy of uncertain significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, paroxysmal paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or PTS�volume amounting segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological violation, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalin, atrophy of the stomach, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic demyelinating polyneuropathy, Dressler syndrome, focal alopecia, total alopecia, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility, sclerodactyly and telangiectasia), male and female autoimmune infertility, e.g., due to antibodies against spermatozoidov, mixed connective tissue disease, Chagas ' disease, rheumatic attack, recurrent abortion, farmer's lung, poliformnaya erythema, postcardiotomy syndrome, Cushing's syndrome, pulmonary Allergy birders, allergic granulomatous vasculitis, benign lymphocytic vasculitis, alport syndrome, alveolitis such as allergic alveolitis and fibrosis alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, cyanosis, schistosomiasis, Ascaris, aspergillosis, syndrome Sumpter syndrome Kaplan, dengue, endocarditis, endomyocardial fibrosis, diffuse interest�social pulmonary fibrosis, interstitial lung fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, elevated persistent erythema, hemolytic disease of the newborn, eosinophilic fasciitis, Shulman syndrome, felty's syndrome, filarias, cycle, such as chronic cycle, heterochronies cycle, iridocyclitis (acute or chronic), or cycle Fuchs, purple Schonlein purpura-Henoch, infection with human immunodeficiency virus (HIV), SCID, acquired immune deficiency syndrome (AIDS), infection by echoviruses, sepsis (systemic inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis, Tiresias, infection with parvovirus, infection by rubella virus infection, post-vaccination syndrome, congenital infection with rubella virus, infection with Epstein-Barr, mumps, Evans syndrome, autoimmune insufficiency of sexual glands, Sydenham chorea, post-streptococcal nephritis, obliterating thromboangiitis, thyrotoxicosis, tabes dorsalis spinal cord, chorionic, the giant cell polymyalgia, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, keratoconjunctivitis sicca, and epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, nephropathy with minimal changes, damage, benign familial and ischemia-reperfusion, reperfu�Oia transplanted organ, the autoimmune reaction in the retina, joint inflammation, bronchitis, chronic obstructive Airways disease/lung, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular insufficiency of the brain) such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermatogenic, autoimmune hemolysis, Beck disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylaxis endophthalmitis, allergic enteritis, nodular leprosy leprosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, a disease of Hemmen-rich, sensorineural hearing loss, the paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis,ophtalmia symphatica(sympathetic ophthalmia, ophthalmia neonatorum, optic neuritis, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, quervain thyroiditis, acquired atrophy of the spleen, cancerous lymphoma, lymphovascular thymus, vitiligo, toxic shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypercalcemia�the nost, mediated by cytokines and T-lymphocytes, diseases involving diabetes leukocyte syndrome, multiple organ lesions, mediated by complexes of antigen-antibody diseases, diseases directed against the membrane of the glomeruli, autoimmune polyneuropathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insult, polyendocrine failure, autoimmune polyglandular syndromes, including autoimmune polyglandular syndrome type I, idiopathic adult hypoparathyroidism (AOIH), cardiomyopathy with dilatation, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, sensut ethmoid bone, frontal bone, maxilla or of the sphenoid bone, associated with eosinophils violation, such as eosinophilia, eosinophilia with infiltration into the lungs, syndrome of eosinophilia-myalgia, Loeffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or containing granuloma eosinophils, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerotium�th cholangitis, chronic mucocutaneous candidiasis sclera and episclera syndrome, Bruton, transient gammaglobulinemia newborns, Wiskott-Aldrich, ataxia-telangiectasia, autoimmune disorders associated with collagen disease, rheumatism such as chronic astronautalis, lymphadenitis, reduction of blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic disorders associated with hypersensitivity, glomerulonephritis, ischemic reperfusion violation, reperfusion injury of the myocardium and other tissues, lymphomatosis tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, cortical necrosis of the kidneys, acute purulent meningitis or other inflammatory disorders of the Central nervous system, ocular and orbital inflammatory disorders, syndromes, associated with the transfusion of granulocytes induced by cytokine toxicity, narcolepsy, acute severe inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis. This document provides that such diseases can be treated by introducing a�of tetela, directed against a marker of B-cell surfaces, such as CD79b, and it includes administration of an unconjugated ("naked") antibody or an antibody conjugated with a cytotoxic agent, as described herein. Also in this document it is envisaged that such diseases can be treated with combined therapy, including antibody against CD79b or conjugate antibodies against CD79b with a medicament according to the invention in combination with another antibody or antibody conjugate and medicinal products, other cytotoxic agent, radiation therapy, or other method of treatment carried out simultaneously or sequentially.

"Treatment" or "treatment" or "alleviation" refers to both therapeutic and prophylactic or preventative measures, where the goal is to prevent or slow down (lessen) scheduled a pathological condition or violation. Individuals in need of treatment include individuals already having a violation, as well as individuals having a predisposition for a disorder, or individuals whose violation is subject to prevention. The individual or mammal is successfully "treated" from expressing CD79b polypeptide malignant tumors, if, after receiving a therapeutic amount of antibodies against CD79b methods according to the present Fig�structure, the patient observed a significant and/or measurable reduction in or absence of one or more of the following: reducing the number of malignant cells or absence of cancer cells; reduce the tumor size; inhibition (i.e., slow to some extent and preferably stop) infiltration of cancer cells into peripheral organs including the spread of a malignant tumor in the soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) metastasis of tumors; inhibition, to some extent, of tumor growth; and/or mitigate, to some extent, of one or more symptoms, associated with the specific cancer; reduced morbidity or mortality, and improving quality of life. Depending on whether the CD79b antibody against to prevent the growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Decrease these signs or symptoms can also be felt by the patient.

The above parameters for assessing successful treatment and improvement of the disease is easy to identify by the conventional methods known to therapist. For therapy of malignant tumors, the efficiency may be determined, for example, by estimating in�Emini to disease progression (TTP) and/or determine the speed of the response (RR). Metastasis can be determined by determining the stage of disease and by bone scan and test the level of calcium and other enzymes to determine the distribution in the bone. Also search for distribution in the pelvis and lymph nodes in this area you can get a CT-image. A chest x-ray and measuring the levels of liver enzymes known methods used to search for metastases in lungs and liver, respectively. Other conventional methods of monitoring disease include transrectal ultrasound echography (TRUS) and transrectal needle biopsy (TRNB).

For bladder cancer, which is a more localized malignant tumor, methods of determining the progression of the disease include cytologic evaluation of the urine cystoscopy, monitoring the presence of blood in the urine, visualization tract mucosa was found with ultrasound or intravenous pyelography, computed tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastases can be evaluated by CT abdomen, chest x-ray or radionuclide imaging of the skeleton.

"Long" introduction refers to the introduction of the means(means) constantly as opposed to short-term mode to donate�rivate initial therapeutic effect (activity) for a long period of time. "Preryvayuscheesya" introduction is a treatment that is not carried out without interruption, but instead is cyclic.

"Individual" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, Pets (such as cats, dogs and horses), primates, mice and rats. In certain embodiments, the mammal is man.

"Mammal" for purposes of treatment, alleviating symptoms of malignant tumor refers to any animal classified as a mammal, including humans, domestic and farm animals, and animals to zoos, sports animals, or pet animals, such as dogs, horses, cats, cows, pigs, rabbits, etc Preferably, the mammal is a human.

Introduction "in combination with" one or more medicinal agents includes simultaneous (joint) and sequential introduction in any order.

As used herein, "carriers" include pharmaceutically acceptable carriers, excipients or stabilizers which are nontoxic to the cell or mammal, subjected� their effects the used dosages and concentrations. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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; chelating agents such as EDTA; sugars alcohols, such as mannitol or sorbitol; soleobrazutaya counterions, such as sodium; and/or nonionic surfactants such as TWEEN®the polyethylene glycol (PEG) or PLURONICS®.

By "solid phase" or "solid substrate" refers to a non-aqueous matrix to which an antibody against CD79b can be attached or contact. Examples of the solid phase, covered by this document include the solid phase formed partially or entirely of glass (such as glass with controlled pore size), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In about�certain embodiments, depending on the context, the solid phase can include a hole tablet for analysis; in other embodiments, it is a column for purification (for example, a column for affinity chromatography). Also, this term includes dispersed solid phase of discrete particles, such as solid phase, as described in U.S. patent No. 4275149.

"Liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactants that are suitable for drug delivery (such as antibodies against CD79b) to a mammal. Components of liposomes usually form a two-layer structure, similar to the placement of the lipids of biological membranes.

"Low molecular weight" connection or "low molecular weight organic compound, as defined herein, has a molecular weight less than about 500 daltons.

"Individual", "subject" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, Pets (such as cats, dogs and horses), primates, mice and rats. In certain embodiments, the mammal is che�avec.

The term "pharmaceutical composition" refers to a drug that has such a form to ensure the effectiveness of the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to the individual to which the composition will be administered. Such a composition may be sterile.

"Sterile" composition is aseptic or contains living microorganisms and their spores.

"Effective amount" of an antibody described herein is an amount sufficient for the implementation of specific goals. "Effective amount" can be determined empirically and generally accepted ways, depending on the goal.

The term "therapeutically effective amount" refers to the amount of antibody or other drug effective to "treat" a disease or disorders in an individual or mammal. In the case of a malignant tumor, a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) infiltration of cancer cells into peripheral organs; inhibit (i.e., slow to some extent and presupposes�plant stop) tumor metastasis; to inhibit, to some extent, tumor growth; and/or alleviate to some extent one or more symptoms associated with malignant tumor. Cm. the definition of "treatment" in this document. Depending on whether the drug is to inhibit growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. "Prophylactically effective amount" refers to the amount effective in the required dosages and within the required time periods to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in individuals before disease or in its early stages, the prophylactically effective amount will be less than therapeutically effective amount.

"Growth inhibitory amount" of an antibody against CD79b is an amount capable of inhibiting growth of a cell, especially tumor, e.g., cancer cells, orin vitroorin vivo. "Growth inhibitory amount" of an antibody against CD79b for the purposes of inhibiting neoplastic cell growth may be determined empirically and in a common manner.

"Cytotoxic amount" of an antibody against CD79b represents the number of capable C�th destruction of cells, especially tumor, e.g., cancer cells, orin vitroorin vivo. "Cytotoxic amount" of an antibody against CD79b for the purposes of inhibiting neoplastic cell growth may be determined empirically and in a common manner.

"Expressing CD79b cell" is a cell that expresses endogenous or transfetsirovannyh CD79b polypeptide either on the cell surface or in a secreted form. "Expressing CD79b malignant tumor is a malignant tumor containing cells that have a CD79b polypeptide presented on the cell surface or that produce and secrete a CD79b polypeptide. "Expressing CD79b malignant tumor" optionally produces sufficient levels of CD79b polypeptide on the surface of its cells, so antibodies against CD79b could contact him and have a therapeutic effect against malignant tumors. In another embodiment of the implementation, "expressing CD79b malignant tumor" optionally produces and secretes sufficient levels of CD79b polypeptide, the antibody antagonist against CD79b could contact him and have a therapeutic effect against malignant tumors. Regarding the latter, the antagonist may be an antisense, oligonuclear�ID, which reduces, inhibits or prevents production and secretion of secreted CD79b polypeptide by tumor cells. Malignant tumor that "sverkhekspressiya" CD79b polypeptide is a tumor that has, or produces and secretes, significantly higher levels of CD79b polypeptide on its cell surface, compared to non-cancerous cell tissue of the same type. Such overexpression may be caused by gene amplification or by increased transcription or translation. Sverkhekspressiya CD79b polypeptide can be identified in the analysis for the detection or prognosis by evaluating increased levels of CD79b protein, present on the cell surface, or secreted by the cell (for example, using immunohistochemical analysis using antibodies against CD79b received allocated against CD79b polypeptide, which can be obtained using recombinant DNA technology from an isolated nucleic acid that encodes a CD79b polypeptide; FACS analysis, etc.). Alternative or additionally, it is possible to measure the levels of the coding CD79b polypeptide nucleic acid or mRNA in the cell, e.g. via fluorescent hybridizationin situusing probe-based nucleic acid corresponding to the coding CD79b nucleic acid or the complementary�Oh her sequence; (FISH; see WO98/45479 published October, 1998), southern blotting, Northern blotting or polymerase chain reaction (PCR), such as quantitative PCR with detection in real-time (RT-PCR). You can also explore sverkhekspressiya CD79b polypeptide by measuring the unattached cells from the antigen in a biological fluid such as serum, e.g., using assays based on antibodies (also see, e.g., U.S. patent No. 4933294, issued June 12, 1990; WO91/05264 published April 18, 1991; U.S. patent 5401638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above tests, the specialist in this field are available in various analyses ofin vivo. For example, you can affect cells in the body of the patient an antibody, which is not necessarily labeled amenable to detection label, e.g. a radioactive isotope, and it is possible to evaluate the binding of an antibody to a patient's cells, e.g., by external scanning for radioactivity or by analyzing biopsy taken from a patient who previously worked the antibody.

As used herein, the term "immunoadhesin" means such antibody molecules that combine the binding specificity of a heterologous protein ("adhesin") with the effector functions of the constant domain of immunoglobulins. �tructure immunoadhesin contain amino acid sequence with the desired binding specificity, different from the site of antigen recognition and binding site of an antibody (i.e., is "heterologous"), merged with the sequence of the constant domain of immunoglobulin. The adhesin part in 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 can be obtained from any immunoglobulin, isotypes such as IgG-1, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

The word "label" when used herein refers to a measurable detection compound or composition that anywherevery directly or indirectly with the antibody, to form a "labeled" antibody. The label itself may be amenable to detection (e.g.,radioisotope labels or fluorescent labels) or, in the case of enzyme tags, it may catalyze chemical alteration of a compound or composition of the substrate that are amenable to detection.

The term "cytotoxic agent" as used herein refers to a substance that inhibits the functioning of cells, or prevented, and/or causes destruction of cells. Assume that the term includes radioactive isotopes (e.g. At211, Isup> 131, I125, Y90That Re186That Re188Sm153, Bi212P32and radioactive isotopes of Lu), chemotherapeutic agents, e.g., methotrexate, adriamycin, Vinca alkaloids (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 of bacteria, fungi, plants or animals, including fragments and/or variants, and various antineoplastic or anti-malignant tumor, as described below. Other cytotoxic funds described below. Destroys tumor cells means causes destruction of tumor cells.

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

"Chemotherapeutic agent" is a chemical compound useful in the treatment of malignant tumors, regardless of the mechanism of its action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, poisonous to the mitotic spindle, the plant alkaloids, cytotoxic/antitumor antibiotics, inhibitors topos�meraz, antibodies, photosensitizers, inhibitors and kinases. Chemotherapeutic agents include compounds used in "targeted therapies" in the conventional chemotherapy. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (CIS-diamine,dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N. J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentacapital[4.3.0]Nona-2,7,9-trien-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-differbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.

Other examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesilate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASARTM, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), g�fitini (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRATM, Johnson & Johnson), ABRAXANETM(that does not contain cremophor), designed to albumin composition of paclitaxel nanoparticles (American Pharmaceutical Partners, Schaumberg, Illinois), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478 effect, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), infospeed (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXANE®, NEOSAR®); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridine, such as bestop, carboquone, maturidade and uredia; ethylenimine and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin and bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its synthetic analogues on the basis of adozelesin, carzelesin and bizelesin); cryptophycin (in particular cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogenous IPrice, such as chlorambucil, chlornaphazine, chlorpropamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novemberin, finestein, prednimustine, trofosfamide, uracil mustard; nitrosoanatabine, such as ka�mustin, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as antianemia antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega (Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, dynemicin A; bisphosphonates, such as clodronate; spiramycin; and chromophore-based neocarzinostatin and similar chromoproteins chromophores based indinavir antibiotics), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, carminomycin, calcination, chromomycin, dactinomycin, daunorubicin, doctorovich, 6-diazo-5-oxo-L-norleucine, morpholinopropan, cyanomethaemoglobin, 2-pyrrolidinecarbonyl and desoxidation), epirubicin, zorubicin, idarubicin, marsellaise, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; analogs of purines, such as fludarabine, 6-mercaptopurine, timipre, thioguanine; analogues of pyrimidines, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, laxiflora�Jn, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenergic means, such as aminoglutetimid, mitotane, trilostane; funds to replenish folic acid, such as prolinnova acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diaziquone; alternity; slipline acetate; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; PSK polysaccharide complex® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin a and unguided); urethane; vindesine; dacarbazine; minomycin; Metaponto; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin�; to produce remissions in childhood; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also in the definition of "chemotherapeutic agent" includes: (i) anti-hormonal remedies, which act by regulating or inhibiting hormone action on tumors such as antiestrogens and selective modulators of estrogen receptors (SERM), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, for example, such as 4(5)-imidazolov, aminoglutetimid, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamid, bikalutamid, leuprolide and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); (iv) inhibitors of protein kinases, such as MEK inhibitors (WO 2007/044515); (v) inhibitors of lipid kinases; (vi) antisense oligonucleotides, particularly oligonucleotides that inhibit expressiion cascades of signal transmission, involved in altered cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as inhibitors of expression of VEGF (e.g., ANGIOZYME®) and inhibitors of HER2 expression; (viii) vaccines such as vaccines for gene therapy, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase inhibitors 1, such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also in the definition of "chemotherapeutic agent" includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Ide), pertuzumab (OMNITARGTM, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and a conjugate of the antibody and the drug, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

"Growth inhibitory agent", when used in this document, refers to the compound or compositions that inhibit cell growth, particularly expressing CD79b cancerous cells, orin vitroorin vivo. Thus, the growth inhibitory agent can be a tool that significantly reduces the percentage of expressing CD79b cells in S-phase. Examples of inhibiting the growth of funds incl�Ute funds which block the cell cycle progression (at a place other than S phase), such as means that induce a stop in G1-phase and a stop in M-phase. Classic blockers M-phases include the Vinca alkaloids (vincristine and vinblastine), taxanes and topoisomerase inhibitors II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those tools that cause a stop in G1-phase, also involve a stop in S-phase, 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 Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. Taxanes (paclitaxel and docetaxel) are the drugs against malignant tumors 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 provides Assembly of microtubules from tubulin dimers and stabilize microtubules, preventing depolymerization, which leads to inhibition of mitosis in cells.

"Doxorubicin" is an anthracycline antibiotic. Full chemical n�the title of doxorubicin is (8S-CIS)-10-[(3-amino-2,3,6-trideoxy-α-L-oxohexanoyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetic)-1-methoxy-5,12-naphthacenedione.

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, human growth hormone N-metionina 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); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and-β; inhibiting substance Mueller; gonadotropin-associated peptide of the mouse; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; 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, IL9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of cytokines with a native sequence.

The term "liner in the package" is used to refer to the instructions that are usually included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, introduction, contraindications and/or warnings concerning the use of such therapeutic products.

The term "intracellular metabolite" refers to a compound formed during the metabolic process or reaction of the conjugate antibody-drug inside the cell (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of the peptide linker in the ADC, or the hydrolysis of functional groups, such as hydrazon, ester or amide. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after penetration, diffusion, capture, or transport into the cell.

The terms "nutriclean� cleaved" and "intracellular cleavage" refer to a metabolic process or reaction inside a cell on the conjugate antibody-drug (ADC), by which covalent bond is destroyed, i.e., the linker, between the group of the drug (D) and the antibody (Ab), causing dissociation of the free drug from the antibody inside the cell. Split ADC group, thus, are intracellular metabolites.

The term "bioavailability" refers to the systemic availability (i.e., levels in blood/plasma) a given quantity of the drug, the patient. Bioavailability is an absolute indicator for introduced dosed form, which indicates the measure as time (speed) and General colista (extent) of drug that reaches

the General circulation.

The term "cytotoxic activity" refers to the effect of killing cells, cytotoxic or growth inhibitory effect of the ADC, or an intracellular metabolite of the ADC. The cytotoxic activity may be expressed as the value of IC50which represents the concentration (molar or mass) per unit volume, which survives half of the cells.

The term "alkyl", as used herein, refers to a saturated linear or branched monovalent hydrocarbon radical of one to twelve carbon atoms (C1-C12), where the alkyl radical optionally can� to be independently substituted by one or more substituents, described below. In another embodiment, the implementation, the alkyl radical has from one to eight carbon atoms (C1-C8), or from one to six 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, -CH(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-pentyl (-CH(CH3)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) ), 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" refers to linear or branched monovalent hydrocarbon radical of two to eight carbon atoms (C2-C8in at least one site of unsaturation, i.e. a carbon-carbon sp2-double bonds, where the alkenyl radical optionally may be independently substituted by one or more substituents described herein, 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=CH2) and etc.

The term "alkenyl" refers to linear or branched monovalent hydrocarbon radical of two to eight carbon atoms (C2-C8in at least one site of unsaturation, i.e. a carbon-carbon sp triple bond, where alkynylaryl radical optionally may be independently substituted by one or more substituents described herein. Examples include, but are not limited to, ethinyl (-C≡CH), PROPYNYL (propargyl, - CH 2C≡CH), etc.

The terms "carbocycle", "carbocyclic", "carbocyclic ring" and "cycloalkyl" refer to a monovalent non-aromatic, saturated or partially unsaturated ring having 3 to 12 carbon atoms (C3-C12) in the form of a monocyclic ring or 7 to 12 carbon atoms in the bicyclic ring. Bicyclic carbocycle, having from 7 to 12 atoms can be represented, for example, as a bicyclo system[4,5], [5,5], [5,6] or [6,6] bicyclic carbocycles having 9 or 10 atoms in the ring can be represented as a system of bicyclo [5,6] or [6,6], or as a system with bridges, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, 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.

"Aryl" means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (C6-C20) formed by removal of one hydrogen atom from a single carbon atom of the original aromatic ring system. Some of the aryl groups present illustrative structures as "Ar". Aryl includes the BIC�licencie radicals, containing an aromatic ring condensed with a saturated, partially unsaturated or aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, radicals formed from benzene (phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl etc Aryl group optionally independently substituted with one or more substituents described herein.

The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used herein interchangeably and they refer to a saturated or partially unsaturated (i.e., having one or more double and/or triple bonds in the ring) carbocyclic radical of 3 to 20 atoms in the ring, in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur and the remaining ring atoms are C, where one or more atoms in the ring optionally substituted independently with one or more substituents, described below. Heterocycle may be a monocycle having 3 to 7 members in the ring (from 2 to 6 carbon atoms and from 1 to 4 heteroatoms selected from N, O, P and S), or Bicycle, having from 7 to 10 members in the ring (4 to 9 carbon atoms and 1 to 6 GE�of eroatoms, selected from N, O, P and S), for example: a bicyclo system[4,5], [5,5], [5,6], or [6,6]. 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. "Heterocyclyl" also includes radicals where heterocyclic radicals are fused with a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuran, tetrahydrothieno, tetrahydropyranyl, dihydropyran, tetrahydrothiopyran, piperidine, morpholino, thiomorpholine, dioxane, piperazinyl, homopiperazine, azetidine, oxetane, titanyl, homopiperazine, oxetanyl, tepanil, oxazepines, diazepines, thiazepines, 2-pyrrolidyl, 3-pyrrolidyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-DIOXOLANYL, pyrazolyl, dithienyl, dithiolane, dihydropyran, dihydrothieno, dihydrofuran, pyrazolopyrimidines, imidazolidinyl ureido, 3-azabicyclo[3.1.0]hexenyl, 3-azabicyclo[4.1.0]heptenyl, azabicyclo[2.2.2]hexanal, 3H-indolealkylamines and N-peredelnye compounds of urea. Also in the scope of this definition includes spirography. Examples g�eroticlinks group, where 2 carbon atoms of the ring substituted by exography (=O), are pyrimidinones and 1,1-diocletianopolis. Heterocyclic group as described herein, optionally substituted independently by one or more substituents described herein.

The term "heteroaryl" refers to a monovalent aromatic radical of 5-, 6 - or 7-membered rings, and includes a condensed ring systems (at least one of which is aromatic) of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridine), imidazolyl, imidazopyridines, pyrimidinyl (including, for example, 4-hydroxypyrimidine), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolin, pyrrolyl, chinoline, ethenolysis, indole, benzimidazole, benzofuranyl, indolinyl, indazoles, indolizinyl, phthalazine, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinol, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furutani, benzofurazanyl, benzothiophenes, benzothiazolyl, benzoxazolyl, chinazoline, chinoxalin, naphthyridines and Phenoperidine. Heteroaryl group optionally independently substituted for one or more�estately, described in this document.

Heterocyclic or heteroaryl groups may be bound atoms of carbon (carbon-linked), or nitrogen (nitrogen-linked) whenever possible. As a non-limiting example associated carbon atoms of the heterocycles or heteroaryl connected in 2, 3, 4, 5 or 6 position of pyridine, 3, 4, 5 or 6 position of pyridine, 2, 4, 5 or 6 position of the pyrimidine, 2, 3, 5, or 6-position of the pyrazine, 2, 3, 4 or 5 position of the furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, 2, 4, or 5 position of oxazole, imidazole or thiazole, 3, 4 or 5 position of isoxazole, pyrazole or isothiazole, 2 or 3 position of aziridine, 2, 3 or 4 position of azetidine, 2, 3, 4, 5, 6, 7 or 8 position of the quinoline or 1, 3, 4, 5, 6, 7 or 8-position of the isoquinoline.

As a non-limiting example associated with nitrogen heterocycles or heteroaryl linked in the 1st position 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, 2 position isoindole or isoindoline, 4-position of the morpholine, and 9-position of carbazole or β-carboline.

"Alkylene" refers to a saturated branched or unbranched or cyclic hydrocarbon rediculous 1-18 carbon atoms, having two centers in the form of a monovalent radical formed by removing two hydrogen atoms from one or two different carbon atoms of the original alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-) 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-) and so on.

"C1-C10alkylene" represents an unbranched saturated hydrocarbon group of the formula -(CH2)1-10. Examples of C1-C1Oalkylene include methylene, ethylene, propylene, butylene, pentile, exile, reptile, octile, Nonlin and decalin.

"Albaniles" refers to an unsaturated branched or unbranched or cyclic hydrocarbon radical of 2-18 carbon atoms having two centers in the form of a monovalent radical formed by removing two hydrogen atoms from the same or two different carbon atoms of the original alkene. Typical alkenylamine radicals include, but are not limited to: 1,2-ethylene (-CH=CH-).

"Akinyan" refers to an unsaturated branched or unbranched or cyclic hydrocarbon radical of 2-18 carbon atoms having two centers in the form of a monovalent radical formed by removing two hydrogen atoms from the same or two different carbon atoms�Yes source alkyne. Typical alkenylamine radicals include, but are not limited to: acetylene (-C≡C-), propargyl (-CH2C≡C-), and 4-pentenyl (-CH2CH2CH2C≡C-).

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

in which the phenyl group can be unsubstituted or substituted by up to four groups including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -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" refers to an acyclic alkyl radical in which one of the hydrogen atoms associated with carbon atom, typically a limit or sp3-a 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, an alkyl group, including albanello, alkenyl or alkylamino group, arylalkyl �the group has from 1 to 6 carbon atoms, and the aryl group has from 5 to 14 carbon atoms.

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

The term "prodrug", as used in this application refers to the form of a precursor or derivative compounds according to the invention, which may be less cytotoxic to cells compared to the reference compound or the drug and can be enzymatically or hydrolytically activated� or converted into the more active original 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 this invention include, but are not limited to, phosphate prodrug containing thiophosphate prodrug containing sulfate prodrugs containing peptide prodrug, the prodrug with modified D-amino acids, glycosylated prodrugs containing β-lactam prodrug containing optionally substituted phenoxyacetamide prodrug containing optionally substituted phenylacetamide prodrug, the prodrug 5-fertilizing and other prodrugs of 5-ptoluidine that can turn into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be converted into a prodrug form for use in this invention include, but are not limited to, compounds according to the invention and chemotherapeutic agents such as described above.

"Metabolite" is a product formed during metabolism in the body a certain compound or its salts. Metabolites of compounds can be identified using conventional methods known in this field, and their activestore determined using tests such as the tests described in this document. Such products can be formed, for example, oxidation, recovery, hydrolysis, Amidala, datamodule, esterification, deesterification, enzymatic degradation, etc., permission for the connection. Thus, the invention includes metabolites of the compounds according to the invention, including compounds produced by a process comprising contacting the compounds according to this invention with a mammal for a time period sufficient for the formation of its metabolic product.

"Liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactants that are suitable for drug delivery to a mammal. Components of liposomes usually form a two-layer structure, similar to the placement of the lipids of biological membranes.

"Linker" refers to a chemical group that contains a covalent bond or a chain of atoms that covalently bind the antibody with a group of medicines. In various embodiments, linkers include a divalent radical such as alkerdeel, areldil, heteroaryl, groups such as -(CR2)nO(CR2)n- repeating elements of alkyloxy (e.g., polietilene, PEG, polymethyl�noxy) and alkylamino (for example, polyethylenimine, JeffamineTM); and esters and amides of dicarboxylic acids, including succinate, succinamide, diglycolate, malonate and caproamide.

The term "chiral" refers to molecules which have the property mismatch with counterparts that are mirror images, while the term "achiral" refers to molecules that are identical to their counterparts that are mirrored mappings.

The term "stereoisomers" refers to compounds which have identical chemical structure, but differ in the arrangement of atoms or groups in space.

"Diastereoisomer" refers to a stereoisomer with two or more chiral centers, whose molecules are not mirror images each other. The diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties and reactivity. Mixtures of diastereoisomers can be divided by analytical high-resolution methods, such as electrophoresis and chromatography.

"Enantiomers" refer to two stereoisomers of compounds that are not matching mirror images each other.

Stereochemical definitions and conventions used in this document, largely correspond to the S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) Mcraw-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 have the ability to rotate the plane pleapositive light. In describing an optically active compound, the prefixes D and L or R and S, are used to denote the absolute configuration of the molecule in the field of its chiral center(s). Prefixes d and l or (+) and (-) are used to designate the sign of rotation pleapositive light by the compound, with (-) or l means that the compound is levorotatory. Connection with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images each other. A specific stereoisomer may also be designated as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. The mixture of enantiomers is 50:50 is called a racemic mixture or a racemate, which may occur only when there is no stereoselective or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric compounds, devoid of optical activity.

The term "tautomer" or "tautomeric form" refers to structural isomers� with different energy which are vzaimoprevrascheny with low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversion by migration of a proton, such as keto-enol and Yiming-Eminova isomerization. Valence tautomers include interconversion through the reorganization of several electrons >

The expression "pharmaceutically acceptable salt", as used herein, refers to pharmaceutically acceptable organic or inorganic salts of the compounds according to the invention. Illustrative salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfate, phosphates, acidic phosphates, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, Pantothenate, bitartrate, ascorbates, succinates, maleate, entityname, fumarate, gluconate, glucuronate, saharty, formiate, benzoates, glutamate, methanesulfonate "mesylate", econsultancy, benzolsulfonat, p-toluensulfonate and pamoate (i.e., 1,1'-methylene-bis(2-hydroxy-3-aftout)). Pharmaceutically acceptable salt can include another molecule, such as acetate ion, succinate ion or other counterion. The counterion may be any organic or inorganic group which stabilizes the charge �and the original connection. Moreover, pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, there may be multiple counterions. Thus, 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 can be obtained by any suitable method available in this area, for example, by treatment of the free base with an inorganic acid such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, etc., or organic acid such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, paranoidly acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, aromatic acid, such as benzo�of ina acid or cinnamic acid, sulfonic acid such as p-toluensulfonate acid or econsultancy acid, or similar.

If the connection according to the invention is an acid, the desired pharmaceutically acceptable salt can be obtained by any suitable method, for example, by treatment of the free acid inorganic or organic base such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide or similar. Illustrative examples of suitable salts include, but are not limited to, organic salts formed 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 formed from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The expression "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients contained in the composition, and/or mammals exposed to treatment them.

"Solvate" refers to an Association or complex of one or more solvent molecules and compounds according to the invention. Examples of solvents that form solvates include, but are not limited to �mi, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to the complex where the solvent molecule is water.

The term "protective group" refers to the Deputy, which is usually used to block or protect certain functional groups during the reaction of other functional groups on the compound. For example, "protective group for amino is a Deputy that is associated with the amino group that blocks or protects the functional amino group in the compound. Suitable protection of the amino group include acetyl, triptorelin, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethoxycarbonyl (Fmoc). Similarly, the "protective group of hydroxy" refers to the Deputy hydroxy-group that blocks or protects the functional hydroxy-group. Suitable protective groups include acetyl and silyl. "Protective group of carboxy" refers to the Deputy carboxypropyl that blocks or protects the functional group carboxymethyl. Common protective groups include carboxymethyl vinylsulfonate, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluensulfonyl)ethyl, 2-(p-nitrobenzylidene)ethyl, 2-(diphenylphosphino)ethyl nitroethyl, etc. For a General description of the protective groups and their use, see T. W. Greene, Protetive Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

"Leaving group" refers to a functional group which can be substituted by another functional group. Certain leaving groups are well known in this field, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluensulfonyl (tosyl), triftormetilfullerenov (triflic) and triftormetilfullerenov.

Reduction

LINKER COMPONENTS:

MC = 6-maleimidomethyl

Val-Cit or "vc" = valine-citrulline (illustrative dipeptide split in the protease linker)

Citrulline = 2-amino-5-freidapinto acid

PAB = p-aminobenzeneboronic (example "smoothshapes" 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 = (can be linked to cysteine residues of the antibody).

CYTOTOXIC drugs:

MMAE = monomethylaniline E (MW 718);

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

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

MMAF-TEG = MMAF with tetraethylene glycol, esterified phenylalanine

MMAF-NtBu = N-t-butyl associated � as an amide to C-by the end of MMAF

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

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

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

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

"Free amino acid residue cysteine" refers to amino acid residue of cysteine, which is built into factory�e antibody has Tilney functional group (-SH) and is coupled via an intramolecular or intermolecular disulfide bridge.

The term "magnitude of the reactivity of the thiol group" represents a quantitative characterization of the reactivity of free amino acid residues of cysteine. The magnitude of the reactivity of thiol groups is the percentage of free amino acid residues of cysteines in the antibody with built in his cysteine residues, which react with reactive toward thiol group reagent, and converted to the maximum value of 1. For example, the free amino acid residue of cysteine on the antibody with built in his cysteine residues, which responds with a 100% yield from reactive to thiol group reagent, such as Biotin-maleimide reagent, with the formation of the Biotin-labeled antibody, is the magnitude of the reactivity of thiol groups, component of 1.0. Another amino acid residue cysteine embedded in the same or another source antibody, which reacts with a reactive to thiol group reagent with yields of 80%, has a value of the reactivity of thiol groups, component of 0.8. Another amino acid residue cysteine embedded in the same or another source antibody that does not react with reactive toward thiol group reagent, has the magnitude of the reactivity of thiol groups, component 0. Determination of the reactivity of the thiol group to a specific cysteine residue can be performed by ELISA, mass spectrometry, liquid chromatography, radioautography or other quantitative analytical tests.

"Reference antibody" is an antibody containing the amino acid sequence in which one or more amino acid residues is replaced by one or more cysteine residues. Source antibody may contain the native sequence or wild-type sequence. The original antibody may have pre-existing modifications of the amino acid sequence (such as insertions, deletions and/or substitutions) relative to another native form, the wild-type form or a modified form of the antibody. The original antibody may be directed against the interest of the antigen target, e.g., a biologically important polypeptide. Also provides antibodies directed against polipeptidnyh antigens (such as associated with tumor glycolipid antigens; see US 5091178).

III. Compositions and methods of the invention

The invention relates to antibodies against CD79b or their functional fragments, and to method of their use for the treatment of hematopoietic tumors.

In one aspect, the invention relates to an antibody which binds, preferably specifically with any of the above or below polypeptides. Optionally, the antibody is a monoclonal antibody, a fragment of the antibody, including Fab, Fab', F(ab')2- and Fv-fragment, ditelo, single-domain antibody, a chimeric antibody, a humanized antibody, single-chain antibody or antibody that inhibits competitive binding of antibodies against CD79b polypeptide with its corresponding antigenic epitope. The antibodies of the present invention can optionally be anywhereman with a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, auristatin, maitan�inoid, derived dolastatin or calicheamicin, an antibiotic, a radioactive isotope, nucleotidase enzyme or similar. The antibodies of the present invention optionally can be produced in CHO cells or bacterial cells and preferably they induce cell death, with which they are associated. For the purposes of detection, the antibodies of the present invention can be labeled amenable to the detection of the label to associate with a firm pad or similar.

In one aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the monovalent affinity of the antibody to CD79b (e.g., affinity antibodies against CD79b in the form of a Fab-fragment) is essentially the same as monovalent affinity of the mouse antibody (e.g., affinity of the antibody against mouse CD79b in the form of a Fab fragment) or a chimeric antibody (e.g. affinity of the chimeric antibody against CD79b in the form of a Fab-fragment, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the monovalent affinity of the antibody to CD79b (e.g., affinity antibodies against CD79b in the form of a Fab-fragment) is lower, for example at least 1, 2 or 3 �Aza below than the monovalent affinity of the mouse antibody (e.g., affinity of the antibody against mouse CD79b in the form of a Fab fragment) or a chimeric antibody (e.g. affinity of the chimeric antibody against CD79b in the form of a Fab-fragment, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the monovalent affinity of the antibody to CD79b (e.g., affinity antibodies against CD79b in the form of a Fab-fragment) exceeds, for example at least 1, 2 or 3 times, the monovalent affinity of the mouse antibody (e.g., affinity of the antibody against mouse CD79b in the form of a Fab fragment) or a chimeric antibody (e.g. affinity of the chimeric antibody against CD79b in the form of a Fab-fragment, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In one aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of a fragment of IgG) is essentially the same as the affinity of the mouse antibody (for example, affine�you mouse antibodies against CD79b in the form of a fragment of IgG) or a chimeric antibody (e.g., affinity chimeric antibodies against CD79b in the form of a fragment of IgG) in its bivalent form, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of a fragment of the IgG is lower, for example at least 1, 2 or 3 times lower than the affinity of the mouse antibody (e.g., affinity of the antibody against mouse CD79b in the form of a fragment of IgG) or a chimeric antibody (e.g. affinity of the chimeric antibody against CD79b in the form of a Fab-fragment) in its bivalent form, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of a fragment of IgG) exceeds, for example, at least 1, 2 or 3 times higher than the affinity of the mouse antibody (e.g., affinity of the antibody against mouse CD79b in the form of a fragment of IgG) or chimeric EN�of icela (e.g. affinity of the chimeric antibody against CD79b in the form of a Fab-fragment) in its bivalent form, containing the sequence of the variable domain of the light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14), comprising or essentially consisting of them.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 2.0 nm. In the following aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 2.0 nm +/- 0.5 in.

In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 1 nm or higher. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 1.5 nm or higher. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 2 nm or greater. In another aspect, the invention relates to humanitarianlaw EN�Italo against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 2.5 nm or above. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is 3 nm or higher. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is between 1 nm and 3 nm. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is between 1.5 nm and 2.5 nm. In another aspect, the invention relates to humanitarianlaw the antibody against CD79b, where the affinity of the antibody in its bivalent form to CD79b (e.g., affinity antibodies against CD79b in the form of IgG) is between 1,75 nm and 2.25 nm.

In one aspect, the monovalent affinity of the antibody against mouse CD79b is substantially the same as the affinity of binding of the Fab-fragment, containing sequences of variable domains of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B). In another aspect, the monovalent affinity of the antibody against mouse CD79b is a mere�TSS is the same as the affinity of binding of the Fab-fragment, containing sequences of variable domains of antibodies obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006, or a chimeric antibody containing the variable domains of the antibodies obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006.

As installed in the field, the binding affinity of a ligand with its receptor can be determined using any of the many tests, and expressed using a variety of quantitative values. Thus, in one embodiment, the implementation, the binding affinity expressed as Kd values and it reflects the actual binding affinity (e.g., with minimized the avidity effects). Typically and preferably, the binding affinity is measuredin vitroor in cell-free conditions or in related cells conditions. As described in more detail herein, the ratio of the difference in binding affinity can be measured quantitatively in the values of the ratio of the magnitude of the monovalent affinity of binding of the humanized antibody (e.g., in Fab form) and the value of the monovalent affinity of binding of a reference/comparative antibody (e.g., in Fab form) (for example, mouse antibody having donor sequence hypervariable region�TEI), where the magnitude of the affinity of binding is determined in the same conditions of analysis. Thus, in one embodiment of the implementation, fold difference in binding affinity is defined as the ratio of Kd values humanized antibody in Fab form and a specified benchmark/comparative Fab antibodies. For example, in one embodiment, the implementation, if an antibody of the invention (A) has affinity that is "3 times lower than the affinity of a reference antibody (M), then if the Kd value for A is 3x, the value of Kd for M is 1x, and the ratio of Kd of A to Kd of M is 3:1. In contrast, in one embodiment, the implementation, if an antibody of the invention (C) has an affinity that is "3 times higher than the affinity of a reference antibody (R), then if the Kd value for C is 1x, the value of Kd for R is 3x, and the ratio of Kd of C to Kd of R is 1:3. To obtain indicators of affinity of binding you can use any of a variety of assays known in this field, including the assays described herein, including, for example, Biacore, radioimmunoassay analysis (RIA) and ELISA.

In one aspect provides an antibody that binds to CD79b, where the antibody comprises: (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-A16 is a KSSQSLDSDGKTYLN (SEQ ID NO:59)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:60)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a WQGTHFPYT (SEQ ID NO:61)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:62)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD (SEQ ID NO:63), and

(vi) HVR-H3 containing the sequence F1-F6, where F1-F10 is a ARNLYL (SEQ ID NO:64).

In one embodiment, the implementation, HVR-L1 of an antibody of the invention contains the sequence of SEQ ID NO:59. In one embodiment, the implementation, HVR-L2 of an antibody of the invention contains the sequence of SEQ ID NO:60. In one embodiment, the implementation, HVR-L3 of an antibody of the invention contains the sequence of SEQ ID NO:61. In one embodiment, the implementation, HVR-H1 of an antibody of the invention contains the sequence of SEQ ID NO:62. In one embodiment, the implementation, HVR-H2 of an antibody of the invention contains the sequence of SEQ ID NO:63. In one embodiment, the implementation, HVR-H3 of an antibody of the invention contains the sequence of SEQ ID NO:64. In one embodiment, the implementation, the antibody according to the invention containing these sequences (in combination as described herein), is a humanized antibody or a human antibody.

In one aspect provides an antibody that concerned�tsya with CD79b, where the 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-A16 is a KSSQSLLDSDGKTYLN (SEQ ID NO:59)

(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:60)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 is a WQGTHFPYT (SEQ ID NO:61)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:62)

(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD (SEQ ID NO:63), and

(vi) HVR-H3 containing the sequence F1-F6, where F1-F10 is a ARNLYL (SEQ ID NO:64); and

(b) at least one variant HVR, where the sequence of the variant HVR contains a modification of at least one residue of the sequence represented in SEQ ID NO.: 59, 60, 61, 62, 63 or 64. In one embodiment, the implementation, HVR-L1 of an antibody of the invention contains the sequence of SEQ ID NO:59. In one embodiment, the implementation, HVR-L2 of an antibody of the invention contains the sequence of SEQ ID NO:60. In one embodiment, the implementation, HVR-L3 of an antibody of the invention contains the sequence of SEQ ID NO:61. In one embodiment, the implementation, HVR-H1 of an antibody of the invention contains the sequence of SEQ ID NO:62. In one embodiment, the implementation, HVR-H2 of an antibody of the invention contains after�outermost SEQ ID NO:63. In one embodiment, the implementation, HVR-H3 of an antibody of the invention contains the sequence of SEQ ID NO:64. In one embodiment, the implementation, the antibody according to the invention containing these sequences (in combination as described herein), is a humanized antibody or a human antibody.

In one aspect, the invention relates to an antibody that contains one, two, three, four, five or six HVR, where each HVR contains a sequence selected from the group consisting of SEQ ID NO: 59, 60, 61, 62, 63 and 64, consists of or essentially consists of, and where SEQ ID NO:59 corresponds to an HVR-L1, SEQ ID NO:60 corresponds to an HVR-L2, SEQ ID NO:61 corresponds to an HVR-L3, SEQ ID NO:62 corresponds to an HVR-H1, SEQ ID NO:63 corresponds to an HVR-H2, and SEQ ID NO:64 corresponds to an HVR-H3. In one embodiment, the implementation, the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each, in order, contains SEQ ID NO: 59, 60, 61, 62, 63 and 64.

Options HVR in the antibody according to the invention can have modifications of one or more residues within the HVR. In one embodiment of the implementation, a variant HVR-L3 contains 1-2 (1 or 2) substitutions in any combination of the following positions: C1 (F) and C8 (F). Letter(s) in parenthesis following each position indicates an illustrative substitution (i.e., replacement) amino acid; as would be clear to the person skilled in the art, suitability of other amino acids as amino acids for Deputy�cies in the context described herein can be assessed using conventional methods known in this field and/or described herein. In one embodiment, the implementation, C1 in a variant HVR-L3 is an F. In one embodiment, the implementation, C8 in a variant HVR-L3 is an F. In one embodiment of the antibody according to the invention contains a variant HVR-L3, where C1 represents F and C8 is an F.

In one embodiment, the implementation, the antibody according to the invention contains a variant HVR-L3, where C1 represents F. In one embodiment, the implementation, the antibody according to the invention contains a variant HVR-L3, where C8 is a F. In some embodiments, the implementation, the specified antibody with a variant HVR-L3 further contains HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, where each of them contains, in this order, the sequence represented in SEQ ID NO: 59, 60, 62, 63 and 64. In some embodiments, these antibodies further contain a consensus sequence frame region of the heavy chain subgroup III person. In one embodiment, the implementation of these antibodies, the consensus sequence of frame area contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is a, A, position 73 is a T and/or position 78 is an A. In one variant�in the implementation of these antibodies, these antibodies further contain a consensus sequence of frame area κI light chain of a human.

In one aspect, the invention relates to an antibody containing sequence HVR presented on figure 9 (SEQ ID NO:18).

A medicament for use in the individual-the owner preferably causes a small immunogenic the answer or not, against funds in specified individual. In one embodiment of implementation, the invention relates to such a tool. For example, in one embodiment of implementation, the invention relates to humanitarianlaw the antibody that causes and/or is expected to cause the response of human antibodies against mouse antibodies (HAMA) is essentially reduced in comparison with the antibody containing the sequence of SEQ ID NO: 10 and 14 in the individual host. In another example, the invention relates to humanitarianlaw the antibody that causes and/or suspected of causing minimal antibody response of human anti-mouse antibodies (HAMA), or not. In one example, the antibody of the invention induces an antibody response against the mouse, which is clinically acceptable level or below.

A humanized antibody according to the invention may contain one or more sequences negitively areas (for example, frame areas) human Il� consensus sequences negitively areas of human rights in its variable domain of the heavy or light chain. In some embodiments, in order negitively areas (for example, frame areas) of a person or consensus sequences negitively areas of a person shows one or more additional modifications. In one embodiment, the implementation, the variable domain of the heavy chain of the antibody according to the invention contains a consensus sequence of frame area of man, which in one embodiment, the implementation represents a consensus sequence of frame area of the subgroup III. In one embodiment of the antibody according to the invention contains a variant of the consensus sequence of frame area of the subgroup III, the modified at least one amino acid position. For example, in one embodiment of the implementation, a variant of the consensus sequence of frame area of the sub-group III may contain substitution at one or more of the provisions of 71, 73 and/or 78. In one embodiment, the implementation, the specified replacement is an R71A, N73T and/or L78A, in any combination.

As is known in the art and as described in more detail herein below, the amino acid position/boundary determining hypervariable region of the antibody can vary, depending on the context and the various definitions known�on the Internet in this area (as described below). Some provisions in the variable domain can be considered as a hybrid hypervariable position in the sense that these provisions can be considered specific to hypervariable region under one set of criteria, and count is not specific to hypervariable region with a different set of criteria. One or more of these provisions can also be extended hypervariable regions (as further defined below). The invention relates to antibodies containing modifications in these hybrid hypervariable positions. In one embodiment, the implementation of these hypervariable provisions include one or more of the provisions 26-30, 33-35B, 47-49, 57 through 65, 93, 94, 101-102 in the variable domain of the heavy chain. In one embodiment, the implementation of these hybrid hypervariable provisions include one or more of the provisions 24-29, 35-36, 46-49, 56 and 97 in the variable domain light chain. In one embodiment, the implementation, the antibody according to the invention contains a variant of the consensus sequence of frame area of human rights, modified in one or more hybrid hypervariable positions.

In one aspect, the antibody according to the invention contains a variable domain of the heavy chain containing the variant consensus sequence of frame area of the subgroup III of man, modified�th in one or more of the provisions 26-30, 33-35, 48-49, 58, 60-63, 93 and 101.

In one aspect, the antibody according to the invention contains a variable domain light chain containing a variant of the consensus sequence of frame area of the sub-group I human Kappa, modified in one or more of the provisions of 24, 27-29, 56 and 97.

In one aspect, the antibody according to the invention contains a variable domain of the heavy chain containing the variant consensus sequence of frame area of the subgroup III, modified in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or all of the provisions 26-30, 33-35, 48-49, 58, 60-63, 93 and 101. In some embodiments, the antibody of the invention contains a variant of the consensus sequence of frame area of the subgroup III, modified at position 71, 73 and/or 78. In one embodiment, the implementation, the specified replacement is an R71A, N73T and/or L78A.

In one aspect, the antibody according to the invention contains a variable domain light chain containing a variant of the consensus sequence of frame area of the sub-group I human Kappa, modified 1, 2, 3, 4, 5 or all of the provisions of 24, 27-29, 56 and 97.

The antibody according to the invention can contain any suitable sequence frame region light chain of a human or a consensus sequence of frame area of the light chain of a human, provided that the antibody has required�bubbled biological characteristics (e.g., desired binding affinity). In one embodiment, the implementation, the antibody according to the invention contains at least a portion of the sequence of frame area of the K light chain (or all of it). In one embodiment, the implementation, the antibody according to the invention contains at least a part of the consensus frame region K subgroup I (or all).

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

In one aspect, an antibody of the invention is a humanized antibody against CD79b, anywhereman with a cytotoxic agent. In one aspect, a humanized antibody against CD79b, anywhereman with a cytotoxic agent inhibits tumor progression in xenograft.

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

In one aspect, the invention relates to an antibody containing the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC presented on figure 13 (SEQ ID NO:31-33). In one embodiment, the implementation, the variable domain contains the sequence F1-HC, FR2-HC, FR3-HC and/or FR4-HC presented on figure 13 (SEQ ID NO:27-30). In one embodiment, the implementation, the antibody contains a sequence of CH1 and/or Fc, is presented in figure 13 (SEQ ID NO:34-35). In one embodiment, the implementation, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 13, SEQ ID NO:31-33), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 13, SEQ ID NO:27-30). In one embodiment, the implementation, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 13, SEQ ID NO:31-33), and the sequence of the CH1 and/or Fc, is presented in figure 13 (SEQ ID NO:34-35). In one embodiment, the implementation, the antibody according to the invention contains a variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and/or HVR3-HC (figure 13, SEQ ID NO:31-33), and the sequence of the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC (figure 13, SEQ ID NO:27-30), and the CH1 and/or Fc (figure 13, SEQ ID NO:34-35).

In one aspect, the invention relates to antibodies�, contains a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC presented on figure 13 (SEQ ID NO:23-25). In one embodiment, the implementation contains a variable domain sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 13 (SEQ ID NO:19-22). In one embodiment, the implementation, the antibody contains a sequence CL1 presented on figure 13 (SEQ ID NO:26). In one embodiment, the implementation, the antibody according to the invention contains a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO:23-25), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO:19-22) presented on figure 13. In one embodiment, the implementation, the antibody according to the invention contains a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO:23-25), and CL1 sequence (SEQ ID NO:26), presented on figure 13. In one embodiment, the implementation, the antibody according to the invention contains a variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and/or HVR3-LC (SEQ ID NO:23-26), and the sequence FR1-LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO:19-22) presented on figure 13, and CL1 sequence presented in figure 13 (SEQ ID NO:26).

In one aspect, the antibodies of the invention include antibodies with the built-in cysteine residues, where one or more amino acids of the original antibody for�enemy free residue of the amino acid cysteine, as described in WO2006/034488; US 2007/0092940 (incorporated herein as references in full). Thus can be designed, i.e., subjected to mutations, any form antibodies against CD79b. For example, the original Fab-fragment antibodies can be modified by engineering methods, to form Fab with built in his cysteine residues, referred to herein as "thio-Fab". Similarly, the monoclonal antibody can be modified by engineering methods so that it formed the "thio-Mab". It should be noted that the mutation in one plot leads to one integrated cysteine residue in thio-Fab, while in thio-Mab mutation in one area leads to two built-in ways of engineered cysteine residues, due to the dimeric structure of the IgG antibody. Antibodies against CD79b with the built-in cysteine residues according to the invention include monoclonal antibodies, humanized or chimeric monoclonal antibodies, and antigen-binding fragments of antibodies, fused polypeptides and analogs that preferentially bind associated with the cell CD79b polypeptides. Antibody with built in his cysteine residues alternative may include an antibody that contains a cysteine in position, as described herein, the antibody or Fab, thanks to the construction sequence and/or selection �Titel, without necessarily changing the original antibody, such as the design and selection of antibodies using phage display or designde novosequence frame region and constant regions of light chain and/or heavy chain. Antibody with built in his cysteine residues contains one or more free amino acid residues of cysteine having the magnitude of the reactivity of thiol groups in the range from 0.6 to 1.0; from 0.7 to 1.0 or 0.8 to 1.0. Free balance the amino acid cysteine is a cysteine residue, which is embedded in the ways of engineering in the original antibody and is not part of a disulfide bridge. Antibodies with the built-in cysteine residues suitable for accession to cytotoxic and/or imaging compounds in the area of the built-cysteine via, for example, maleimide or haloacetic. Nucleophilic reactivity of the thiol functional group of the Cys residue in relation maleimide group approximately 1000 times higher than that of any functional group of another amino acid in the protein, such as amino group of lysine residues or N-terminal amino group. Specific to the thiol group of the functional group in iodoacetyl and maleimide reagents can react with amino groups, however, require a higher pH (>9,0) and a longer reaction time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

In one aspect, a CD79b antibody against with built in his cysteine residues according to the invention contains a built-in ways engineered cysteine in any of the following provisions, where the position is numbered according to Kabat light chain (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) and according to EU numbering in the heavy chain (including the Fc-region) (see Kabat et al. (1991), above), where the constant area light chain indicated by underlining in figure 17A and 18A, starts at position 109 (Kabat numbering), and the constant plot heavy chain, indicated by the underline in the figures 17B and 18B, begins at position 118 (EU numbering). Also the position can be designated by its position in the numbering sequence for a full-sized amino acid light chain and the heavy chain shown in figures 17-18. According to one embodiment of the invention, the CD79b antibody against contains embedded in it the cysteine at LC-V205C (number according to Kabat: Val 205; number by sequence 210 in figure 18A, the modified methods engineering at Cys in this position). Built-cysteine in a light chain is indicated in bold double underline in figure 18A. According to one variant of implementation, the CD79b antibody against has an internal cysteine at HC-A118C (number EU: Ala 118; �Omer Kabat 114; number by sequence 118 figure 17B, the modified methods engineering at Cys in this position). Built-cysteine in the heavy chain is shown in bold double underline in figure 17B. According to one embodiments, the antibody against CD79b has an internal cysteine in the Fc-S400C (number EU: Ser 400; Kabat number 396; number by sequence 400 figure 17-18B, modified by Cys in this position). In other embodiments, the integrated cysteine of the heavy chain (including the Fc region) is located in any of the following provisions (according to the numeration according to Kabat EU numbering given in brackets): 5, 23, 84, 112, 114 (118 when EU numbering), 116 (120 in EU numbering), 278 (282 in EU numbering), 371 (375 in EU numbering) or 396 (400 in EU numbering). Thus, changes of amino acids in these positions for the source of humanized antibodies against CD79b according to the invention are: V5C, A23C, A84C, S112C, A114C (A118C in EU numbering), T116C (T120C in EU numbering), V278C (V282C in EU numbering), S371C (S375C in EU numbering) or S396C (S400C in EU numbering). Thus, changes of amino acids in these positions for a source of chimeric antibodies against CD79b according to the invention are: Q5C, K23C, S84C, S112C, A114C (A118C in EU numbering), T116C (T120C in EU numbering), V278C (V282C in EU numbering), S371C (S375C in EU numbering) or S396C (S400C in EU numbering). In other embodiments,the integrated light chain cysteine is found in any of the following provisions (according to the numbering according to Kabat): 15, 110, 114, 121, 127, 168, 205. Thus, changes of amino acids in these positions for the source of humanized antibodies against CD79b according to the invention are: V15C, V110C, S114C, S121C, S127C, S168C or V205C. Thus, changes of amino acids in these positions for a source of chimeric antibodies against CD79b according to the invention are: I15C, V110C, S114C, S121C, S127C, S168C or V205C.

In one aspect, the invention includes antibodies against CD79b with built in his cysteine residues, containing one or a few amino acid residues of cysteine, where antibodies against CD79b with built in his cysteine residues binds to a CD79b polypeptide and is obtained by a process comprising replacing one or more amino acid residues of the original antibody against CD79b on cysteine in which the original antibody comprises at least one HVR sequence selected from:

(a) HVR-L1 containing the sequence A1-A15, where A1-A16 is a KSSQSLLDSDGKTYLN (SEQ ID NO:59)

(b) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:60)

(c) HVR-L3, containing the sequence C1-C9, where C1-C9 is a WQGTHFPYT (SEQ ID NO:61) or FQGTHFPFT (SEQ ID NO:79)

(d) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:62)

(e) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD(SEQ I NO:63), and

(f) HVR-H3 containing the sequence F1-F6, where F1-F10 is a ARNLYL (SEQ ID NO:64).

In a specific aspect, the invention relates to antibodies against CD79b with built in his cysteine residues containing the amino acid sequence having at least about 80% identity amino acid sequence, alternative, at least approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the identity of the amino acid sequence of the antibody with built in his cysteine residues, having a full-sized amino acid sequence as described herein, or an antibody with built in his cysteine residues, having an amino acid sequence lacking the signal peptide as described herein.

In another aspect, the invention relates to the selected antibody against CD79b with built in his cysteine residues containing the amino acid sequence that is encoded by a nucleotide sequence which hybridise with the molecule, the complementary DNA molecule that encodes (a) an antibody with built in his cysteine residues, having a full-sized amino acid sequence as described herein, (b) an antibody with built in his remnants of cysts�ina, having an amino acid sequence lacking the signal peptide as described herein (c) an extracellular domain of a transmembrane protein antibodies with built in his cysteine residues with a signal peptide, or without it, as described in this document (d) the amino acid sequence encoded by any of the nucleic acid sequences described herein, or (e) any other specifically defined fragment of a full-sized amino acid sequence of the antibody with built in his cysteine residues, as described herein.

In a particular aspect, the invention relates to the selected antibody against CD79b with built in his cysteine residues without N-terminal signal sequence and/or without the first methionine encoded by a nucleotide sequence that encodes such an amino acid sequence as described herein. Its production method is also described herein, and these methods include cultivating a host cell containing a vector that contains the corresponding encoding the nucleic acid molecule under conditions suitable for expression of antibodies with built in his cysteine residues, and isolation of antibodies with built in his remnants of cysts�ina from cell culture.

Another aspect of the invention relates to the selected antibody against CD79b with built in his cysteine residues, in which the transmembrane domain is either removed or inactivated. Its production method is also described herein, and these methods include cultivating a host cell containing a vector that contains the corresponding encoding the nucleic acid molecule under conditions suitable for expression of antibodies with built in his cysteine residues, and isolation of antibodies with built in his cysteine residues from cell culture.

In other aspects, the invention relates to isolated chimeric antibodies against CD79b with the built-in cysteine residues, comprising any of the products described in this document antibodies with built in his cysteine residues, fused to a heterologous polypeptide (not CD79b). Examples of such chimeric molecules comprise any of the products described in this document antibodies with the built-in cysteine residues, fused with a heterologous polypeptide, such as sequence epitope tag or an Fc-region of immunoglobulin.

Antibodies against CD79b with built in his cysteine residues may be a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-stranded EN�Italo or antibody, which competitive inhibits the binding of antibodies against CD79b polypeptide with its corresponding antigenic epitope. The antibodies of the present invention can optionally be anywhereman with 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 similar. The antibodies of the present invention optionally can be produced in CHO cells or bacterial cells and preferably they inhibit the growth or proliferation of the cell with which they are associated, or induce cell death. For diagnostic purposes, the antibodies of the present invention can be labeled amenable to detection label associated with the solid substrate, or similar.

In other aspects of the present invention, the invention relates to vectors containing DNA encoding any of the products described in this document antibodies against CD79b and antibodies against CD79b with the built-in cysteine residues. Also provides the host cell containing any such vector. As an example, the host cell may be a CHO cell, the cells ofE. colior yeast cells. Also provides the process of any of the products described in this �we polypeptides and it includes culturing host cells under conditions suitable for expression of the desired polypeptide, and secretion of the desired polypeptide from the cell culture.

Antibodies with the built-in cysteine residues may be suitable for the treatment of malignant tumors and include antibodies specific to cell surface receptors and transmembrane receptors, and associated with tumor antigens (TAA). Such antibodies can be used in the form of simple antibodies (unconjugated with drug or group of tags) or in the form of conjugates antibody-drug (ADC). Antibodies with the built-in cysteine residues according to the invention can be site-specific and effectively communicate with reactive toward thiol group reagent. Reactive to thiol group reagent may be a multifunctional linker reagent, the reagent labels for capture, the fluorophore reagent or intermediate connection of a drug-linker. Antibody with built in his cysteine residues can be labeled amenable to detection label, immobilizative on a solid phase substrate and/or konjugierte with a group of medicines. The reactivity of the thiol group can be shared to any antibody where substitution of amino acids reac�active amino acids cysteine within the light chain, selected from the ranges of amino acid residues: L10-L20, L105-L115, L109-L119, L116-L126, L122-L132, L163-L173, L200-L210; and within the heavy chain selected from amino acid residues ranges: H1-H10, H18-H28, H79-H89, H107-H117, H109-H119, H111-H121, and in the Fc region within the ranges selected from H270-H280, H366-H376, H391-401, where the amino acid numbering of provisions begins in 1 position according to 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 further proceeds as described in WO2006034488; US 2007/0092940. The reactivity of the thiol group can also be shared on specific domains of the antibody, such as a constant domain of the light chain (CL) and constant domains of the heavy chain, CH1, CH2 and CH3. Replacement of cysteine, leading to the values of the reactivity of thiol groups, amounting to 0.6 and more, 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 the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Such antibodies and their use is described in WO2006/034488; US 2007/0092940.

Antibodies with the built-in cysteine residues according to the invention preferably retain the antigen-binding ability of their counterparts representing the original wild-type antibody. Thus, antibodies with the built-in cysteine residues able to bind, preferably specifically with the Antiga�AMI. Such antigens include, for example, associated with tumor antigens (TAA), proteins of cell surface receptors and other cell surface molecules, transmembrane proteins, proteins of signal transmission, regulatory factors of survival of cells, regulatory factors of cell proliferation, molecules associated with growth and differentiation of tissues (e.g., known or believed they participate in them functionally), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in the formation of blood vessels, and molecules associated with angiogenesis (e.g., known or guessing they are involved in it). Associated with a tumor antigen may be a cluster differentiation factor (i.e., protein CDS, including, but not limited to CD79b). Antibodies against CD79b with the built-in cysteine residues according to the invention retain the antigen-binding ability of their counterparts representing the source CD79b antibody. Thus, antibodies against CD79b with the built-in cysteine residues according to the invention is able to bind, preferably specifically with the CD79b antigens, including isoforms CD79b beta and/or alpha, including when such antigens are expressed on the surface of cells, including, but not limited to, B-cell

In one aspect, the antibodies of the invention can be konjugierte with any group of labels that can be covalently linked to the antibody through a reactive group, an activated group or reactive Tilney 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 label can perform the functions of: (i) providing a measurable detection signal; (ii) interact with a second label to modify amenable to the detection signal provided by the first or second label, e.g. to obtain FRET (resonance energy transfer fluorescence); (iii) stabilize interactions or increase affinity of binding, with antigen or ligand; (iv) impact on mobility, e.g. electrophoretic mobility or the ability to penetrate into the cell, by charge, hydrophobicity, shape, or other physical parameters, or (v) enable the group to capture, modulating the affinity to the ligand, binding of the antibody/antigen or the formation of complexes with ions.

Labeled antibodies with the built-in cysteine residues can be used in diagnostic assays, e.g., for detecting expression of interest antigen specific to�edah, tissues, or serum. For diagnostic applications, the antibody typically mark beyond detection group. There's a set of tags that, in General, can be divided into the following categories:

Radioisotopes (radionuclides), such as3H,11C,14C,18F,32P,35S,64Cu,68Ga86Y99Tc,111In123I,124I,125I,131I,133Xe177Lu,211At or213Bi. Labelled radioisotope antibody useful in experiments aimed at receptor visualization. The antibody can be labeled with reagents ligands that are bound, form chelates or other complexes with a radioisotope metal where the reagent is reactive towards thiol group built-cysteine antibody, using methods described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. (1991). Chelating ligands, which can form complex with metal ion, include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). Radionuclides can be targeted by means of complex formation with the conjugate antibody-drug according to the invention (Wu et al. (2005) Nature Biotechnology 23(9):1137-1146).

Linker reagents such as DOTA-maleimide (4-maleimidomethyl-DOTA) can be obtained by the reaction aminobenzyl-DOTA with 4-maleimidomethyl acid (Fluka) activated from�propylchloride (Aldrich), with the subsequent way Axworthy et al. (2000) Proc. Natl. Acad. Sci. USA 97(4): 1802-1807). DOTA-maleimide reagents react with free amino acid residues of cysteine antibodies with the built-in cysteine residues and provide forms a complex with the metal ligand on the antibody (Lewis et al. (1998) Bioconj. Chem. 9:72-86). Chelating linker reagents for labeling, such as DOTA-NHS (mono-N-hydroxysuccinimidyl ester 1,4,7,10-tetraazacyclododecane-1,4,7,10-vs acid), commercially available (Macrocyclics, Dallas, TX). Focused on receptor imaging using radionuclide-labeled antibodies may provide a marker for the cascade is triggered by the detection and quantitative determination of the gradual accumulation of antibodies in tumor tissue (Albert et al. (1998) Bioorg. Med. Chem. Lett. 8: 1207-1210). Conjugated radioactive metals may remain inside the cell after lysosomal degradation.

Complexes the chelate-metal, suitable as a label for antibodies for visualization experiments described in: US 5342606; US 5428155; US 5316757; US 5480990; US 5462725; US 5428139; US 5385893; US 5739294; US 5750660; US 5834456; 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. Cancer1990, 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. 5:4221-4226; 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 include labels, such as chelates of rare earth elements (europium chelates), fluorescein, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; types rhodamine, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; Texas red; and analogs thereof. Fluorescent labels can be konjugierte with antibodies using the methods described, for example, in Current Protocols in Immunology, above. Reagents fluorescent dyes and fluorescent label reagents include, which are commercially available from Invitrogen/Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

Various enzyme-substrate labels are available or described (US 4275149). The enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured using different methods. For example, an enzyme may catalyze a color change of the substrate, which can be measured spectrophotometrically. Alternative enzyme may alter the fluorescence or x�illuminescence of the substrate. Methods of quantification of fluorescence changes described above. The chemiluminescent substrate becomes electronically excited chemical reaction and it can emit light that can be measured (using, for example, chemiluminometer) or he becomes a donor to the energy acceptor fluorescence. Examples of enzymatic labels include luciferases (e.g., Firefly luciferase and bacterial luciferase; US 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, macavinta, a peroxidase, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, oxidase saccharides (e.g., glucose oxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclic oxidase (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides, etc. Methods of conjugation of enzymes to antibodies are described in 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 enzyme-substrate include, for example:

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

(ii) alkaline phosphatase (AP) with para-nitrophenols�that as chromogenic substrate; and

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

Specialists in this field are available in numerous combinations of enzyme-substrate. For a General review, see US 4275149 and US 4318980.

The indirect label can be konjugierte with a side chain of an amino acid, an activated side chain of an amino acid, an antibody with built in his cysteine residues, etc. for Example, the antibody can be konjugierte with Biotin and any of the three broad categories of labels mentioned above can be konjugierte with Avidya or streptavidin, or Vice versa. Biotin binds selectively with streptavidin and thus, the label can be anywhereman with the antibody in this indirect manner. Alternative to achieve indirect conjugation of the label with the variant polypeptide, the variant polypeptide kongugiruut with a small hapten (e.g., digoxin) and one of various types of labels mentioned above, kongugiruut with the variant polypeptide against hapten (for example, an antibody against digoxin). Thus, it is possible to achieve indirect conjugation of the label with the variant polypeptide (Hermanson, G. (1996), Bioconjugate Techniques Academic Press, San Diego).

The antibody of the present invention can be used in any known assay method, such as ELISA, con�Urantia binding assays, direct and indirect sandwich assays, and immunoprecipitation analyses (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp.147-158, CRC Press, Inc.).

Detecting the label can be suitable for localization, visualization and quantification of binding or recognition. Labeled antibodies of the invention can carry out the detection of cell surface receptors. Another application of labeled amenable to the detection of labeled antibodies is the immune method of carbon granules comprising the conjugation of granules with a fluorescently labeled antibody and detection of the fluorescence signal upon binding of the ligand. In similar ways the detection of binding is used, the effect of surface plasmon resonance (SPR) measurement and detection of interactions antibody-antigen.

Detecting markers, such as fluorescent dyes and chemiluminescent dyes (Briggs et al. (1997) "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids", J. Chem. Soc, Perkin Trans. 1:1051-1058) provide measurable detection signal, and, as a rule, is applicable for the labeling of antibodies, preferably with the following properties: (i) the labeled antibody should produce a very high signal with low background so that it was possible to selectively detect small amounts of antibodies in cell-free and cellular assays; and (ii) the labeled antibody should be photostable�th, to observe, monitor and record the fluorescent signal without significant discoloration by light. For applications involving binding to cell surface labeled antibodies to membranes or cell surfaces, especially living cells, the labels preferably (iii) have good solubility to achieve effective conjugate concentration and the sensitivity of dadzie and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.

Direct quantification of the fluorescence intensity of the cells and counting of cases, fluorescent labeling, e.g., for binding to cell surface conjugates peptide-dye can be done on the system (FMAT® 8100 HTS System, Applied Biosystems, Foster City, Calif.), which automates the blending and reading in the non-radioactive assays with live cells or with beads (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 include analyses of the binding of cell surface receptors, immune analyses of capture-related fluorescence immunosorbent assays (FLISA), analysis of cleavage by caspase (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events asociated with Fas-apoptosis also been other ideas where in vivo", (1998) Proc. Natl. Acad. Sci. USA 95:618-23; US 6372907), analyses 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 cytotoxicity assays. Technology fluorometric micro-volume analysis can be used to identify positive or negative regulation by molecules, which focuses on the cell surface (Swartzman, "A homogeneous and multiplexed down immunoassay for high-throughput screening using fluorometric microvolume assay technology", (1999) Anal. Biochem. 271:143-51).

Labeled antibodies of the invention are suitable as biomarkers for imaging and probes in various ways and technologies of biomedical and molecular imaging such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography), Chen et al. (2004) Bioconjugate Chem. 15:41-49; (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is a visualization technique in which antibodies labeled with radioactive substances are administered to an animal or patient person and take a picture of areas of the body where localized antibody (US 6528624). Biomarkers for imaging can be objectively measured and used as an indicator of normal biological processes,pathogenic processes or pharmacological responses to a therapeutic intervention. Biomarkers can be of several types: type 0 biomarkers are natural markers of the disease and they directly correlate with known clinical indices, such as the evaluation by MRI of inflammation of the synovial membrane in rheumatoid arthritis; markers type I give information about the effect of intervention in accordance with the mechanism of action, even though this mechanism may not be associated with clinical outcome; markers type II function as alternate outcomes, where the change in the biomarker or biomarker signal predicts clinical benefit for "confirmation" of the directional response, such as measured by CT bone erosion in rheumatoid arthritis. Markers for visualization, thus, can provide pharmacodynamic (PD) therapeutic information on: (i) the expression of the target protein, (ii) the binding of drugs to protein targets, i.e., selectivity, and (iii) pharmacokinetic data on excretion and half-life. The advantages of imaging biomarkers forin vivoregarding laboratory markers include: the introduction of non-invasive, quantifiable assessment of the body, repetitive dosing and evaluation, i.e., evaluation at multiple time points, and potentially portable effects from doclines�x (small animals) to clinical (person) results. For some applications, biological visualization replaces or minimizes the number of experiments on animals in preclinical studies.

Methods of labeling peptides is well known. 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, FIa.); 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 groups, a fluorescent reporter and Navy, located close enough, undergo resonance energy transfer fluorescence (FRET). Reporter group, as a rule, are fluorescent dyes that are excited by light at a certain wavelength and transfer the energy to the acceptor group, or Navy, with the corresponding Stokes shift for the emission at maximum brightness. Fluorescent dyes include molecules with large aromatic group�and, such as fluorescein and rhodamine, and their derivatives. Fluorescent reporter may be partly or largely stew a group of Navy in the intact peptide. The cleavage of the peptide peptidase or protease, it is possible to measure amenable to the detection of increased fluorescence (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34).

Labeled antibodies of the invention can also be used as a means for affinity purification. In this process, the labeled antibody is immobilized on a solid phase, such as Sephadex resin or filter paper, using methods well known in the field. Immobilized antibody is contacted with a sample containing the antigen to be cleaned, and then the substrate is washed with a suitable solvent that removes essentially all the material in the sample except the antigen to be treated, which is associated with immobilized variant polypeptide. In the end, the substrate is washed with another suitable solvent, such as glycine buffer, pH 5.0, that releases the antigen from the variant polypeptide.

Reagents for labeling, as a rule, possess a reactive functional group which may react (i) directly with the thiol group of cysteine in the antibody with built in his cysteine residues with the formation of labeled antibodies�, (ii) with a linker reagent with the formation of the intermediate linker-label, or (iii) a linker with the antibody with the formation of labeled antibodies. Reactive functional group reagents for labeling include: maleimide, haloacetic, Succinimidyl ester of iodated (e.g. NHS, N-hydroxysuccinimide), isothiocyanate, sulphonylchloride, 2,6-dichlorotriazinyl, panafcortelone ester and phosphoramidate, although you can also use other functional groups.

Illustrative reactive functional group is a N-hydroxysuccinimidyl ester (NHS) Deputy carboxyl groups amenable to the detection of the label, such as Biotin or a fluorescent dye. Ester NHS and labels can previously obtain, allocate, clear, and/or oharakterizovat, or it is possible to formin situand subjecting the reaction with the nucleophilic group of an antibody. Typically, the carboxyl form of the label is activated by reaction with some combination of a carbodiimide reagent, e.g., dicyclohexylcarbodiimide, diisopropylcarbodiimide or uranium reagent, such as TSTU (O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea of tetrafluoroborate, HBTU (O-benzotriazole-1-yl)-N,N,N',N'-tetramethylurea of hexaflurophosphate) or HATU (O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea of hexaflurophosphate, activator, such as 1-hydroxy-benzotriazole (HOBt), and N-hydroxysuccinimide with obtaining NHS ester and labels. In some cases, the label and the antibody can bind via activation of thein situlabels and reaction with the antibody to form a conjugate of a label-antibody in a single step. Other activating and binding reagents include TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate), TFFH (N,N',N",N'"-tetramethyluronium 2-forexceptional), PyBOP (benzotriazole-1-yloxy-Tris pyrrolidinone hexaflurophosphate, EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and arylsulfonate, for example, .

Connections albumin-binding peptide-Fab of the invention:

In one aspect, an antibody of the invention is fused with an albumin-binding protein. Protein binding plasma can be an effective method for improving the pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Binding to serum albumin peptides (ABP) can alter the pharmacodynamics merged with the active domain proteins, including change capture, penetration and diffusion in the tissue. These pharmacodynamic pairs�meters can be modulated by a special choice of the appropriate serum albumin binding peptide sequences (US 20040001827). The number of albumin-binding peptides were identified by screening phage display (Dennis et al. (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Protrins" J Biol Chem. 277:35035-35043; WO 01/45746). Compounds of the invention include sequences ABP described: (i) Dennis et al. (2002) J Biol Chem. 277:35035-35043 in tables III and IV, page 35038; (ii) US 20040001827 in SEQ ID nos:9-22; and (iii) WO 01/45746 on pages 12-13, all of which are incorporated herein as references. Albumin-binding peptide(ABP)-Fab design merge albumin-binding peptide from the C-end of the heavy chain Fab in a stoichiometric ratio of 1:1 (1 ABP/1 Fab). It was shown that the binding of these ABP-Fab to albumin increases the half-life of antibodies more than 25 times in rabbits and mice. Thus, the above described reactive Cys residues can be embedded in these ABP-Fab and used for site-specific conjugation with cytotoxic drugs followed by animal studiesin vivo.

Illustrative sequence, the albumin-binding peptides include, but are not limited to, the amino acid sequence shown in SEQ ID NO:80-84:

CDKTHTGGGSQRLMEDICLPRWGCLWEDDFSEQ ID NO:80
QRLMEDICLPRWGCLWEDDFSEQ ID NO:81
QRLIEDICLPRWGCLWEDDFSEQ ID NO:82
RLIEDICLPRWGCLWEDDSEQ ID NO:83
DICLPRWGCLWSEQ ID NO:84

Conjugates of the antibody-drug

In another aspect, the invention relates to immunoconjugates, or conjugates antibody-drug (ADC) containing the antibody, anywhereman with a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal, or its fragments), or a radioactive isotope (i.e., radioconjugates). In another aspect of the invention, moreover, there will be ways to apply immunoconjugates. In one aspect immunoconjugate contains any of the above antibodies against CD79b, covalently associated with a cytotoxic agent, or capable of detection by the connection.

In one aspect, a CD79b antibody against according to the invention binds to the same epitope CD79b, which is bound by another CD79b antibody against. In another embodiment, the implementation, the antibody against CD79b according to the invention binds to the same epitope on CD79b, which is bound Fab fragment of the monoclonal antibodies obtained from hybridomas deposited�s in ATCC as PTA-7712 on July 11, 2006 a monoclonal antibody containing the variable domains of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B) or a chimeric antibody containing either the variable domains of the antibodies obtained from hybridomas PTA-7712 deposited with the ATCC on July 11, 2006, and constant domains from IgG1, or the variable domains of monoclonal antibody comprising sequences of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B). In another embodiment, the implementation, the antibody against CD79b according to the invention binds to the same epitope on CD79b, which is bound by another CD79b antibody against (i.e., CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT 107-2 (AbD Serotec, catalog #MCA2209), the antibody against human CD79b (BD Biosciences, catalog #557592; San Jose, CA)).

In another aspect, a CD79b antibody against according to the invention binds to an epitope on CD79b distinct from the epitope, bound by another CD79b antibody against. In another embodiment, the implementation, the antibody against CD79b according to the invention binds to an epitope on CD79b distinct from the epitope, bound Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006, monoclonal antibodies containing the variable domains of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B), or chimeric antibodies containing either the variable domains of the antibodies obtained from hybridomas PTA-7712 deposited with the ATCC 11 and�La 2006 and constant domains from IgG1, or the variable domains of monoclonal antibody comprising sequences of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B). In another embodiment, the implementation, the antibody against CD79b according to the invention binds an epitope on CD79b distinct from the epitope on CD79b, bound by another CD79b antibody against (i.e., CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT 107-2 (AbD Serotec, catalog #MCA2209), the antibody against human CD79b (BD Biosciences, catalog #557592; San Jose, CA)).

In another aspect, a CD79b antibody against according to the invention differs from (i.e., not them) Fab fragment of the monoclonal antibodies obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006, monoclonal antibodies containing the variable domains of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B), or chimeric antibodies containing either the variable domains of the antibodies obtained from hybridomas PTA-7712 deposited with the ATCC on July 11, 2006, and constant domains from IgG1, either the variable domains of monoclonal antibody comprising sequences of SEQ ID NO:10 (figure 7) and SEQ ID NO:14 (figures 8A-B). In another embodiment, the implementation, the antibody against CD79b according to the invention differs from (i.e., is not) a Fab fragment of the other antibodies against CD79b (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), the antibody p�of otiv human CD79b (BD Biosciences, catalogue #557592; San Jose, CA)).

In one aspect, an antibody of the invention binds to specific CD79b first animal species, and is not associated with specific CD79b in a second species. In one embodiment, the implementation, the first type of animal is the person and/or Primate (e.g., cynomolgus monkey), and the second animal species is murine (e.g., mouse) and/or dog. In one embodiment, the implementation, the first animal species is human. In one embodiment, the implementation, the first type of animal is a Primate, such as cynomolgus macaques. In one embodiment, the implementation, the second animal is a mouse, such as mouse. In one embodiment, the implementation, the second animal is a dog.

In one aspect, the invention relates to compositions containing one or more antibodies according to the invention and a carrier. In one embodiment, the implementation, the carrier is pharmaceutically acceptable.

In one aspect, the invention relates to nucleic acids encoding antibodies against CD79b according to the invention.

In one aspect, the invention relates to vectors containing the nucleic acid according to the invention.

In one aspect, the invention relates to the cell host containing a nucleic acid or vector according to the invention. The vector may be a vector of any type, for example, �recombinantly vector, such as expressing vector. You can use any of the many host cells. In one embodiment of implementation, the host cell is a prokaryotic cell, for example,E. coli. In one embodiment of implementation, the host cell is a eukaryotic cell, e.g. a mammalian cell, such as cell Chinese hamster ovary (CHO).

In one aspect, the invention relates to methods for producing antibodies according to the invention. For example, the invention relates to a method of producing antibodies against CD79b (which, as defined herein, includes full-size antibody and its fragments), and said method includes the expression in a suitable the host cell a recombinant vector of the invention encoding the indicated antibody (or its fragment), and the allocation of the specified antibodies.

In one aspect, the invention relates to a product containing container; and a composition contained within the container, where the composition contains one or more antibodies against CD79b according to the invention. In one embodiment, the implementation, the composition contains a nucleic acid according to the invention. In one embodiment, the implementation, the composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the implementation�t, the product according to the invention additionally includes instructions for introduction of the composition (e.g., antibodies) to the individual.

In one aspect, the invention relates to a kit containing a first container containing a composition comprising one or more CD79b antibody of the invention; and a second container containing a buffer. In one embodiment, the implementation, the buffer is pharmaceutically acceptable. In one embodiment, the implementation, the composition comprising an antibody antagonist further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the implementation, the kit further includes instructions for introduction of the composition (e.g., antibodies) to the individual.

In one aspect, the invention relates to the use of antibodies against CD79b according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic Le�goat (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to the use of nucleic acids according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to the use of expressing the vector according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymph�s, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to the use of host cell according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to the use of the product according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, opuholi/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to the use of the kit according to the invention for the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases such as malignant tumor, the tumor and/or cell-proliferative disorder. In one embodiment of the implementation, a malignant tumor, the tumor and/or cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to a method of inhibiting growth of a cell that expresses CD79b, pricemoney method comprises contacting the specified cell with the antibody according to the invention, thereby causing inhibition of growth of the specified cell. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of medical treatment of a mammal having a malignant tumor containing a cell that expresses CD79b, wherein said method includes introduction to the specified mammal a therapeutically effective amount of the antibody according to the invention, thereby effectively carrying out the treatment of the specified mammal. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method for the treatment or prevention of cell proliferative disorders associated with increased expression of CD79b, wherein said method comprises administering to the individual in need of such treatment, an effective amount of an antibody according to the invention, thereby effectively carrying out the treatment or prevention of a specified cell-proliferative disorders. In one embodiment, the implementation of the specified proliferative infringement is a malignant tumor. In about�nom variant of implementation, antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of inhibiting cell growth, where the growth of the indicated cells at least partially depends on the effect of CD79b on strengthening growth, and said method comprises contacting cells with an effective amount of an antibody according to the invention, thereby inhibiting the growth of the specified cell. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method of medical treatment of a tumor in a mammal, where the specified growth of the tumor is at least partially dependent on the effect of CD79b on strengthening growth, and said method comprises contacting cells with an effective amount of an antibody according to the invention, thereby effectively carrying out the treatment of the indicated tumor. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

In one aspect, the invention relates to a method for treating malignant tumors, comprising administering to the patient a pharmaceutical grade�algebraic structure, containing immunoconjugate described in this document acceptable diluent, carrier or excipient. In one embodiment, the implementation, the malignant tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone. In one embodiment, the implementation, the patient is administered a cytotoxic agent in combination with a connection-conjugate antibody-drug.

In one aspect, the invention relates to a method of inhibiting the proliferation of B-cells, including the impact on the cell immunoconjugates containing the antibody according to the invention, under conditions that allow binding immunoconjugate with CD79b. In one embodiment, the implementation, the proliferation of B-cells is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone. � one variant of implementation, B-cell is a xenograft. In one embodiment, the implementation, the effect occursin vitro. In one embodiment, the implementation, the effect occursin vivo.

In one aspect, the invention relates to a method for determining the presence of CD79b in a sample suspected to contain CD79b, wherein said method includes the impact on the specified sample with the antibody according to the invention, and determining binding of the indicated antibody to CD79b in a specified sample, where the specified binding of the antibody to CD79b in a specified sample indicates the presence of said protein in a given sample. In one embodiment, the implementation, the sample is a biological sample. In the next version of the implementation, the biological sample contains B-cells. In one embodiment, the implementation, the biological sample is a sample from a mammal suffering from or suspected to suffer B-cell violation and/or B-cell-proliferative violation, including, but not limited to, lymphoma, nehodgkinski lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma of glue�oak mantle zone.

In one aspect, the invention relates to a method of diagnosing a cellular proliferative disorders associated with an increase in the number of cells, such as B-cells expressing CD79b, wherein the method comprises kontaktirovanije test cells in a biological sample with any of the above antibodies; determining the level of antibody bound to test cells in the sample by detecting binding of an antibody to CD79b; and comparing the level of antibody bound to cells in a control sample, where the level of bound antibodies normalize relative to the number of cells expressing CD79b, in test and control samples and where a higher level of bound antibody in the sample compared to a control sample indicates the presence of cell-proliferative disorders associated with cells expressing CD79b.

In one aspect, the invention relates to a method for the detection of soluble CD79b in blood or serum, the method comprises contacting the test sample of blood or serum from the mammal, presumably suffering from B-cell-proliferative violation, with an antibody against CD79b according to the invention and the detection of increased levels of soluble CD79b in the sample relative to the control sample of blood or serum from ZD�global mammal. In one embodiment, the implementation, the detection method is suitable as a method of diagnosing a B-cell proliferative disorders associated with increased content of soluble CD79b in the blood or serum of a mammal.

In one aspect, the invention relates to a method of binding antibodies according to the invention with a cell that expresses CD79b, wherein said method comprises contacting the specified cell with the antibody of the invention. In one embodiment, the implementation, the antibody anywhereman with a cytotoxic agent. In one embodiment, the implementation, the antibody anywhereman with a growth inhibitory agent.

The methods of the invention can be applied to affect any suitable pathological state, for example, cells and/or tissue associated with the expression of CD79b. In one embodiment, the implementation, the cells targeted by the method according to the invention are hematopoietic cells. For example, hematopoietic cell can be a cell selected from the group consisting of a lymphocyte, leukocyte, platelet, erythrocyte and natural killer cells. In one embodiment, the implementation, the cell, which is directed to the method of the invention, is a B-cell or T-cell. In one embodiment, the implementation, the cell, which is directed to the method of the invention, p�establet a malignant cell. For example, the malignant cell can be a cell selected from the group consisting of a lymphoma cell, leukemia cells, or myeloma cells.

The methods according to the invention, moreover, may include additional stages of treatment. For example, in one embodiment, the implementation, the method additionally includes the stage, where the target cell and/or the target tissue (e.g., malignant cell) is subjected to radiation or effects of chemotherapeutic agents.

As described herein, CD79b is a component of the transmission signal B-cell receptor. Thus, in one embodiment of the methods according to the invention, the cell, which is targeting (for example, a malignant cell) is a cell in which is expressed CD79b, compared with a cell that expresses not CD79b. In the next version of the implementation, a target cell is a malignant cell, in which expression of CD79b is enhanced compared to normal non-cancerous cell tissue of the same type. In one embodiment of implementation, the method of the invention causes the death of the target cell.

In other aspects of the present invention, the invention relates to vectors containing DNA encoding any of the antibodies described herein. Also pre�seen a host, containing any such vector. As an example, the host cell may be a CHO cell, the cells ofE. colior yeast cells. It also includes the process of production of the antibodies described herein, and it includes culturing host cells under conditions suitable for expression of the desired antibody and the selection of the desired antibody from the cell culture.

In another aspect, the invention relates to compositions containing antibodies against CD79b, as described herein, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to the use of antibodies against CD79b polypeptide, as described herein, for the manufacture of a medicinal product suitable for the treatment of a condition that responds to antibodies against CD79b polypeptide.

Another aspect of the invention relates to compositions containing a mixture of compounds antibody-drug of formula I where the average drug load on the antibody ranges from about 2 to about 5, or from about 3 to about 4.

Another aspect of the invention relates to pharmaceutical compositions comprising a compound of the ADC of formula I, mixture of compounds of the ADC of formula I, or their pharmacist�Cesky acceptable salt or solvate, and a pharmaceutically acceptable diluent, carrier or excipient.

Another aspect relates to a pharmaceutical combination, comprising a compound of the ADC of formula I and a second compound having properties against malignant tumors, or other therapeutic effects.

Another aspect relates to a method of destroying or inhibiting the proliferation of tumor cells or malignant cells, comprising treating cells such amount of the conjugate antibody-drug of formula I, or its pharmaceutically acceptable salt or solvate, which is effective in destroying or inhibiting the proliferation of tumor cells or malignant cells.

Another aspect relates to a method for treating malignant tumors, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising the ADC of formula I.

Another aspect relates to products, i.e. sets containing conjugate antibody-drug, container, or liner in the packaging or label, with notes, introduction.

One aspect of the invention relates to a method for producing compounds of the conjugate antibody-drug of formula I, which includes stages: (a) the reaction of a group of built-cysteine antibodies with built in his �the shortcomings of cysteine with the linker reagent with the formation of intermediate compounds antibody-linker Ab-L; and (b) the reaction of Ab-L with an activated group of medicines D; thereby obtaining the conjugate antibody-drug; or comprising steps of: (c) reaction of a nucleophilic group in the group of the drug with a linker reagent with the formation of the intermediate drug-linker D-L; and (d) the reaction of D-L with the group embedded cysteines in the antibody with built in his cysteine residues; thereby obtaining the conjugate antibody-drug.

One aspect of the invention relates to the analysis for the detection of malignant cells, comprising: (a) effect on the cells of the conjugate antibodies against CD79b with built in his cysteine residues and the drug; and (b) the extent of binding of the compounds of the conjugate antibodies against CD79b with built in his cysteine residues and drugs to cells.

A. Antibodies against CD79b

In one embodiment, the implementation, the present invention relates to antibodies against CD79b, which can be used herein as medicines. Illustrative antibodies include polyclonal, monoclonal, humanized, bespecifically antibodies and heteroconjugate antibodies.

1. Polyclonal antibodies

Polyclonal antibodies are pre�occhialino induce in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Can be the appropriate conjugation of the corresponding antigen (especially when using synthetic peptides) with a protein which is immunogenic in the species to be immunized. For example, the antigen can be konjugierte with keyhole lymph snails (KLH), serum albumin, bovine thyroglobulin, or trypsin inhibitor soybean, using a bifunctional or forming a derivative of the agent, for example, ester of (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2or R1N=C=NR, where R and R1are different alkyl groups.

Immunize animals against 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 of freind and injecting the solution intradermally at multiple areas. After one month the animals are re-immunized with 1/5 to 1/10 the original amount of peptide or conjugate in complete adjuvant of frand by subcutaneous injections in several areas. Through seven to 14 days, the animals performed blood sampling and serum analyzed in relation to the titer of antibodies. The animals are re-immunization d� achieve titers plateau. Conjugates can also be obtained from recombinant cell culture as protein fused molecules. Also, to enhance the immune response in a suitable manner of use causing the aggregating agents such as alum.

2.Monoclonal Antibodies

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

In the hybrid method, a mouse or other suitable animal host, such as a hamster, immunize, as described above, to obtain lymphocytes that produce or are capable of producing antibodies that are specific bind to the protein used for immunization. Alternative lymphocytes can immunizein vitro. After immunization, lymphocytes are isolated and then subjected to fusion with myeloma cell line using a suitable agent that causes cell fusion, such as polyethylene glycol, to obtain a hybrid cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The thus obtained hybrid cells are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused source of myeloma cells (t�activate called partners for a merger). For example, if the original myeloma cells lack the enzyme hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically includes substance hypoxanthine, to produce remissions in childhood and thymidine (Wednesday HAT), which inhibit the growth of cells deficient in HGPRT.

Preferred myeloma cells-partners represent cells that are effectively merge, support stable high level production of antibodies selected antitelomerase cells and are sensitive to a selective medium that performs a select against unfused original cells. Preferred myeloma cell lines are lines of myeloma mice, such as lines derived from tumors of mice MOPC-21 and MPC-11, available at Salk Institute Cell Distribution Center, San Diego, California USA, cells and SP-2 and derivatives e.g., X63-Ag8-653, available in American Type Culture Collection, Manassas, Virginia, USA. Also for the production of human monoclonal antibodies described myeloma cell line and the line heteromalla mouse-human (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. About 51 To 63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium, which produces hybrid cells, the rate of production of monoclonal antibodies directed against the antigen. Preferably, specificness� binding of monoclonal antibodies, produced by hybrid cells is determined by immunoprecipitation or analysis of binding ofin vitrosuch as radioimmune assay (RIA) or enzyme-linked immunoassay (ELISA).

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

After identification of hybrid cells which produce antibodies with the desired specificity, affinity and/or activity, the clones can be subclinical ways of limiting dilutions and grown by standard methods (Goding, Monoclonal antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable for this purpose, the culture medium include, for example, medium (D-MEM or RPMI-1640. In addition, the hybrid cells can be grownin vivoin animals as ascitic tumors, for example, via injection of cells to mice.

Monoclonal antibodies that are excreted by subklonov, suitable means is isolated from the culture medium, ascites fluid or serum by the conventional methods of purification of antibodies, such as affinity chromatography (e.g., chromatography system using protein A - or protein G-sepharose) or ion-exchange chromatography, chromatography with hydroxyapatite, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal� antibodies, easily allocate and sequeiros using conventional methods (e.g., by using oligonucleotide probes that are capable of specific contact with the genes encoding the heavy and light chains of antibodies of the mouse). Hybrid cells serve as a preferred source of such DNA. To ensure the synthesis of monoclonal antibodies in the recombinant cell host, after DNA extraction can be integrated into expression vectors, which are then transferout in the host cell, such as cells ofE. coli, COS cells monkeys, cells of the Chinese hamster ovary (CHO) or myeloma cells that otherwise do not produce protein antibodies. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al.,Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

In the next version of the implementation, monoclonal antibodies or fragments of antibodies can be isolated from phage libraries of antibodies created using the methods described in McCafferty et al.,Nature, 348:552-554 (1990). In Clackson et al.,Nature, 352:624-628 (1991) and Marks et al.,J. Mol. Biol., 222:581-597 (1991) described the selection of antibodies of mouse and human, respectively, using phage libraries. In later publications describe the production of high-affinity (nm-order) human antibodies by permutation circuits (Marks t al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and recombinationin vivoas a strategy for constructing very large phage libraries (Waterhouse et al.,Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these methods represent a viable alternative to traditional methods hybrid monoclonal antibodies for isolation of monoclonal antibodies.

Also the DNA that encodes the antibody, can be modified to obtain chimeric or fused polypeptides antibodies, for example, by replacing sequences of the constant domain of the heavy and light chains (CHand CL) human homologous sequences of the mouse (U.S. patent No. 4816567; and Morrison, et al.,Proc. Natl Acad. Sci USA, 81:6851 (1984)), or merger of the encoding immunoglobulin sequences from the entire coding sequence for a polypeptide, non-immunoglobulin, or part of it (heterologous polypeptide). Sequences of polypeptides that are not immunoglobulins, replace the constant domains of an antibody, or they replace the variable domains of one antigen-binding center of the antibody with obtaining a chimeric bivalent antibody comprising one antigen-binding center with specificity for one antigen and another antigen-binding center with specificity for a different antigen.

3.Antibodies che�of owaka and humanized antibodies

Antibodies against CD79b, in addition, may include humanized antibodies or human antibodies. "Humanized" forms of antibodies are not human (e.g., mouse) are chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies) which contain minimal sequence, formed from immunoglobulin is not human. Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues the CDR of non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the remains of a frame Fv region of human immunoglobulin replace the corresponding residues are not human. Humanized antibodies may also contain residues that are not found neither in the recipient antibody nor in the imported CDR sequences or frame area. Generally, a humanized antibody will contain essentially all of at least one, and typically two, variable domains in which all or essentially all of the CDR regions correspond to the CDR of an immunoglobulin is not human, � all or essentially all of the areas FR represent region FR of the consensus sequence of human immunoglobulin. A humanized antibody is also preferably will contain at least a portion of the constant portion (Fc) of an immunoglobulin, typically a constant portion of human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)].

Methods of humanizing antibodies, non-human, is described in this field. Generally, a humanized antibody has one or more built in his amino acid residues from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization mostly can be performed in accordance with the method of Winter and colleagues [Jones et al.,Nature, 321:522-525 (1986); Riechmann et al.,Nature,332:323 to 327 (1988); Verhoeyen et al.,Science,239:1534-1536 (1988)], by replacing the CDRs or CDR sequences of the rat, corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where essentially a less variable regions of a person substituted by the corresponding sequence not related to the person. In practice, humanized antibodies, as a rule, represent an�of icela person in which some CDR residues and possibly some FR residues substituted by residues from analogous sites of antibodies rodents.

The choice of the variable domains of a human, both light and heavy chains, for use in obtaining humanized antibodies is very important to reduce antigenicity and HAMA response (antibodies against mouse antibodies) when the antibody is intended for therapeutic use in humans. The reduction or elimination of the HAMA response is an essential aspect of clinical development of suitable medicines. 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 herein, the invention relates to antibodies which are humanized, so that the HAMA response was reduced or eliminated. Variants of these antibodies, in addition, can be obtained using conventional methods known in this field, some of which are further described below. Under the so-called method of "best match", the sequence of the variable domain of the antibody rodent analyze regarding entire library of known sequences of the variable domain�in person. Identify the sequence of the V-domain of man, which is the most similar to the sequence of the rodent, and a frame region (FR) of a person it take for the humanized antibody (Sims et al.,J. Immunol, 151:2296 (1993); Chothia et al.,J. Mol. Biol., 196:901 (1987)). In another method uses a particular frame region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The 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 as described herein can serve as a starting (initial) sequence to ensure the diversity of the sequence(s) of a frame region and/or hypervariable sequence(s). Selected sequence frame region, which is associated with the source hypervariable sequence, referred to herein acceptor of a frame region of a person. Although the acceptor frame region can be made, or may be formed from, of human immunoglobulin (VL and/or VH-regions), preferably acceptare frame region is taken, or formed, from the consensus sequences�spine skeleton of human rights, such as frame areas, which, as shown, have minimal immunogenicity in patients-people, or not have it.

When the acceptor is formed from human immunoglobulin, optionally you can select a sequence of frame area of human rights, which were selected on the basis of its homology with the donor sequence frame region by aligning donor sequence frame region with different frame sequences of human rights in the collection of the sequences of frame areas of human rights, and the most homologous sequence frame can be selected as acceptor.

In one embodiment of implementation, the consensus frame region of a person described in this paper was derived, or formed from a consensus sequence frame region VH subgroup III and/or VL Kappa subgroup I.

Thus, frame acceptor VH region of a person may contain one, two, three or all of the following sequences of frame fields:

FR1 containing EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:69),

FR2 containing WVRQAPGKGLEWV (SEQ ID NO:70),

FR3 containing RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYC (SEQ ID NO:73), where X1represents A or R, X2is a T or N, and X3represents A or L,

FR4 containing WGQGTLTVSS (SEQ ID NO:72).

Examples of consensual frame of the VH regions include:

the consensus frame region VH subgroup I of a person without CDR by Kabat (SEQ ID NO:36);

the consensus frame region VH subgroup I of a person without extended hypervariable regions (SEQ ID NO:37-39);

the consensus frame region VH subgroup II human without CDR by Kabat (SEQ ID NO:40);

the consensus frame region VH subgroup II human without extended hypervariable regions (SEQ ID NO:41-43);

the consensus frame region VH subgroup III a man without a CDR according to Kabat (SEQ ID NO:44);

the consensus frame region VH subgroup III person without extended hypervariable regions (SEQ ID NO:45-47);

acceptor skeleton of the VH region of a person without CDR by Kabat (SEQ ID NO:48);

acceptor skeleton of the VH region of a person without extended hypervariable regions (SEQ ID NO:49-50);

accepting frame area 2 VH man without CDR by Kabat (SEQ ID NO:51); or

accepting frame area 2 VH man without extended hypervariable regions (SEQ ID nos:52-54).

In one embodiment, the implementation, frame acceptor VH region contains one, two, three or all of the following sequences of frame fields:

FR1 containing EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:69),

FR2 containing WVRQAPGKGLEWV (SEQ ID NO:70),

FR3 containing RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:71),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO:74),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:75),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCS (SEQ ID NO:76), or

RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO:77)

FR4 containing WGQGTLVTVSS (SEQ ID NO:72).

Acceptor frame region VL of human can contain one, two, three or all of the following sequences of frame fields:

FR1 containing DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:65),

FR2 containing WYQQKPGKAPKLLIY (SEQ ID NO:66),

FR3 containing GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:67),

FR4 containing FGQGTKVEIKR (SEQ ID NO:68).

Examples of consensual frame of the VL regions include:

the consensus frame region VL Kappa subgroup I (SEQ ID NO:55);

the consensus frame region VL Kappa subgroup II (SEQ ID NO:56);

the consensus frame region VL Kappa subgroup III (SEQ ID NO:57); or

the consensus frame region VL Kappa subgroup IV of human (SEQ ID NO:58)

Although the acceptor sequence may be identical to a selected sequence of frame area of a person, regardless of whether taken whether it is from human immunoglobulin, or from a consensus frame region of a human, the present invention provides that the acceptor sequence may contain pre-existing amino acid substitutions relative to the sequence of human immunoglobulin or a consensus sequence of frame area of the person. These previously existing replacement preferably�individual are minimal, as a rule, accounting for only four, three, two or one difference in the amino acids relative to the sequence of human immunoglobulin or a consensus sequence of frame area.

The remains of the hypervariable region of the antibody, non-human antibody include acceptor in the frame region VL and/or VH of a human. For example, you can include residues corresponding to residues of CDR according to Kabat, the remnants of the hypervariable loops by Chothia, Abm residues, and/or contact residues. Not necessarily, include the following balances 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 inclusion of residues hypervariable region considered in the present document, it will be understood that it can be achieved in many different ways, for example, nucleic acid encoding the desired amino acid sequence, can be obtained by mutation of the nucleic acid coding sequence of the variable domain of the mouse so that it remains of the frame region were replaced with residues of the acceptor skeleton of human rights, or mutation of nucleic acid that encodes the sequence of the variable domain of the human, so that the remains of the hypervariable domain was replaced with residues that are not human, or the synthesis of NAA�einevoll acid, encoding the desired sequence, etc.

In the examples of the present description, received options with a transplanted hypervariable region by mutagenesis method of Kunkel nucleic acid encoding the acceptor sequence of a person using a separate oligonucleotide for each hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Appropriate changes can be made in the frame region and/or hypervariable region using conventional methods, for correction and rehabilitation of appropriate interactions hypervariable region of the antigen.

Phage (formigny) display (also referred to herein rahovym display in some contexts) can be used as a convenient and fast way of getting and screening of many different potential variants of the antibodies in the library obtained by the randomization sequence. However, a specialist is available and other methods for producing and screening for modified antibodies.

The technology of phage (fahmideh) display provides a powerful tool for obtaining and selection of new proteins that bind to a ligand such as an antigen. Use of the methods of phage (fahmideh) display allows to obtain large libraries of protein variants that can quickly sort by �eating sequences, are associated with the molecule-target with high affinity. Nucleic acids that encode variants of the polypeptide as a rule, subjected to fusion with a nucleic acid sequence that encodes a viral envelope protein, such as protein gene III protein or gene VIII. Were developed monovalent system fahmideh display where the nucleic acid sequence that encodes a protein or polypeptide is subjected to fusion with a nucleic acid sequence that encodes a part of the protein III gene. (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In a monovalent system fahmideh display fused gene is expressed at low levels and also expressed protein III gene of the wild type, so that the infectivity of the particles is preserved. How to create peptide libraries and screening these libraries are described in many patents (e.g., U.S. patent No. 5723286, U.S. patent No. 5432018, U.S. patent No. 5580717, U.S. patent No. 5427908 and U.S. patent No. 5498530).

Libraries of antibodies or the antigen-binding polypeptides obtained in various ways, including changing a single gene by embedding a random DNA sequence or by cloning of a family of similar genes. Ways display antibodies or antigen-binding fragments using phage (fahmideh) display is described in U.S. patent No. 5750373, 733743, 5837242, 5969108, 6172197, 5580717 and 5658727. Then the library was subjected to screening for the expression of antibodies or antigen-binding proteins with the desired characteristics.

Ways to replace the selected amino acids in a matrix of nucleic acid are well known in this field, and some of them are described in this document. For example, the remains of the hypervariable region can be replaced using the Kunkel method. See, for example, Kunkel et al., Methods Enzymol. 154:367-382 (1987).

The sequence of oligonucleotides includes one or more of the constructed sets of codons for residues of the hypervariable region to be changed. A set of codons is a set of different nucleotide triplet sequences used to encode the required options of amino acids. Sets of codons can be represented using symbols to indicate specific nucleotides or equimolar mixtures of nucleotides, as presented below, according to 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 the set of codons DVK, D can be nucleotides A or G or T; V can be A G or C; and K can be G or T. This set of codons can correspond to 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 using standard methods. It is possible to synthesize a set of oligonucleotides, e.g., using solid-phase synthesis comprising sequences that correspond to all possible combinations of nucleotide triplets provided by the set of codons and which encode the desired group of amino acids. Synthesis of oligonucleotides with certain "degeneracy" of nucleotides at certain positions is well known in this field. Such sets of nucleotides having certain sets of codons, can be synthesized using commercial devices for nucleic acid synthesis (available, e.g., from Applied Biosystems, Foster City, CA), or they can be obtained commercially (e.g., from Life Technologies, Rockville, MD). Thus, the set of synthesized oligonucleotides with a specific set of codons, as a rule, includes a variety of oligonucleotides with different sequences, and the differences are determined by the set of codons for the whole sequence. The oligonucleotides used according to the invention have sequences that allow hybrid�organization with the matrix nucleic acid variable domain, and can also include sites for restriction enzymes for cloning purposes.

In one method, nucleic acid sequences that encode amino acid variants, you can create oligonucleotide-mediated mutagenesis. This method is well known in this field, as described by Zoller et al. Nucl Acids Res. 10: 6487-6504(1987). In brief, nucleic acid sequences that encode amino acid variants, creating by hybridizing set of oligonucleotides encoding the desired set of codons, DNA-matrix, where the matrix is a single-stranded form of the plasmid containing the sequence of the matrix nucleic acid of the variable segment. After hybridization using DNA polymerase to synthesize a second circuit, the complementary matrix, which thus includes the oligonucleotide primer, and contains sets of codons, provided a set of oligonucleotides.

As a rule, use oligonucleotides with a length of at least 25 nucleotides. Optimal oligonucleotide has 12 to 15 nucleotides that are completely complementary to the matrix on either side of the nucleotide(s) encoding the mutation(s). This ensures proper hybridization of the oligonucleotide with single-stranded DNA-matrix. Oligonucleotides are easy to synthesize using �ways, known in this field, such as methods described by Crea et al., Proc. NAT'l. Acad. Sci. USA, 75:5765 (1978).

DNA-matrix are given either with vectors, which are vectors formed from the bacteriophage M13 (suitable commercially available vectors M13mpl8 and M13mpl9) or by using vectors that contain the origin of replication of single-stranded phage, as described by Viera et al., Meth. Enzymol, 153:3 (1987). Thus, DNA is subject to mutations that can be embedded into one of these vectors to obtain a single-stranded matrix. Obtaining single-stranded matrix is described in sections 4.21-4.41 Sambrook et al., above.

To change the native DNA sequence, the oligonucleotide hybridized with single-stranded matrix in appropriate hybridization conditions. Then add the enzyme for polymerization of DNA, usually DNA polymerase T7 or fragment maple DNA polymerase I to synthesize a circuit, complementary matrix, using the oligonucleotide as a primer for synthesis. Thus, there is heteroduplex molecule, such that one strand of DNA encodes the mutated form of gene 1, and the other chain (the original matrix) encodes the native unaltered sequence of gene 1. Then this heteroduplex molecule is transformed into a suitable host cell, typically, prokaryotic cell, such asE. coliJM101. After cell cultivation, sown nagarathna tablets and subjected to screening using oligonucleotide primer, radioactively labeled with 32-phosphate to identify the bacterial colonies that contain the mutated DNA.

The method described above can be modified so as there were homoduplex molecule, where both strands of the plasmid contain the mutation(s). Modifications are as follows: single-stranded oligonucleotide is subjected to annealing to single-stranded matrix as described above. A mixture of three deoxyribonucleotides, desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTT), combined with a modified timezonebias called dCTP-(aS) (which can be obtained in Amersham). This mixture was added to the complex matrix of the oligonucleotide. When adding DNA polymerase to this mixture, form a chain of DNA that is identical to the matrix, except for the mutated bases. In addition, this new strand of DNA contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease cleavage. After cutting matrix-chain of double-stranded heteroduplex appropriate restriction enzyme, matrix chain can be split by ExoIII nuclease or another appropriate nuclease behind the field that contains the plot(plots) subject to mutagenesis. Then the reaction is stopped to leave a molecule that is only partially single-stranded. Then form a double-stranded th�duplexing DNA using DNA polymerase in the presence of all four deoxyribonucleotides, ATP and DNA ligase. Then this homoduplex molecule can be transformed into a suitable host cell.

As stated previously, the sequence of the oligonucleotides in the set has a length sufficient for hybridization with the matrix nucleic acid, and may, but not necessarily, contain the restriction sites. DNA-matrix can be obtained using vectors, which are formed by any of the vectors bacteriophage M13 or vectors which contain the origin of replication of single-stranded phage, as described by Viera et al. Meth. Enzymol., 153:3 (1987). Thus, DNA is subject to mutations, should be integrated into one of these vectors to obtain a single-stranded matrix. Obtaining single-stranded matrix is described in sections 4.21-4.41 Sambrook et al., above.

According to another method, you can restore the binding to the antigen in the process of humanization of antibodies through the selection preparirovannyh hypervariable regions (see application No. 11/061841, filed February 18, 2005). The method includes embedding hypervariable regions, non-human, in the acceptor skeleton of the region and, in addition, the introduction of one or more amino acid substitutions in one or more hypervariable regions without modifying the sequence of the acceptor skeleton of the region. Alternative depositing one or more amino acid�on the Internet at: substitutions may be accompanied by modifications in the sequence of the acceptor skeleton of the region.

According to another method, a library can be created by providing sets of upstream and downstream oligonucleotides, wherein each set has a plurality of oligonucleotides with different sequences, and different sequences are defined by sets of codons represented in the sequence of the oligonucleotides. The sets of upstream and downstream oligonucleotides, together with the sequence of the matrix nucleic acid variable domain, can be used in PCR to generate libraries of PCR products. PCR products can be called "cassettes nucleic acids" because they can be subjected to fusion with other related or unrelated sequences of nucleic acids, e.g., viral envelope proteins and dimerization domains, using conventional methods of molecular biology.

The sequence of primers for PCR includes one or more constructed sets of codons for available for solvent and highly diverse positions 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 amino acid variant.

Powergorilla antibodies, which satisfy the required criteria, because they are selected through the corresponding stage of the screening/selection can be extracted and cloned using standard recombinant methods.

In addition, it is important that antibodies were humanized with high affinity for the antigen and other beneficial biological properties. To achieve this objective, in accordance with the preferred method, humanized antibodies are prepared using a process of analysis of the source sequences and various alleged humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins are widely available and known to specialists in this field. Available are computer programs which illustrate and display possible three-dimensional conformational structures of selected sequences of immunoglobulins candidates. The study of these displayed data allows the analysis of the possible role of the residues in the functioning of the anticipated sequences of immunoglobulins, i.e., the analysis of residues that influence the ability of the immunoglobulin candidate to bind its antigen. Thus, it is possible to take away the remains of FR and to combine them from impact will�a combined and import sequences so to achieve the required properties of antibodies, such as increased affinity for the antigen(s) target. Typically, the remains of the hypervariable region have a direct and significant effect on binding to the antigen.

There are different forms of humanized antibodies against CD79b. For example, a humanized antibody may be an antibody fragment such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) to obtain immunoconjugate. Alternative a humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, it is possible to obtain human antibodies. For example, currently it is possible to obtain transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full set of human antibodies in the absence of endogenous production of immunoglobulins. For example, it was described that the homozygous deletion of the gene of the joint section of heavy chain antibodies (JH) in chimeric mice and mutant mice germ line results in complete inhibition of endogenous antibody production. The transfer of immunoglobulin genes of the germline of the person in such mutant mice germ line leads to products�AI human antibodies upon antigenic stimulation. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al.,Nature,362:255-258 (1993); Bruggermann et al.,Year in Immuno., 7:33 (1993); and U.S. patent No. 5545806, 5569825, 5591669 (all of GenPharm), 5545807; and WO 97/17852.

Alternative for obtaining human antibodies and fragments of antibodies toin vitroyou can use the technology of phage display (McCafferty et al., Nature 348:552-553 (1990)), of the sets of genes of variable (V) domains of immunoglobulins unimmunized donors. In accordance with this method, the genes of the V-domain antibody clone in frame read either the main or secondary gene sheath of the filamentous bacteriophage, such as M13 or fd, and they are exhibited as functional fragments of the antibodies on the surface of phage particles. Because filamentous particle contains a single-stranded DNA copy of the phage genome, selection on the basis of the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting such properties. Thus, the phage mimics some of the properties of B-cells. Phage display can be performed in various forms, are considered e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). For phage display can be used multiple sources of segments V-genes. Clackson et al.,Nature, 352:624-628 (1991), identified a diverse set of antibodies against oxazolone from a small random combinatorial �of biblioteki V-genes obtained from spleens of immunized mice. It is possible to construct a set of V genes from unimmunized donors-people and can produce antibodies against a broad set of antigens (including against its own antigens), mainly in accordance with the methods described by 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 by means of activated B-cellsin vitro(see U.S. patents 5567610 and 5229275).

4.Fragments of antibodies

In some cases, predominant is the use of fragments of antibodies, instead of whole antibodies. The smaller size of the fragments allows for rapid removal, and can provide improved access to solid tumors.

To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al.,Science, 229:81 (1985)). However, these fragments can now be obtained directly from recombinant host cells. Fab, Fv and scFv fragments of antibodies can be expressed and secreted inE. colithus allowing a simple method of producing large quantities of these fragme�comrade. Fragments of antibodies can be isolated from phage libraries of antibodies described above. Alternative fragments, Fab'-SH can be allocated directly fromE. coliand chemically bind with obtaining fragments F(ab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, the fragments F(ab')2it is possible to allocate directly from recombinant cell culture host. Fab and F(ab')2-a fragment with an increased half-life ofin vivocontaining residues binding a receptor of salvation epitope described in U.S. patent No. 5869046. Other methods of obtaining fragments of antibodies will be obvious to a qualified specialist. In other embodiments, preferred to select the 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 are the only types with the intact antigen-binding regions that are devoid of constant plots; thus, they are suitable for reduced nonspecific binding during usein vivo. Can be designed sFv fused with proteins with obtaining an effector protein at either N-or C - end of the sFv. Cm. Antibody Engineering, ed. Borrebaeck, above. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. patent 5641870. These linear fragments of antibodies can be monospecifičeskoj�and or bespecifically.

5.Bespecifically antibodies

Bespecifically antibodies are antibodies that have the binding specificity of at least two different epitopes. Illustrative bespecifically antibodies can bind to two different epitopes of the CD79b polypeptide described herein. Other such antibodies may combine the above plot CD79b binding and the section(s) of binding of other proteins. Alternative shoulder against CD79b can be combined with a shoulder, which is associated with a triggering molecule on a leukocyte such as a molecule T-cell receptor (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to expressing CD79b cells. Bespecifically antibodies can also be used to localize cytotoxic funds in cells that Express CD79b. These antibodies possess a CD79b binding shoulder and arm that binds the cytotoxic agent (e.g., saporin, an antibody against interferon-α, a Vinca alkaloid, A-chain of ricin, methotrexate or hapten with a radioactive isotope). Bespecifically antibodies can be obtained as intact antibodies or fragments of antibodies (e.g., F(ab')2-bespecifically antibodies).

In WO 96/16673 described bi�spetsificheskoe antibody against ErbB2/FcγRIII, and in U.S. patent No. 5837234 described bespecifically antibody against ErbB2/FcγRI. Bespecifically antibody against ErbB2/Fcα presented in WO98/02463. In U.S. patent No. 5821337 described bespecifically antibody against ErbB2/CD3.

Methods of obtaining bespecifically antibodies known in this field. A common way of obtaining intact bespecifically antibodies based on simultaneous 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)). Because of the random Assembly of heavy and light chains of immunoglobulins, such hybridomas (quadroma) potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out by stages affinity chromatography, is quite time-consuming and the yield is low. Similar methods are described in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to another approach, carried out a merger of the variable domains of the antibody with the desired binding specificity (plots of binding of the antibody-antigen) with sequences of the constant domains of immunoglobulins. Preferably carried out the merger with the constant domain of the heavy chain Ig, containing at least a portion of Sharn�world region, CH2 and CH3. Preferably, at least one of the components to be merged, had the first constant domain of the heavy chain (CH1) containing the site necessary for light chain binding. DNA encoding to be merged components of the heavy chains of immunoglobulins and, if desirable, the light chain of the antibody, inserted into separate expression vectors, and cotransfected in a suitable organism, the host. This provides significant flexibility in the regulation of the ratios of the three polypeptide fragments with each other in the variants of implementation, where unequal ratios of the three polypeptide chains in construction provides an optimum yield of the desired especifismo antibodies. However, it is possible to embed two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high product yield or when the ratios have no significant impact on the required combination circuits.

In a preferred embodiment of the implementation of this approach, bespecifically antibodies comprise the heavy chain hybrid immunoglobulin single binding specificity on one shoulder, and a pair of heavy chain-light chain hybrid immunoglobulin (about�adusei a different binding specificity) in the other shoulder. It was found that this asymmetric structure facilitates the separation of the desired especifismo compounds and unwanted combinations of chains of immunoglobulin, as the presence of light chain immunoglobulin in only one half bespecifically molecules provides an easy way of separation. This approach is described in WO 94/04690. For a more detailed description of obtaining bespecifically antibodies see, for example, Suresh et al.,Methods in Enzymology,121:210 (1986).

According to another approach described in U.S. patent No. 5731168, to maximize the percentage of heterodimers which are recovered from recombinant cell cultures can construct the area of contact between a pair of antibody molecules. The preferred contact area comprises at least part of CH3-domain of the constant domain of the antibody. In this way, one or more small side chains of the amino acids from the contact area of the first antibody molecule replaced with larger side chains (e.g. tyrosine or tryptophan). In the contact area of the second molecule antibodies create compensatory "cavities" of identical or similar with a large side chain(s) size by replacing the large side chains of the amino acids on the chain smaller (e.g., alanine or threonine). This provides a mechanism to increase exposure�and heterodimer compared to other unwanted end-products, such as homodimer.

Bespecifically antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate may be associated with Avidya and the other with Biotin. Such antibodies, for example, been proposed to target immune system cells against unwanted cells (U.S. patent No. 4676980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be obtained using any suitable method cross-linkage. Suitable means for cross-linkage are well known in this field and are described in U.S. patent No. 4676980, together with several ways of cross-linkage.

Methods of obtaining bespecifically antibodies of fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained using the formation of chemical bonds. In Brennan et al., Science, 229: 81 (1985) describes a method where an intact antibody proteoliticeski split with obtaining F(ab')2-fragments. These fragments regenerate in the presence of substances forming complexes with dithiolate, sodium arsenite, to stabilize neighboring dithiolo and prevent the formation of intermolecular disulfide. Then, the resulting Fab'-fragments are in turn derived from dinitrobenzoate (TNB). One of the derivatives of Fab'-TNB then repeat�RNO converted into Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB with getting especifismo antibodies. Received bespecifically antibodies can be used as tools for the selective immobilization of enzymes.

Recent advances have simplified direct allocation fromE. colifragments, Fab'-SH, which can chemically bind with getting bespecifically antibodies. In Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the preparation of a fully humanized molecule F(ab')2especifismo antibodies. Implemented the secretion ofE. colieach Fab'fragment separately and were subjected to direct chemical compoundin vitrowith the formation of especifismo antibodies. Bespecifically antibody, thus obtained, had the ability to contact the cells, sverkhekspressiya receptor ErbB2, and normal T-cells, as well as to start the lytic activity of cytotoxic lymphocytes against targets representing breast cancer.

We also discussed various ways of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, were obtained bespecifically antibodies using latinovich lightning. Kositelny et al.,J. Immunol.,148(5):1547-1553 (1992). Peptides latinboy zipper proteins Fos and Jun bound to the Fab'portions of two different antibodies by fusing genes. Homodimeric antibodies Voss�enableval articulated in the field of obtaining monomers, and then re-oxidized with getting heterodimers of antibody. This method can also be used to obtain homodimeric antibodies. Method "dimer antibody" as described by Hollinger et al.,Proc. Natl. Acad. Sci USA, 90:6444-6448 (1993) provides an alternative mechanism for obtaining bespecifically fragments of antibodies. The fragments contain VHassociated with VLby means of a linker that is too short to allow pairing between the two domains on the same chain. Thus, the domains VHand VLone fragment are forced to pair with complementary domains of VLand VHanother fragment, thereby forming two antigen-binding center. Also described another strategy to obtain fragments bespecifically antibodies using the dimer of single-chain Fv (sFv). Cm. Gruber et al.,J. Immunol, 152: 5368 (1994).

Also provides antibodies with more than two valencies. For example, you can get thespecific antibodies. Tutt et al.J. Immunol. 147: 60 (1991).

6. Heteroconjugate antibodies

Heteroconjugate antibodies also belong to the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies, for example, been proposed to target immune system cells to unwanted cells [U.S. patent No. 4676980],and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is envisaged that the antibodies can be obtainedin vitrousing known methods of synthetic chemistry of proteins, including methods involving cross-linking agents. For example, immunotoxins can be designed using the reaction of disulfide exchange or through the formation of thioether linkages. Examples of suitable reagents for this purpose include aminothiols and methyl-4-mercaptopyrimidine and reagents are described, for example, in U.S. patent No. 4676980.

7.Polyvalent antibodies

Multivalent antibody may internalizations (and/or metaboliziruetsa) faster than a bivalent antibody by a cell expressing an antigen to which antibodies bind. The antibodies of the present invention can be multivalent antibodies (which can refer to a class different from that of the IgM class) with three or more antigen-binding sites (e.g. tetravalent antibodies), which can easily be obtained by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. Multivalent antibody may contain the dimerization domain and three or more antigen-binding sites. The preferred dimerization domain comprises (or consists of) an Fc-phase or a hinge region. In this scenario, the antibody will contain� Fc region and three or more antigen-binding site from the N-Terminus of the Fc-plot. Preferred multivalent antibody provided herein comprises (or consists of) three to about eight, but preferably four, antigen-binding site. Multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain(s) contains two or more variable domains. For example, the polypeptide chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polipeptidnoi chain Fc-region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide chain(s) may contain: chain VH-CH1-flexible linker-VH-CH1-Fc-plot; or chain VH-CH1-VH-CH1-Fc-plot. Multivalent antibody provided herein preferably further comprises at least two (and preferably four) of the polypeptide variable domain of the light chain. Multivalent antibody provided herein, for example, may contain from about two to about eight polypeptides variable domain of the light chain. Polypeptides variable domain of the light chain considered in this document, contain variable home�n light chain and, optionally, further contain a CL domain.

8.The change in effector function using techniques of genetic engineering

It may be desirable modification of the antibodies according to the invention in respect to effector function, e.g., 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-plot of antibody. Alternative or additionally, in the Fc-plot you can pay the remainder(and) cysteine, providing, thus, the formation of disulfide bonds between the chains in this area. Thus obtained homodimeric the antibody may possess enhanced ability to internalize and/or increased complement-dependent destruction 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 can also be obtained using heterobifunctional Poperechnaya of linkers as described in Wolff et al.Cancer Research 53:2560-2565 (1993). Alternatively you can construct an antibody that has a redundant Fc-sections and, thus, may have an enhanced ability in relation to lysis of complemen�ohms and ADCC. Cm. Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989). To increase the half-life of antibodies in serum, can be inserted into the antibody (especially an antibody fragment) binding to the receptor of salvation epitope, as described, for example, in U.S. patent 5739277. As used in the context of the present application, the term "receptor binding of salvation epitope" refers to an epitope of the Fc-region of IgG molecules (e.g., IgG1, IgG2, IgG3or IgG4), which is responsible for increasing the half-life in serum IgG moleculesin vivo.

9.Immunoconjugate

The invention also relates to immunoconjugates (interchangeably referred to as "antibody conjugates-drug" or "ADC"), comprising the antibody, anywhereman with a cytotoxic agent such as a chemotherapeutic agent, growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal, or its fragments), or a radioactive isotope (i.e., radioconjugates).

In certain embodiments, immunoconjugate contains the antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents suitable for obtaining such immunoconjugates described above. Enzymatically active toxins and fragments thereof that can be used include the A-chain of diphtheria toxin, n�svyazyvaysya active fragments of diphtheria toxin, A-chain, exotoxin (fromPseudomonas aeruginosa), A-chain of ricin A-chain abrina, A-chain of medecine, alpha sarcin, proteinsAleurites fordii, proteins deantoni, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), inhibitor ofmomordica charantiaCurtin, krotin, inhibitorsapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, analyzin and trichothecenes. To obtain radioconjugates antibodies available for various radionuclides. Their examples include the212Bi131I,131In90Y and186Re. Conjugates of the antibody and cytotoxic tools can be accessed using a variety of bifunctional binding proteins substances such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCL), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), the diisocyanates such as 2,6-toluene diisocyanate), and secondary active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, immunotoxin on the basis of ricin can be obtained as described in Vitetta et al.Science 238: 1098 (1987). Labeled with carbon-14 1-isothiocyanatobenzene-3- acid (MX-DTPA) is an �illustratively chelating agent for conjugation of radionucleotide with the antibody. Cm. WO94/11026.

Also in this document provides conjugates of the antibody and one or more low molecular weight toxins, such as calicheamicin, peptides auristatin, such as monomethylaniline (MMAE) (a synthetic analog of dolastatin), maytansinoid, such as DM1, trichoton and CC1065, and the derivatives of these toxins that have activity of toxins.

Illustrative immunoconjugate - conjugates antibody-drug

Immunoconjugate (or "conjugate antibody-drug" ("ADC")) of the invention can have the formula I below, where the antibody conjugative (i.e., covalently linked) with one or more groups of the drug (D) through an optional linker (L). The ADC may include conjugates thio-MAb and drugs ("TDC").

Ab-(L-D)p(I)

Thus, the antibody may be anywhereman with a drug, either directly or via a linker. In formula I, p is the average number of groups of the drug to the antibody, which can vary, for example, from approximately 1 to approximately 20 groups of the drug to the antibody, and in certain embodiments, from 1 to about 8 grouplisting funds for the antibody. The invention relates to compositions containing a mixture of compounds of antibody-drug compounds of formula I where the average drug load on the antibody ranges from about 2 to about 5, or from about 3 to about 4.

a. Illustrative linkers

The linker may contain one or more linker components. Illustrative linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzeneboronic ("PAB"), and linker components obtained by conjugation with linker reagents: N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", also referred to herein as "MCC"), and N-Succinimidyl-(4-iodoacetyl)aminobenzoate ("SIAB"). In this area there are various linker components, some of which are described below.

The linker may be a "split linker" facilitating release of the drug in the cell. For example, you can use sensitive to acid linker (e.g., hydrazon) sensitive to proteases (e.g., sensitive to peptidase) linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., CancerResearch 52: 127-131 (1992); U.S. patent No. 5208020).

In certain embodiments, a linker is as shown in the following formula II:

-Aa-Ww-Yy-(II)

where A represents the extension piece, and a is an integer from 0 to 1; W is an amino acid element, and w is an integer from 0 to 12; Y is a GS spacer element, and y is 0, 1 or 2; and Ab, D, and p are as defined above for formula I. Illustrative embodiments of such linkers are described in US 2005-0238649 A1, which is incorporated herein by reference in full.

In some embodiments, a linker component may contain "extension piece" that binds the antibody with a different linker component or group of drugs. Illustrative of the extension elements is presented below (where the wavy line indicates the sites of covalent attachment to the antibody):

In some embodiments, a linker component may contain amino acid element. In one such variant of implementation, amino acid element allows the cleavage of the linker by the protease, the� ease the drug release from immunoconjugate under the influence of intracellular proteases such as lysosomal enzymes. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21: 778-784. Illustrative amino acid elements include, but are not limited to, dipeptide, Tripeptide, tetrapeptide and Pentapeptide. Illustrative dipeptides include: 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). Illustrative tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid element can contain amino acid residues which occur in nature, as well as minor amino acids and naturally occurring analogues of amino acids such as citrulline. Amino acid elements can be designed and optimized in their selectivity for enzymatic cleavage of a specific enzyme, for example, associated with a tumor protease, cathepsin B, C and D, or protease plasmin.

In some embodiments, a linker component may contain "GS spacer" element that binds the antibody with a group of medicinal products, either directly or by means of the extension element and/or the amino acid element. The GS spacer element can be "slotsplaying" or "not capable of self-cleavage". "Not capable of self-cleavage" GS spacer element �predstavljaet an element, in which part of the GS spacer element or all of the GS spacer element remains associated with the group of the drug by enzymatic (e.g., proteolytic) the splitting of the ADC. Examples are not capable of self-cleavage of the GS spacer elements include, but are not limited to, glycine GS spacer element and the glycine-glycine GS spacer element. Also provides other combinations of peptide spacers that are sensitive to specific to a sequence of enzymatic cleavage. For example, enzymatic digestion ADC containing a glycine-glycine GS spacer element associated with the tumor cells protease may result in detaching the group of glycine-glycine-drug from the rest of the ADC. In one of these embodiments, the group of glycine-glycine-drug are then subjected to a separate hydrolysis step in the tumor cell, thus Tsaplya glycine-glycine GS spacer element from the group of drugs.

"Smoothshapes" GS spacer element allows the release of the group of medicines without a separate hydrolysis step. In certain embodiments, the GS spacer linker element contains a p-aminobenzyl element. In one such variant of implementation, the p-aminobenzoyl alcohol with�anywayt element with an amino acid via an amide bond and get the carbamate, methylcarbamate, or a carbonate between benzyl alcohol and a cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103. In one embodiment, the implementation, the GS spacer element is a u-aminobenzylpenicillin (PAB). In certain embodiments, the phenylene portion of a p-aminoaniline element replaced by Qm, where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-cyano; and m is an integer in the range 0-4. Examples slotsplaying GS spacer elements further include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, e.g., US 2005/0256030 A1), derivatives such as 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para-aminobenzoate. You can use the spacers that undergo cyclization upon amide hydrolysis, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc, 1972, 94, 5815); and amides of 2-aminophenylamino acid (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). The removal of amine-containing drugs that are substituted in a-position of glycine (Kingsbury, t al., J. Med. Chem., 1984, 27, 1447) are also an example slotsplaying spacers suitable for the ADC.

In one embodiment, the implementation, the GS spacer unit is a branched bis(gidroximetil)styrene (BHMS) element, shown below, which can be used to incorporate and release multiple drugs.

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

In another embodiment, the implementation, the linker L may be a dendritic linker type for covalent joining more than one group of medicines through branching multifunctional linker group to an antibody (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic linkers can increase the molar ratio of the drug and the antibody, i.e. the load that is associated with the efficiency of the ADC. Thus, when the antibody with built in his cysteine residues has only one reactive thiol group of cysteine, many groups of drugs can be associated through a dendritic linker.

Illustrative Linke�related components and their combinations are presented below in the context of ADC of formula II:

Linker components, including lengthening, GS spacer elements and amino acid, can be synthesized by methods known in this field, such as the methods described in US 2005-0238649 A1.

b. Illustrative of the group of medicines

(1) Maytansine and maytansinoids

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

Maytansinoids group medicines are an attractive group of the drug in the conjugate antibody-drug, because they: (i) relatively accessible to the p�of producing fermentation or chemical modification or transformation in a derived fermentation products, (ii) may be converted to derivatives with functional groups suitable for conjugation through disulfide and nevalidni linkers to antibodies, (iii) are stable in plasma, and (iv) are effective against various tumor cell lines.

Connection maytansine suitable for use as maytansinoid groups of drugs that are well known in this field and they can be isolated from natural sources according to known methods or obtained using the methods of genetic engineering and fermentation (US 6790952; US 2005/0170475; Yu et al. (2002) PNAS 99: 7968-7973). Maytansine and analogues maytansine can also be obtained synthetically by known methods.

Illustrative maytansinoids group of medicines include groups having a modified aromatic ring, such as: C-19-Dehler (U.S. patent No. 4256746) (obtained by reconstruction using the case of lithium ansamitocins P2); C-20-hydroxy (or C-20 desmethyl) +/-C-19-Dehler (U.S. patent No. 4361650 and 4307016) (produced by demethylation usingStreptomycesorActinomycesor dechlorination using LAH); and C-20 dimethoxy, C-20-acyloxy (-OCOR), +/-Dehler (U.S. patent No. 4294757) (obtained by acylation using acylchlorides), and groups having modifications at other positions.

�llustrative maytansinoids group of drugs also include groups, having modifications such as: C-9-SH (U.S. patent No. 4424219) (obtained by reaction maytansine with H2S or P2S5); C-14-alkoxymethyl(dimethoxy/CH2OR)(US 4331598); C-14-gidroximetil or acyloxymethyl (CH2OH or CH2OAc) (U.S. patent No. 4450254) (derived from Nocardia); C-15-hydroxy/acyloxy (US 4364866) (obtained by conversion of maytansine byStreptomyces); C-15-methoxy (U.S. patent No. 4313946 and 4315929) (extracted fromTrewia nudlflora); C-18-N-desmethyl (U.S. patent No. 4362663 and 4322348) (produced by demethylation maytansine by Streptomyces); and 4,5-deoxy (US 4371533) (produced by restoration maytansine the titanium trichloride/LAH).

It is known that many of the provisions of the compounds of maytansine are appropriate as provisions for binding, depending on the type of communication. For example, for the formation of ester bonds, suitable position C-3 having a hydroxyl group, a position C-14, modified gidroximetil, position C-15, modified hydroxyl group and position C-20, with a hydroxyl group (US 5208020; US RE39151; US 6913748; US 7368565; US 2006/0167245; US 2007/0037972).

Maytansinoids group of medicines include groups having the structure:

where the wavy line indicated the covalent attachment of the sulfur atom maytansinoids group of the drug to the linker� on ADC. R can independently represent H or C1-C6alkyl. Allenova chain linking amide group with a sulfur atom, can represent metanil, etanol or propyl, i.e. 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).

For compounds according to the invention provides all stereoisomers maytansinoids group of the drug, i.e. any combination of R - and S-configurations at the chiral carbons in D. In one embodiment, the implementation, maytansinoids group of drugs has the following stereochemistry:

Illustrative embodiments of maytansinoid groups of drugs include: DM1; DM3; and DM4, having the structures:

where the wavy line indicated the covalent attachment of the sulfur atom of the drug to the linker (L) conjugate antibody-drug. (WO 2005/037992; US 2005/0276812 A1).

Other illustrative conjugates antibody-maytansinoid drug have the following structures and abbreviations (where Ab represents the antibody and p is from 1 to about 8):

Fig�administrative conjugates antibody-drug where DM1 is linked through a BMPEO linker to a thiol group of the antibody have the following structure and reduction:

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, in Erickson, et al. (2006) Cancer Res. 66(8):4426-4433; U.S. patent No. 5208020, 5416064, US 2005/0276812 A1, and European patent EP 0 425 235 B1, the disclosures of which are incorporated herein as references in full.

Conjugates of the antibody-maytansinoid get the chemical binding of an antibody to a molecule maytansinoid without a substantial reduction of the biological activity of either the antibody or the molecule maytansinoid. See, for example, U.S. patent No. 5208020 (the description of which is incorporated herein by reference in its entirety). Maytansinoid can be synthesized by known methods or isolated from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 in the other patents and nonpatent publications referred to herein above, such as maytansine and analogues maytansine modified in the aromatic ring or at other positions of the molecule maytansine, such as various esters maytansine.

To obtain conjugates antibody-maytansinoid things�meets a variety of linker groups, known in this field, including, for example, the linker groups described in U.S. patent No. 5208020 or in patent EP 0 425 235 B1; Chari et al. Cancer Research 52:127-131 (1992); and US 2005/016993 A1, the disclosures of which are incorporated herein as references in full. Conjugates of the antibody-maytansinoid containing linker component SMCC, can be obtained as described in US 2005/0276812 A1, "Antibody-drug conjugates and Methods". Linkers that contain a disulfide group, a simple thioether group unstable to acids, photolabile group, not resistant to peptidases group, or unstable to granulocyte esterases group, as described in the aforementioned patents. Additional linkers are described and illustrated herein.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional means for attaching proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of complex imidapril (such as dimethylacetamide HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-dies�the cyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). In certain embodiments, the binding agent is an N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) or N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), providing a disulfide bond.

The linker can be linked to the molecule maytansinoid in different positions, depending on the type of communication. For example, the ester linkage can be formed by reaction with a hydroxyl group using conventional methods of joining. The reaction can occur in the position C-3 having a hydroxyl group, position C-14, modified gidroximetil, position C-15, a modified hydroxyl group, and the position C-20, with a hydroxyl group. In one embodiment, the implementation, the bond is formed at the position C-3 maytansine or equivalent maytansine.

(2) Auristatin and dolastatin

In some embodiments, immunoconjugate contains antibody, anywhereman with dolastatin or peptide analog, or derivative of dolastatin, for example, auristatin (U.S. patent No. 5635483; 5780588). It was shown that dolastatin and auristatin violate the dynamics of microtubules, GTP hydrolysis, and division of nuclei and cells (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have an act�the emergency supply against malignant tumors (U.S. patent No.5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Group medicines in the form of dolastatin or auristatin can be linked to the antibody through the N(amino)-end or the C(carboxyl)-end of the peptide group of the drug (WO 02/088172).

Illustrative embodiments of auristatin include communicating via the N-end group of the drug in the form of monomethylaniline DE and DF (US2005/0238649 described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004, the description of which is incorporated herein by reference in its entirety).

Peptide group of drugs may be selected from formulas (DEand DFbelow:

where the wavy line in DEand DFspecify the site of covalent binding with the antibody or antibody-linker component, and independently at each position:

R2selected from H and C1-C8alkyl;

R3selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl, C1-C8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl, C1-C 8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

or R4and R5together 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-C8alkyl;

R7selected from H, C1-C8alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl, C1-C8alkyl-(C3-C8carbocycle), C3-C8of the heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8alkyl;

R10selected from aryl or C3-C8heterocycle;

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

R11selected from H, C1-C20of alkyl, aryl, C3-C8heterocycle, -(R13O)m-R14or -(R13O)m-CH(R15)2; m is an integer in the range 1-1000;

R13represents C2-C8alkyl;

R4 represents H or C1-C8alkyl;

each occurrence of R15independently represents H, COOH, -(CH2)n-N(R16)2, -(CH2)n-SO3H or -(CH2)n-SO3-C1-C8alkyl;

each occurrence of R16independently 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 in the range from 0 to 6.

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

In another variant implementation, each of R2and R6is methyl, and R9represents-H.

In another embodiment of the implementation, each occurrence of R8represents-OCH3.

Illustrative variant implementation, each of R3and R4represents isopropyl, each of R2and R6/sup> is methyl, R5represents-H, R7represents sec-butyl, each occurrence of R8represents-OCH3and R9represents-H.

In one embodiment, the implementation, Z represents-O - or-NH-.

In one embodiment, the implementation, R10represents aryl.

In the illustrative embodiment of the, R10represents-phenyl.

Illustrative variant implementation, when Z represents-O-, R11represents-H, methyl or tert-butyl.

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

In another variant implementation, when Z is-NH, R11represents-CH(R15)2where R15represents -(CH2)n-SO3H.

Illustrative variant implementation of auristatin formula DEis a MMAE, where the wavy line indicated the covalent attachment to the linker (L) conjugate antibody-drug:

Illustrative variant implementation of auristatin formula DFis �Wallpaper MMAF, where the wavy line indicated the covalent attachment to the linker (L) conjugate antibody-drug (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124):

Other illustrative options for implementation include compounds monomethylamine having a carboxymethyl-modified phenylalanine at the C-end of the Pentapeptide group medicines in the form of auristatin (WO 2007/008848), and connections monomethylamine with modifications to the side chain of phenylalanine at the C-end of the Pentapeptide group medicines in the form of auristatin (WO 2007/008603).

Other groups of drugs include the following MMAF derivatives, where the wavy line indicated the covalent attachment to the linker (L) conjugate antibody-drug:

and

In one aspect, the group of drugs you can associate a hydrophilic group, including, but not limited to, esters of triethyleneglycol (TEG), as shown above, R11. Without relation to any particular theory, the hydrophilic groups contribute to the internalization and inhibit agglomeration of the group of medicines./p>

Illustrative embodiments of the ADC of formula I containing auristatin/dolastatin or their derivative described in US 2005-0238649 and Doronina et al. (2006) Bioconjugate Chem. 17, 114-124, which are incorporated herein as references in full. Illustrative embodiments of the ADC of formula I containing MMAE or MMAF and various linker components have the following structures and abbreviations (where "Ab" is an antibody, p is from 1 to about 8, "Val-Cit" or "vc" is a dipeptide valine-citrulline, and "S" represents a sulfur atom. It should be noted that in certain descriptions of structure linked through sulfur ADC in this document, the antibody is represented as "Ab-S", just to indicate the sign of the presence of communication through the sulfur, but not to specify that a particular atom of sulfur has several groups of the linker-drug. Left bracket in the following structures can also be placed to the left of the sulfur atom, between Ab and S, which is equivalent to the description of the ADC according to the invention described in this document.

Illustrative embodiments of the ADC of formula I containing MMAF and various linker components include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, it has been shown that immunoconjugate containing MAF, associated with the antibody via a linker that is not proteoliticeski contain no cleavable, have activity comparable to immunoconjugate containing MMAF associated with the antibody through proteoliticeski contain no cleavable linker. See, Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. In such cases, it is believed that the drug release is provided by the degradation of the antibody in the cell. Ibid.

Typically, groups of drugs based on peptides can be obtained by the formation of peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, by way of the liquid synthesis (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the chemistry of peptides. Group medicines in the form of auristatin/dolastatin can be obtained according to the methods of: US 2005-0238649 A1; U.S. patent No.5635483; U.S. patent No.5780588; 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, groups of the drug in the form of auristatin/dolastatin formula DFsuch as MMAF and derivatives thereof, can be obtained using the methods described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. Group medicines in the form of AU�of estatina/dolastatin formula D Esuch as MMAE and its derivatives, can be obtained using the methods described in Doronina et al. (2003) Nat. Biotech. 21:778-784. Group drug-linker MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be a convenient way to synthesize conventional ways, for example as described in Doronina et al. (2003) Nat. Biotech. 21:778-784, and the publication of the patent application No. US 2005/0238649 A1, and then konjugierte with interest the antibody.

(3) Calicheamicin

In other embodiments, immunoconjugate contains antibody, anywhereman with one or more molecules calicheamicin. Antibiotics family calicheamicin capable of forming double-stranded DNA breaks when subpicomolar concentrations. To obtain conjugates of the family calicheamicin, see U.S. patent No.. 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all issued by American Cyanamid Company). Structural analogues calicheamicin that can be used include, but are not limited to, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θI1(Hinman et al. Cancer Research 53: 3336-3342 (1993), Lode et al. Cancer Research 58: 2925-2928 (1998) and the aforementioned U.S. patents issued to American Cyanamid). Another anticancer drug, which can be konjugierte antibody is QFA which is an antifolate. As calicheamicin, so QFA, have intracellular effects, and they do not easily pass through the plasma membrane. Thus, the cell capture these substances through antibody mediated internalization greatly enhances their cytotoxic effects.

c. Other cytotoxic funds

Other antitumor agents that can be konjugierte with the antibody include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of funds, collectively known as the complex of LL-E33288 described in U.S. patent No. 5053394, 5770710 and espiramicina (U.S. patent No. 5877296).

Enzymatically active toxins and fragments thereof that can be used include the A-chain of diphtheria toxin, non-binding active fragments of diphtheria toxin a-chain, exotoxin (fromPseudomonas aeruginosa), A-chain of ricin A-chain abrina, A-chain of medecine, alpha sarcin, proteinsAleurites fordii, proteins deantoni, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), inhibitor ofmomordica charantiaCurtin, krotin, inhibitorsapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, analyzin and trichothecenes. See, for example, WO 93/21232 published October 28, 1993.

In addition, the present invention relates to immunoconjugate formed between the antibody and the connection with nucleotidase activity (e.g. a ribonuclease or a DNA endonuclease such as desoxyribonuclease�; Tnkase).

In certain embodiments, immunoconjugate may contain a radioactive atom. For the production of radioactive conjugated antibodies available for various radioactive isotopes. Their examples include At211, I131, I125, Y90That Re186That Re188Sm153, Bi212P32, Pb212and radioactive isotopes of Lu. In the case of immunoconjugate for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99mor I123or a spin label for image acquisition of nuclear magnetic resonance (NMR) (also known as magnetic resonance 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 incorporated in a conjugate of the known methods. For example, the peptide can be biologically synthesized or can be synthesized by chemical amino acid synthesis using suitable precursors of amino acids, including, for example, fluorine-19 instead of hydrogen. Labels such as tc99mor I123That Re186That Re188and In111can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80 49-57) can be used to incorporate iodine-123. In "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) describes in detail other ways.

In certain embodiments, immunoconjugate can contain an antibody, anywhereman with activating the prodrug by the enzyme which converts a prodrug (e.g., peptidyl chemotherapeutic agent, see WO 81/01145) to an active drug, such as a drug against malignant tumors. Such immunoconjugate fit in mediated antibody-dependent enzyme prodrug therapy ("ADEPT"). Enzymes that can be konjugierte with the antibody include, but are not limited to, alkaline phosphatase, which are suitable for converting phosphate prodrug into the free drug; arylsulfatase, which are suitable for transformation containing the sulfate salt of the prodrug into the free drug; sitoindosides which is suitable for converting non-toxic 5-fertilizin in the drug against malignant tumors, 5-fluorouracil; proteases, such as proteaseSerratiathermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins B and L), which are suitable for transformation containing peptide prodrug into free drugs; D-alanismorissette, which are suitable for the conversion of the prodrug, which �will win the substituents in the form of D-amino acids; breaks down carbohydrates enzymes such as β-galactosidase and neuraminidase, which are suitable for converting glycosylated prodrug into the free drug; β-lactamase, which is suitable for the transformation of medicines, converted into a derivative with β-lactams into free drugs; and penicillinases, such as amidase penicillin V and amidase of penicillin G, which are suitable for turning of medicines transformed into their derivatives nitrogen atoms of amino groups with phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Enzymes can be covalently bind with the antibodies by methods of recombinant DNA, are well known in this field. See, for example, Neuberger et al., Nature 312:604-608 (1984).

d. Load drug

The drug load corresponds to p, the average number of groups of the drug to the antibody in a molecule of the formula I. the drug Load may be in the range from 1 to 20 groups of medicines (D) antibody. The ADC of formula I include collections of antibodies conjugated with a number of groups of the drug in the range from 1 to 20. The average number of groups of the drug to the antibody in preparations of ADC after reactions of conjugacy can be�ü characterized by conventional methods, such as mass spectrometry, ELISA and HPLC. Quantitative distribution of the ADC values in p can also be determined. In some instances, separation, purification, and characterizatio homogeneous ADC, where p is a certain value for the ADC with another load of drugs, can be conducted by means, such as reversed-phase HPLC or electrophoresis. Pharmaceutical compositions of the conjugates antibody-drug of formula I, thus, may represent a heterogeneous mixture of conjugates in which the antibody is linked to 1, 2, 3, 4 or more groups of drugs.

For some conjugates antibody-drug p may be limited by the number of sites on the antibody. For example, when the binding is established through Tilney group of cysteine, as an illustrative embodiment of the disclosed above, the antibody may have only one or a few tylnej groups of cysteine, or it may have only one or several sufficiently reactive tylnej groups, through which they may be attached to the linker. In certain embodiments, a higher drug load, for example p > 5, can lead to aggregation, insolubility, toxicity or loss of ability to penetrate into cells defined�Lenno conjugates antibody-drug. In certain embodiments, the drug load for the ADC according to the invention is in the range from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it was shown that for certain ADC optimum ratio of groups of the drug to the antibody may be less than 8, and it can be from approximately 2 to approximately 5. Cm. US 2005-0238649 A1.

In certain embodiments, during the reaction of conjugation conjugation occurs fewer drug groups than theoretical amount. An antibody may contain, for example, lysine residues that do not react with the intermediate connection of a drug-linker or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive tylnej groups of cysteine, which can be associated with a group of the drug; indeed, most tylnej of cysteine residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody can be restored by reducing agents, such as dithiothreitol (DTT) or tricarbonylchromium (TCEP), partially or fully reducing conditions, to obtain the reaction�about-able tylnej groups of cysteine. In certain embodiments, the antibody is subjected to denaturing conditions to detect reactive nucleophilic groups such as lysine or cysteine.

The load (the ratio of drug/antibody) of the ADC can be controlled in various ways, for example by: (i) limiting the molar excess of the intermediate drug-linker or linker reagent relative to antibody, (ii) a time limit or temperature reactions of conjugation, and (iii) partial or limiting recovery conditions for the modification of the thiol group of cysteine.

It should be understood that when more than one nucleophilic group reacts with the intermediate connection of a drug-linker or linker reagent followed by reaction with a reagent of the medicinal product, then the product obtained is a mixture of compounds ADC with one or more distributed groups of drugs associated with the antibody. The average number of groups of the drug to the antibody in the mixture can be calculated using double ELISA with antibodies that are specific to the specific antibody and a drug. Individual ADC molecules fashionable to identify in a mixture of mass spectrometry and divide by HPLC, for example, �cromatografia hydrophobic interactions (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, KJ., et al. "Effect of drug loading on the phatmacology, 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 certain embodiments, a mixture of conjugates is possible to allocate a homogeneous ADC with a single load value by electrophoresis or chromatography.

e. Some ways to get immunoconjugates

The ADC of formula I can be obtained in several ways using, reaction conditions and reagents of organic chemistry, known to specialists in this area, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent with the formation of Ab-L, via a covalent bond, followed by reaction with a group of drug D; and (2) reaction of a nucleophilic group in a group of drugs with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Illustrative methods of obtaining the ADC of formula I of the latter method is described in US 2005-0238649 A1, which is incorporated herein by reference in full.

Well�lesopilnye groups on antibodies include, but are not limited to: (i) N-terminal amino group, (ii) the amino group of the side chains, e.g. lysine, (iii) thiol groups of the side chains, e.g. cysteine, and (iv) a hydroxyl or amino sugars, where the antibody is glycosylated. Amino, thiol and hydroxyl groups are nucleophilic and capable of reacting with the formation of covalent bonds with electrophilic groups on linker groups and linker reagents including: (i) active esters such as NHS esters, HOBt esters, halogenfree and acid halides; (ii) alkyl - and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group. Certain antibodies have recoverable by disulfides between the chains, i.e. cysteine bridges. Reactivity of the antibody for conjugation with linker reagents can be enhanced by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylchromium (TCEP), so that the antibody partially or completely restored. Each cysteine bridge, thus, can form, in theory, two reactive tylnej nucleophil. Dopolnitelnye nucleophilic groups can be introduced in the antibody by modification of lysine residues, for example, by reaction residues l�Zina with 2-aminothiophenol (reagent trot), resulting in conversion of an amine into a thiol. Reactive thiol groups can be made to the antibody by embedding one, two, three, four, or more cysteine residues (e.g., by obtaining variants of antibodies containing one or more negativnyh amino acid residue cysteine).

Conjugates of the antibody-drug according to the invention can also be obtained by the reaction between the electrophilic group on the antibody, such as a carbonyl group of an aldehyde or ketone with a nucleophilic group on 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, the implementation, the antibody is modified by introducing an electrophilic group that can react with nucleophilic substituents on the linker reagent or drug. In another embodiment, the implementation of sugar glycosylated antibodies can be oxidized, for example, oxidizing reagents on the basis of periodate, with the formation of groups of aldehydes or ketones, which can react with the amine group of linker reagents or drug groups. Received aminogroup Schiff bases can form a stable bond, or theiryou recover, for example, borohydride reagents to form stable amine linkages, through. In one embodiment, the implementation, the reaction of the carbohydrate portion of a glycosylated antibody with either galactosialidosis or metaperiodate sodium, can lead to carbonyl groups (groups of aldehydes and ketones) in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, the implementation, the antibody containing N-terminal residues of serine or threonine can react with metaperiodate sodium, which leads to the formation of aldehyde instead of the first amino acid (Geoghegan &Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852). This aldehyde can be subjected to reaction with a group of drug or linker nucleophile.

Nucleophilic groups on a group of drugs include, but are not limited to: an amino group, Tilney, hydroxyl groups, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazines, able to react with the formation of covalent bonds with electrophilic groups on linker groups and linker reagents including: (i) active esters such as NHS esters, HOBt esters, californiathe and acid halides; (ii) alkyl - and benzylchloride, such as halogenated;(iii) aldehydes, ketones, carboxyl, and maleimide group.

Compounds according to the invention fully include, but are not limited to, ADC obtained with the following cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate) which are commercially available (e.g., from 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, can also be obtained using a variety of bifunctional means for attaching proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of complex imidapril (such as dimethylacetamide HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-diakonia (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, immunotoxin ricin can be obtained as described in Vitetta et al., Science 238: 1098 (1987). Labeled in�leerdam-14 1-isothiocyanatobenzene-3- acid (MX-DTPA) is an illustrative chelating agent for conjugation of the radionuclide to the antibody. Cm. WO94/11026.

Alternative protein containing the antibody and cytotoxic agent, can be obtained, for example, by recombinant methods or peptide synthesis. The recombinant DNA molecule may contain areas that encode a portion of an antibody and a cytotoxic portion means of the conjugate either adjacent each other or separated by land, encoding a linker peptide which does not violate the desired properties of the conjugate. In another embodiment, the implementation, the antibody can be konjugierte with the "receptor" (such as streptavidin) for use in pre-targeting the tumor, where the conjugate antibody-receptor is administered to the patient, followed by removal not bound conjugate from the circulation using a means for deducing, and then the introduction of a "ligand" (e.g., the avidin) which is conjugated to a cytotoxic agent (e.g., radionuclide).

Illustrative immunoconjugate - thio conjugates-antibody-drug

a. Antibodies against CD79b with the built-in cysteine residues

DNA encoding amino acid sequence variants of antibodies against CD79b with the built-in cysteine residues and source of antibodies against CD79b according to the invention, obtained in various ways, which include, but are not limited to, the allocation of �arodnogo source (in the case of naturally occurring variants of the amino acid sequence), getting by using site-directed (or oligonucleotide-mediated) mutagenesis (Carter (1985) et al. Nucl Acids Res. 13: 4431-4443; Ho et al. (1989) Gene (Amst.) 77:51-59; Kunkel et al. (1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al. (1998) J. Biol. Chem. 273:20252-20260), mutagenesis by PCR (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 Valletta et al. (1989) Nuc. Acids Res. 17:723-733), and cassette mutagenesis (Wells et al. (1985) Gene 34:315-323) previously obtained DNA encoding the polypeptide. The mutagenesis protocols, kits and reagents are commercially available, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations also contribute, by oligonucleotide-directed mutagenesis using double stranded plasmid DNA as template using mutagenesis based on PCR (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al. (1983) Methods Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500). Variants of recombinant antibodies also can be designed by manipulating with restriction enzymes or by PCR overlapping extension with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon to be replaced(replacements). To obtain DNA encoding such mutant antibodies with the built-in cysteine residues, you can use the methods of mutagenesis (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).

To obtain human antibodies against CD79b and fragments of antibodies toin vitrofrom the sets of genes of variable (V) domains of immunoglobulins unimmunized donors can use the technology of phage display (McCafferty et al., Nature 348:552-553 (1990)). In accordance with this method, the genes of the V-domain antibody clone in frame read either the main or secondary gene sheath of the filamentous bacteriophage, such as M13 or fd, and they are exhibited as functional fragments of the antibodies on the surface of phage particles. Because filamentous particle contains a single-stranded DNA copy of the phage genome, selection on the basis of the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting such properties. Thus, the phage mimics some of the properties of B-cells (Johnson et al. (1993) Current Opinion in Structural Biology 3:564-571; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; Griffith et al. (1993) EMBO J. 12:725-734; US 5565332; US 5573905; US 5567610; US 5229275).

Antibodies against CD79b can chemically be synthesized using known methods of synthesis of oligopeptides or you can obtain them and clean using recombinant technology. The corresponding amino acid sequence, or part thereof, can be obtained by direct peptide synthesis using solid-phase with�osobov (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 synthesisin vitrocan be carried out using methods or by using automation. Automated solid-phase synthesis can be performed, for example, using protected by t-BOC or Fmoc amino acids and using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. The various parts of antibodies against CD79b or CD79b polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the required antibodies against CD79b or CD79b polypeptide.

To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al.(1992) Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al. (1985)Science, 229:81) or to get them directly from recombinant host cells. Fab, Fv and scFv fragments of antibodies against CD79b can be expressed and secreted inE. colithus, providing a simple method for producing large amounts of these fragments. Fragments of antibodies can be isolated from phage libraries of antibodies described above. Alternative fragments, Fab'-SH can be allocated directly fromE. coliand Khimich�ski link with obtaining fragments F(ab') 2(Carter et al. (1992) Bio/Technology 10:163-167), or they can be isolated directly from recombinant cell culture host. Antibodies against CD79b may be a single-chain Fv fragment (scFv) (WO 93/16185; U.S. patent No. 5571894; and U.S. patent No. 5587458). Fragment antibodies against CD79b could also be a "linear antibody" (US 5641870). These linear fragments of antibodies can be monospecifičeskoj or bespecifically.

The following description relates primarily to production of antibodies against CD79b by culturing cells transformed or transfected with a vector containing the encoding CD79b antibody against the nucleic acid. DNA encoding antibodies against CD79b, can be obtained from a cDNA library derived from tissue allegedly possessing mRNA antibodies against CD79b and expressing it on beyond the detection level. Thus, DNA antibodies against CD79b person or CD79b polypeptide can be conveniently obtained from a cDNA library derived from human tissue. The gene encoding the antibody against CD79b, also can be obtained from a genomic library or by known methods of synthesis (e.g., automated nucleic acid synthesis).

The methods of construction, selection, and receiving according to the invention provide antibodies against CD79b with the built-in remnants of the cyst�on, which are reactive toward electrophilic functional groups. In addition, these methods provide compounds of conjugates of antibodies, such as compounds of conjugates of the antibody-drug (ADC) with molecules of the drug in certain prescribed selective areas. Reactive cysteine residues on the surface of antibodies allow specific conjugation with a group of medicines via Tilney reactive group, such as maleimide or haloacetic. Nucleophilic thiol reactivity funkcionalnoe group of the Cys residue to maleimide group approximately 1000 times higher than the reactivity to any other functional group of the amino acid in the protein, such as amino group of lysine residues or N-terminal amino group. Specific to the thiol group of the functional group in iodoacetyl and maleimide reagents may react with amino groups, however, require a higher pH (>of 9.0) and longer reaction time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The number of free tylnej groups in the protein can be assessed by standard analysis of an Elman. Immunoglobulin M represents one of the examples linked by a disulfide of pentamera, and immunoglobulin G is an example of a protein with the inner�mi disulfide bonds, linking the subunits together. In proteins such as these, requires reduction of the disulfide bonds in such a reagent, as dithiotreitol (DTT) or selenol (Singh et al. (2002) Anal. Biochem. 304: 147-156) for the formation of reactive free thiol group. This approach may lead to the loss of tertiary structure of the antibody and the specificity of binding antigen.

Analysis PHESELECTOR (phage ELISA for selection of reactive tylnej groups) allows the detection of reactive groups of cysteine antibodies in phage ELISA format, thereby facilitating the design of antibodies with the built-in cysteine residues (Junutula, J. R. et al. (2008) J Immunol Methods 332:41-52; WO 2006/034488; US 2007/0092940). Antibody with built in his cysteine residues deposited on the surface of the wells, followed by incubation with fagbemi particles, the addition of HRP labeled secondary antibody and detection by absorption. Mutant proteins displayed on the phage, can be subjected to screening is fast, reliable and efficient manner. You can get a library of antibodies with the built-in cysteine residues and throw their selection of binding using the same approach to identify the appropriate inclusion of reactive sites of free Cys in a random phage protein libraries of antibodies or other proteins. This method includes reaccumulate proteins with cysteine, exposed on the phage, with an affinity reagent or reporter group which is reactive towards thiol group.

Analysis PHESELECTOR allows screening reactive tylnej groups in antibodies. An example is the identification of an option A121C this way. You can conduct an effective search for a Fab molecule in order to identify more options thio-Fab with reactive toolname groups. For identification and quantitative determination of accessibility of solvent to the amino acid residues in the polypeptide used the parameter representing the relative accessibility of the surface. The availability of the surface can be expressed in surface area (Å2), which can be contacted with the solvent molecule such as water. The space occupied by water, is approximately a sphere with a radius of 1.4 Å. Freely available or is licensed software (Secretary to CCP4, Daresbury Laboratory, Warrington, WA4 4AD, United Kingdom Fax: (+44) 1925 603825, or by Internet: www.ccp4.ac.uk/dist/html/INDEX.html as software package CCP4 Suite of crystallography, which uses algorithms to calculate the distance of the surface of each amino acid in the protein obtained with the known x-ray crystallography coordinates ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Act. Cryst. D50:760-763). Two illustrative module software which do not calculate the distance of the surface, represent "AREAIMOL and SURFACE", on the basis of algorithms B. Lee and F. M. Richards (1971) J. Mol. Biol. 55:379-400. AREAIMOL defines available for solvent surface protein as the location of a probe sphere (corresponding to the solvent molecule) as it rolls over the van der Waals surface of the protein. AREAIMOL calculates the surface area available for solvent, by creating points on the surface approximately every atom of the extended sphere (the distance from the center of the atom, equal to the sum of the radii of the atom and probe), and by eliminating those points that lie in equivalent areas associated with neighboring atoms. AREAIMOL finds accessible to the solvent, the size of the atoms in the PDB file coordinates, and summarizes the available area for the remainder, the chain or the whole molecule. Available area (or difference of squares) for individual atoms can be written to the results file pseudo-PDB. AREAIMOL assumes a single radius for each element, and recognizes only a limited number of distinct elements.

AREAIMOL and SURFACE give the absolute availability, i.e. the number of square angstroms (Å). The relative accessibility of the surface is calculated relative to the standard state corresponding to the amino acid�those in the polypeptide. The standard state is a Tripeptide Gly-X-Gly, where X represents the interest an amino acid, and a standard condition should be an "extended" conformation, i.e. similar conformation in the beta chains. The elongated conformation maximizes the availability of X. the Computed available area is divided into available space in the standard state of the Tripeptide Gly-X-Gly, and is provided to the portion that represents the relative ease of access. The percentage availability is a relative availability multiplied by 100. Another illustrative algorithm for calculating the surface distance based on the module SOLV program xsae (Broger, C., F. Hoffman-LaRoche, Basel), which calculates the relative accessibility of amino acid residues for the water sphere on the basis of x-coordinates of the polypeptide. The relative surface accessibility of each amino acid in the antibody can be calculated using available information on the crystal structure (Eigenbrot et al. (1993) J Mol. Biol. 229: 969-995).

DNA encoding the antibodies with the built-in cysteine residues, are easily identified and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specific contact with the genes encoding the heavy and light chains of antibodies of the mouse). To�etki of hybridomas serve as a source of such DNA. After isolation, the DNA can be placed into expression vectors, which are then transferout in the host cell, such as cells ofE. coliof COS cells monkeys, cells of the Chinese hamster ovary (CHO), or other cells-the hosts are mammalian cells such as myeloma (US 5807715; US 2005/0048572; US 2004/0229310) that otherwise do not produce protein antibodies, providing a synthesis of monoclonal antibodies in the recombinant cell host.

After the design and selection of antibodies with the built-in cysteine residues, for example thio-Fab, with the built-in highly reactive unpaired Cys residues, it is possible to get free residues of the amino acid cysteine by: (i) expression in a bacterial system, such asE. coli(Skerra et al. (1993) Curr. Opinion in Immunol. 5:256-262; Pluckthun (1992) Immunol. Revs. 130: 151-188) or cell culture system of mammals (WO 01/00245), for example, in cells of the Chinese hamster ovary (CHO); and (ii) purification using conventional methods of protein purification (Lowman et al. (1991) J. Biol. Chem. 266(17): 10982-10988).

Embedded thiol group of Cys react with electrophilic linker reagents and intermediate compounds, the drug-linker with the formation of conjugates of antibodies with built in his cysteine residues and medicines and other labeled antibodies with the built-in cysteine residues. �the gang Cys in antibodies with the built-in cysteine residues, and Cys residues present in the original antibody, which form a pair and constitute mesapotamia and vnutriserdechne disulfide bonds, do not possess any reactive toolname groups (until they are treated with reducing agent) and does not react with electrophilic linker reagents or intermediate compounds, the drug-linker. Newly built Cys residue may remain unpaired, and are able to respond, i.e., to form a conjugate with an electrophilic linker reagent or intermediate connection of a drug-linker, such as a drug-maleimide. Illustrative of the intermediate drug-linker include: MC-MMAE, MC-MMAF, MC-vc-PAB-MMAE and MC-vc-PAB-MMAF. Structural provisions built Cys residues of the heavy and light chains are numbered according to the numbering system for consistency. This system of numbering by sequence correlates with the numbering system of Kabat (Kabat et al., (1991) Sequences of Proteins of Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), starting from N-Terminus, and is different from the numbering system of Kabat (bottom row) inserts specified by a, b, c. Using the numbering system of Kabat, the true linear amino acid sequence may contain fewer or additional amino acids amino acid�you corresponding to a shortening of, or insertion into FR or CDR of the variable domain. Altered areas of the heavy chain with embedded cysteine residues are indicated using the numbering schemes for the sequence and numbering according to Kabat.

In one embodiment, the implementation, the antibody against CD79b with built in his cysteine residues obtained by the process including:

(a) replacing one or more amino acid residues of the original antibody against CD79b cysteine; and

(b) determining the reactivity of thiol groups in the antibody against CD79b with built in his cysteine residues by reaction of the antibody with built in his cysteine residues with reactive toward thiol group reagent.

Antibody with built in his cysteine residues may be more reactive than the original antibody, reactive to thiol group reagent.

Free cysteine amino acid residues may be located in heavy or light chains, or in constant or variable domains. Fragments of antibodies, such as Fab, also can be designed with one or more amino acids cysteine substituting amino acid fragment of the antibody, with the formation of fragments of antibodies with the built-in cysteine residues.

Another embodiment of the invention relates to a method p�of producing (manufacturing) antibodies against CD79b with built in his cysteine residues, including:

(a) embedding one or more residues of the amino acid cysteine in the original CD79b antibody against to obtain antibodies against CD79b with built in his cysteine residues; and

(b) determining the reactivity of thiol groups in the antibody with built in his cysteine residues to reactive to thiol group reagent;

where the antibody with built in his cysteine residues is more reactive than the original antibody with a reactive to thiol group reagent.

Stage (a) of the method of producing antibodies with built in his cysteine residues may include:

(i) mutagenesis nucleic acid sequence that encodes the antibody with built in his cysteine residues;

(ii) the expression of antibodies with built in his cysteine residues; and

(iii) isolation and purification of antibodies with built in his cysteine residues.

Stage (b) of the method of producing antibodies with built in his cysteine residues may include the expression of antibodies with built in his cysteine residues on the viral particle selected from ragovoy or fahmideh particles.

Stage (b) of the method of producing antibodies with built in his cysteine residues may also include:

(i) reaction of the antibody with built in his cysteine residues with reaction�-capable thiol group affinity reagent with getting affine labeled antibodies with built in his cysteine residues; and

(ii) measuring the binding affinity of the labeled antibody with built in his cysteine residues with particulate media.

Another embodiment of the invention relates to a method of screening for antibodies with the built-in cysteine residues with a highly reactive unpaired amino acids cysteine in relation to the reactivity of thiol groups, including:

(a) embedding one or more residues of the amino acid cysteine in the original antibody to obtain antibodies with built in his cysteine residues;

(b) reaction of the antibody with built in his cysteine residues with reactive towards the thiol group of an affinity reagent with the formation of the affine labeled antibody with built in his cysteine residues; and

(c) measuring the binding affinity labeled antibody with built in his cysteine residues with particulate media; and

(d) determining the reactivity of thiol groups in the antibody with built in his cysteine residues to reactive to thiol group reagent.

Stage (a) of the method of screening for antibodies with the built-in cysteine residues may include:

(i) mutagenesis nucleic acid sequence that encodes the antibody with built in his cysteine residues;

(ii) the expression of antibodies with built - �mi in him cysteine residues; and

(iii) isolation and purification of antibodies with built in his cysteine residues.

Stage (b) of the method of producing antibodies with built in his cysteine residues may include the expression of antibodies with built in his cysteine residues on the viral particle selected from ragovoy or fahmideh particles.

Stage (b) of the method of producing antibodies with built in his cysteine residues may also include:

(i) reaction of the antibody with built in his cysteine residues with reactive towards the thiol group of an affinity reagent with getting affine labeled antibody with built in his cysteine residues; and

(ii) measuring the binding affinity labeled antibody with built in his cysteine residues with particulate media.

b. Options IgG anti-CD79b with built in his cysteine residues

The cysteine embedded in the section 118 of the heavy chain (EU numbering) (equivalent to position 118 of the heavy chain, the numbering sequence) of intact chimeric source of monoclonal antibodies against CD79b or plot light chain 205 (Kabat numbering) (equivalent to position 210 in the light chain, the numbering sequence) of intact chimeric source of monoclonal antibodies against CD79b ways of embedding cysteine, as described in this document.

The obtained antibody with the TSA�m ilitary them in cysteine residues with a cysteine at position 118 of the heavy chain (EU numbering) were: (a) thio-hu2F2.D7-HC (A118C) with the sequence of the heavy chain (SEQ ID NO:85) and light chain sequence (SEQ ID NO:86), figure 17.

The obtained antibody with the built-in cysteine residues with a cysteine at position 205 of the light chain (numbering according to Kabat) were: (a) thio-hu2F2.D7-LC (V205C) with the sequence of the heavy chain (SEQ ID NO:87) and the light chain sequence (SEQ ID NO:88), figure 18.

These monoclonal antibodies with the built-in cysteine residues expressible in cells CHO (Chinese hamster ovary) by temporarily fermentation in a medium containing 1 mm cysteine.

According to one variant of implementation, humanized antibodies 2F2 against CD79b with the built-in cysteine residues contain one or more of the following sequences of the heavy chain with a free amino acid residue cysteine (SEQ ID NO:91-99, table 2).

Table 2:
Comparison of the numbering of the heavy chain according to the sequence, Kabat and EU for variants of a humanized antibody 2F2 against CD79b with built in his cysteine residues
SEQUENCEThe NUMBERING SEQUENCENUMBERING ACCORDING to KabatThe EU NUMBERINGSEQ ID NO:
EVQLCESGGGV5C V5C91
LRLSCCASGYTA23CA23C92
MNSLRCEDTAVA88CA84C93
TLVTVCSASTKS112CS112C94
VTVSSCSTKGPA114CA114CA118C95
VSSASCKGPSVT116CT116CT120C96
WYVDGCEVHNAV278CV278CV282C97
KGFYPCDIAVES371CS371CS375C98
PPVLDCDGSFFS396CS36C S400C99

According to one variant of implementation, the chimeric antibody 2F2 against CD79b with the built-in cysteine residues contain one or more of the following sequences of the heavy chain with a free amino acid residue cysteine (SEQ ID NO:100-108, table 3).

Table 3:
Comparison of the numbering of the heavy chain according to the sequence, Kabat and EU to variants of the antibody ch2F2 against CD79b with built in his cysteine residues
SEQUENCEThe NUMBERING SEQUENCENUMBERING ACCORDING to KABATThe EU NUMBERINGSEQ ID NO:
QVQLCQPGAEQ5CQ5C100
VKLSCCASGYTK23CK23C101
LSSLTCEDSAVS88CS84C102
TSVTVCLASTKS112CS112C103
VTVSSCSTKGPA114CA114CA118C104
VSSASCKGPSVT116CT116CT120C105
WYVDGCEVHNAV278CV278CV282C106
KGFYPCDIAVES371CS371CS375C107
PPVLDCDGSFFS396CS396CS400C108

According to one variant of implementation, humanized antibodies 2F2 against CD79b with the built-in cysteine residues contain one or more of the following light chain sequences with a free amino acid residue cysteine (SEQ ID NO:109-115, table 4).

Table 4:
Comparison of the numbering of the light chain of the chain in sequence, and Kabat for variants of a humanized antibody 2F2 against CD79b with built in his cysteine residues
SEQUENCEThe NUMBERING SEQUENCENUMBERING ACCORDING to KABATSEQ ID NO:
SLSASCGDRVTV15CV15C109
EIKRTCAAPSVV115CV110C110
TVAAPCVFIFPS119CS114C111
FIFPPCDEQLKS126CS121C112
DEQLKCGTASVS132CS127C113
VTEQDCKDSTYS173CS168C114
GLSSPCTKSFNV210CV205C 115

According to one variant of implementation, the chimeric antibody 2F2 against CD79b with the built-in cysteine residues contain one or more of the following light chain sequences with a free amino acid residue cysteine (SEQ ID NO:116-122, table 5).

Table 5:
Comparison of the numbering of the light chain sequence and Kabat options for antibodies 2F2 against CD79b with built in his cysteine residues
SEQUENCEThe NUMBERING SEQUENCENUMBERING ACCORDING to KABATSEQ ID NO:
TLSVTCGQPASI15CI15C116
EIKRTCAAPSVV115CV110C117
TVAAPCVFIFPS119CS114C118
FIFPPCDEQLKS126CS121C119
DEQLKCGTASV S132CS127C120
VTEQDCKDSTYS173CS168C121
GLSSPCTKSFNV210CV205C122

c. Labeled antibodies against CD79b with the built-in cysteine residues

Antibodies against CD79b with the built-in cysteine residues according to the invention can be site-specific and effectively communicate with reactive toward thiol group reagent. Reactive to thiol group reagent may be a multifunctional linker reagent, the reagent labels for capture, i.e., the labeled reagent (e.g., reagent Biotin-linker), amenable to detection label (e.g. a fluorophore reagent), reagent for solid phase immobilization (e.g., SEPHAROSETM, polystyrene or glass), or the intermediate drug-linker. One example reactive to thiol group reagent is N-ethylmaleimide (NEM). In the illustrative embodiment, the implementation, the reaction of thio-Fab with the reagent Biotin-linker leads to biotinylation thio-Fab, which can be used to carry out the detection of the presence and measurement of the reactivity sun�rooyen cysteine residue. The reaction of thio-Fab with a multifunctional linker reagent leads to thio-Fab functionalized with a linker, which can then be subjected to reaction with a reagent of the group of medicinal products or with a different label. The reaction of thio-Fab with intermediate connection of a drug-linker leads to the conjugate thio-Fab and medicines.

The illustrative methods described herein can be applied mainly for identification and production of antibodies, and, more generally, of other proteins using stages of constructing and screening described in this document.

This approach can be used for conjugation of other reactive towards thiol group reagents, in which the reactive group is, for example, maleimide, iodoacetamide, pyridylsulfonyl or other reactino-capable thiol group partner for 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). Reactive to thiol group reagent may represent a group of the drug, a fluorophore such as a fluorescent dye such as fluorescein or rhodamine, a chelating agent for in�salesale or metal for radiation therapy, peptidyl or dipeptidyl label or capable of detecting the label, or modifying a removal tool, such as various isomers of polyethylene glycol, a peptide that binds to a third component, or another carbohydrate or lipophilic agent.

d. The use of antibodies against CD79b with the built-in cysteine residues

Antibodies against CD79b with the built-in cysteine residues and their conjugates can be used as therapeutic and/or diagnostic agents. In addition, the present invention relates to methods of prevention, control, cure or mitigate one or more symptoms associated with an associated with B-cell disorder. In particular, the present invention relates to methods of prevention, control, cure or mitigate one or more symptoms associated with cell-proliferative violation, such as a malignant tumor, e.g., lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone. In addition, the present izobreteny� relates to methods of diagnosis associated with CD79b disorders or predisposition to the development of such violation, and also to methods of identifying antibodies and antigen-binding fragments of antibodies, that preferentially bind associated with B-cells CD79b polypeptides.

Another embodiment of the present invention relates to the use of antibodies against CD79b with built in his cysteine residues to obtain the drug suitable for the treatment of a condition, that is responsible for associated with B-cells of the violation.

e. Conjugates of the antibody with built in his cysteine residues and medicines (thio conjugates-antibody-drug (TDC))

Another aspect of the invention is a compound of the conjugate antibody-drug containing antibodies against CD79b with built in his cysteine residues (Ab), and a group of medicines auristatin (D), where the antibody with built in his cysteine residues are linked through one or a few amino acid residues of cysteine linker group (L) with D; wherein the compound has the formula I:

Ab-(L-D)p(I)

where p is 1, 2, 3, or 4; and where an antibody with built in his cysteine residues obtained by a process comprising replacing one or more amino acids�'s the remnants of the original antibodies against CD79b one or more free amino acid residues of cysteine.

Another aspect of the invention relates to compositions containing a mixture of compounds antibody-drug of formula I where the average drug load on the antibody ranges from about 2 to about 5, or from about 3 to about 4.

In the figures 17-18 presents embodiments of conjugates of antibodies against CD79b with built in his cysteine residues and medicines (ADC), where a group of medicines that represents auristatin, associated with an integrated group of cysteine: light chain (LC-ADC) or heavy chain (HC-ADC).

The potential benefits of conjugates of antibodies against CD79b with built in his cysteine residues and medicines include increased safety (higher therapeutic index), improved PK parameters, the preservation vnutrisemejnyh disulfide bonds of the antibody, which may stabilize the conjugate and retain its active binding conformation, certain sections of conjugation of the drug, and that the receipt of conjugates of antibodies with built in his cysteine residues and drugs by conjugation of the antibody with the built-in cysteine residues with reagents drug-linker leads to a more homogeneous product.

Linkers

p> "Linker", "Linker element" or "link" means a chemical group that contains a covalent bond or a chain of atoms that covalently bind the antibody with a group of medicines. In various embodiments, the linker is referred to as L. "Linker" (L) is a bifunctional or multifunctional group, which can be used to associate one or more groups of the drug (D) and item-antibodies (Ab) to form conjugates antibody-drug (ADC) of formula I. Conjugates antibody-drug (ADC) can be conveniently obtained by use of a linker having a reactive functional group for binding with the drug and the antibody. Thiol group of cysteine in the antibody with built in his cysteine residues (Ab) can form a bond with an electrophilic functional group of the linker reagent, a group of medicinal products or intermediate connection of a drug-linker.

In one aspect, a linker has a reactive site which has an electrophilic group that is reactive to the nucleophilic cysteine, present in the antibody. Thiol group of cysteine in the antibody is reactive to an electrophilic group�e on the linker and forms a covalent bond with the linker. Suitable electrophilic groups include, but are not limited to, maleimido and haloacetic group.

Linkers include a divalent radical such as alkerdeel, Allen, heteroaryl, groups such as -(CR2)nO(CR2)n- recurring items from alkyloxy (e.g., polietilene, PEG, polymethylenes) and alkylamino (e.g., polyethylenimine, JeffamineTM); and esters and amides of dicarboxylic acids, including succinate, succinamide, diglycolate, malonate and caproamide.

Antibodies with the built-in cysteine residues react with linker reagents or intermediate compounds, the drug-linker with electrophilic functional groups such as maleimide or α-halocarbonyl, according to the conjugation method at page 766 Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773, and according to the Protocol of example 6.

The linker may consist of one or more linker components. Illustrative linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzeneboronic ("PAB"), N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"), N-Succinimidyl-(4-iodoacetyl)aminobenzoate ("SIAB"), ethyleneoxy-CH2CH2O - �the image quality of one or more repetitive elements ("EO" or "PEO"). Additional linker components known in the field and some of them are described in this document.

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

-Aa-Ww-Yy-

where:

-A - represents the extension piece, covalently linked to the thiol group of cysteine in the antibody (Ab);

a is 0 or 1;

each W independently represents an amino acid element;

w independently represents an integer in the range 0 to 12;

-Y - is a GS spacer element is covalently linked to the group of the drug; and

y is 0, 1 or 2.

Lengthening the

The extension piece (-A-), when present, is capable of binding element of the antibody with an amino acid element (-W-). In this respect, the antibody (Ab) has a functional group that can form a bond with a functional group of the extension element. Suitable functional groups that may be present on the antibody, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. In one aspect, the functional groups of the antibody are sulfhydryl or amino. Sulphide�date the group can be extracted by reduction of the intramolecular disulfide bonds of the antibody. Alternative sulfhydryl group can be obtained by reaction of the amino group of lysine in the antibody using 2-aminothieno (trot reagent) or other forming sulfhydryl reagent. In one embodiment, the implementation, the antibody (Ab) has a free thiol group of cysteine, which can form a bond with an electrophilic functional group of the extension element. Illustrative of the extension elements in the conjugates of formula I represented by formulas II and III, where Ab-, -W-, -Y-, -D, w and y are as defined above, and R17is a divalent radical selected from (CH2)r, C3-C8carbocycle, O-(CH2)rarylene, (CH2)rarylene, -aralen-(CH2)r-, (CH2)r-(C3-C8carbocycle), (C3-C8carbocyclic)-(CH2)r, C3-C8heterocyclyl, (CH2)r-(C3-C8heterocyclyl), -(C3-C8heterocyclyl)-(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(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH CH2O)r-CH2- and -(CH2CH2O)rC(O)NRb(CH2)r-; where Rbrepresents H, C1-C6alkyl, phenyl or benzyl; and r independently is an integer in the range 1-10.

Arilin includes divalent aromatic hydrocarbon radicals of from 6-20 carbon atoms formed by removal of two hydrogen atoms from aromatic ring system. A typical group of aryleno include, but are not limited to, radicals formed from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc.

Heterocyclyl groups include ring system in which one or more ring atoms are a heteroatom, for example nitrogen, oxygen and sulfur. Heterocyclyl radical contains from 1 to 20 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S. Heterocycle may be a monocycle having 3 to 7 members in the ring (from 2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or Bicycle, having from 7 to 10 members in the ring (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: a bicyclo system[4,5], [5,5], [5,6] or [6,6]. 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 �burden), in particular volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566.

Examples of heterocycles include, as non-limiting example, pyridyl, dihydropyridin, tetrahydropyridine (piperidyl), thiazolyl, tetrahydrothiophene, tetrahydrothiophene with oxidized sulfur, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thionaphthene, indole, indoline, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydropyranyl, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6H-1,2,5-thiadiazine, 2H,6H-1,5,2-diazenyl, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine, 2H-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indole, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, chinoxalin, chinazoline, cinnoline, pteridine, 4Ah-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, furutani, phenoxazines, isopropanol, bromanil, imidazolidinyl ureido, imidazolyl, pyrazolidine, pyrazolyl, piperazinyl, indolinyl, isoindolyl, hinokitiol, morpholinyl, ox�original, benzotriazolyl, benzisoxazoles, oxindoles, benzoxazolyl and satanail.

Carbonitrile groups include saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as Bicycle. The monocyclic carbocycle have from 3 to 6 atoms in the ring, more specifically 5 or 6 atoms in the ring. Bicyclic carbocycle have from 7 to 12 atoms in the ring, for example, placed in order of how bicyclo system[4,5], [5,5], [5,6] or [6,6], or 9 or 10 atoms in the ring, placed in order of how system bicyclo [5,6] or [6,6]. Examples of monocyclic carbocycles include 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, cycloheptyl and cyclooctyl.

You should understand all of the illustrative embodiment of the ADC of formula I, such as II-VI, that even when not explicitly stated, with the antibody bound from 1 to 4 drug groups (p=1-4), depending on the number of embedded cysteine residues.

Illustrative of the extension element of the formula II is formed from maleimido-caproyl (MC), where R17represents -(CH2)5-:

Illustrative of the extension element of the formula II formed �W maleimido-propanol (MP), where R17represents -(CH2)2-:

Another illustration of the extension element of the formula II is an extension piece, where R17represents -(CH2CH2O)r-CH2-, and r = 2:

Another illustration of the extension element of the formula II is an extension piece, where R17represents -(CH2)rC(O)NRb(CH2CH2O)r-CH2-, where Rbrepresents H and each r is 2:

Illustrative of the extension element of the formula III is an extension piece, where R17represents -(CH2)5:

In another embodiment, the implementation of the extension element is associated with the antibody against CD79b with built in his cysteine residues via a disulfide bond between the sulfur atom, with a built-in cysteine in the antibody and a sulfur atom extension element. A representative of the extension element of this embodiment represented by formula IV, where R17Ab-, -W-, -Y-, -D, w and y are as defined above.

In another embodiment of the reactive group of the extension element contains reactive with�asobou to the thiol group of a functional group. which can form a bond with a free thiol group of cysteine in the antibody. Examples of reactive to thiol group of functional groups include, but are not limited to, maleimide, α-haloacetic, activated esters, such as succinimide esters, 4-nitroaniline esters, pentafluorophenyl esters, tetraterpenoids esters, anhydrides, acid chlorides, sulphonylchloride, isocyanates and isothiocyanates. A representative of the extension elements of this embodiment are represented by formulas Va and Vb, where-R17-, Ab-, -W-, -Y-, -D, w and y are as defined above:

In another embodiment, the implementation, the linker may be a dendritic linker type for covalent joining more than one group of medicines through branching multifunctional linker group to an antibody (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768; King (2002) Tetrahedron Letters 43:1987-1990). Dendritic linkers can increase the molar ratio of the drug and the antibody, i.e. the load that is associated with the efficiency of the ADC. Thus, when the antibody with built in his cysteine residues has only one reactive thiol group of cysteine, many groups �cartonnage means you can link through a dendritic linker.

Amino acid

The linker may contain amino acid residues. Amino acid element (-Ww-), when present, links the antibody (Ab) with a group of the drug (D) in the conjugate antibodies with built in his cysteine residues and drug (ADC) according to the invention.

-Ww- is a dipeptide, Tripeptide, tetrapeptide, Pentapeptide, Hexapeptide, heptapeptide, octapeptide, nonapeptide, Decapeptide, undecapeptide or dodecapeptide element. Amino acid residues which comprise an amino acid element, include a naturally occurring amino acid residues, as well as minor amino acids and naturally occurring analogues of amino acids such as citrulline. Each element W is independently has the formula shown below in square brackets, and w is an integer in the range 0 to 12:

where R19represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH 2, - (CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH22-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

or

When R19is different from hydrogen, the carbon atom is linked to R19is chiral. Each carbon atom is linked to R19independently is (S)- or (R)-configuration, or a racemic mixture. Amino acid elements, therefore, may be enantiomerically pure, racemic or diastereomeric.

Illustrative amino acid elements-Ww- include a dipeptide, a Tripeptide, a tetrapeptide or a Pentapeptide. Illustrative dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Illustrative tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues that contain a component in the form of amino linker, include naturally occurring amino acid residues, as well as minor amino acids and naturally occurring analogues of amino acids such as citrulline.

Amino acid element can fermentati�but to split one or more enzymes, including associated with tumor protease, releasing a group of medicinal products (-D), which in one embodiment, the implementation protheroesin vivoif released with the formation of the drug (D). Components in the form of amino acid linkers can be designed and optimized in their selectivity for enzymatic cleavage by specific enzymes, for example, associated with a tumor protease, cathepsin B, C and D or protease plasmin.

The GS spacer

The GS spacer element (-Yy-), when present (y = 1 or 2), links an amino acid element (-Ww-) with the group of medicinal products (D) when an amino acid element is present (w=1-12). Alternatively, the GS spacer element connects the extension piece with a group of medicines that when amino acid element is missing. The GS spacer element also associates a group of medicines with the element of the antibody, amino acid element and the extension element are absent (w, y = 0). The GS spacer elements represent the elements of two basic types: smoothshapes and not capable of self-cleavage. Not capable of self-cleavage of the GS spacer element is an element in which a part of the GS spacer element or all of the GS spacer element stauts� associated with a group of medicines after cleavage, in particular enzymatic, amino acid element from the conjugate antibody-drug or group drug-linker. When the ADC containing a glycine-glycine GS spacer element or a glycine GS spacer element undergoes enzymatic digestion associated with tumor cell protease associated with cells of a malignant tumor protease or associated with lymphocyte protease, the group glycine-glycine-drug or group of glycine-drug is cleaved from Ab-A3-Ww-. In one embodiment of the implementation, there is an independent hydrolysis reaction in the target cell that breaks down communication glycine-group of the drug and releasing the drug.

In another embodiment of the implementation, -Yy- is a u-aminobenzeneboronic (PAB) the element, part phenylene which is substituted with Qmwhere Q represents-C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-cyano; and m is an integer in the range 0-4.

Illustrative options for implementation is not capable of self-cleavage of the GS spacer element (-Y-) are: -Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one embodiment, the implementation group provides drug-linker or ADC in which the GS spacer element is absent (y=0), or their pharmaceutically acceptable salt or solvate.

Alternative ADC containing smoothshapes GS spacer element can release-D. In one embodiment of the implementation, -Y - is a PAB group that is linked with the-Ww- via the nitrogen atom of the amino group in the PAB group, and connected directly to-D via a carbonate, carbamate or a simple ester group, where the ADC has an illustrative structure:

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

Other examples slotsplaying spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), heterocyclic analogs PAB (US 2005/0256030) beta-glucuronide (WO 2007/011968), and ortho - or para-aminobenzoate. You can use the spacers that undergo cyclization upon amide hydrolysis, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo ring system[2.2.1] and bicyclo[2.2.2] (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and amide 2-aminophenylamino acid (Amsberry, et al. (1990) J. Org. Chem. 55:587). Elimination of amine containing drugs that are substituted by glycine (Kingsbury et al. (1984) J. Med. Chem. 27: 1447) are also an example of smoothshapes of the spacer fit into the ADC.

Illustrative of the GS spacer elements (-Yy-) correspond to the formulas X-XII:

Dendritic linkers

In another embodiment, the implementation, the linker L may be a dendritic linker type for covalent joining more than one group of medicines through branching multifunctional linker group to an antibody (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic linkers can increase the molar ratio of the drug and the antibody, i.e. the load that is associated with the efficiency of the ADC. Thus, when the antibody with built in his cysteine residues has only one reactive thiol group of cysteine, many groups of drugs can be associated through a dendritic linker. Illustrative embodiments of the branched dendritic linkers include 2,6-bis(gidroximetil)-p-kretly and 2,4,6-Tris(gidroximetil)phenolic gendarmerie elements (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, is�the implementation, the GS spacer unit is a branched bis(gidroximetil)styrene (BHMS), which can be used to incorporate and release multiple drugs having the structure:

containing 2-(4-aminobenzylidene)propane-1,3-diol dendrimeric element (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 between 0-4; n is 0 or 1; and p ranges from 1 to 4.

Illustrative embodiments of the compounds of the conjugate antibody-drug of formula I include XIIIa (MC), XIIIb (val-cit), XIIIc (MC-val-cit) and XIIId (MC-val-cit-PAB):

Other illustrative embodiments of the compounds of the conjugate antibody-drug formula Ia include XIVa-e:

where X is a

or

Y represents

or

and R independently represents H or C1-C6alkyl; and n is from 1 to 12.

In other�Ohm variant of implementation, the linker has reactionsyou functional group, which has a nucleophilic group that is reactive to an electrophilic group present in the antibody. Suitable electrophilic group on the antibody include, but are not limited to, the carbonyl group of aldehydes and ketones. Heteroatom of a nucleophilic group of a linker can react with an electrophilic group on the antibody and form a covalent bond element in antibodies. Suitable nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and originated. The electrophilic group on an antibody provides a convenient site for binding with the linker.

Typically, the peptide linkers of the type can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, in accordance with the method of liquid-phase synthesis (E. Schroder and K. Lubke (1965) "The Peptides", volume 1, pp 76-136, Academic Press) that is well known in the chemistry of peptides. Linker intermediate compounds can be obtained by any combination or sequence of reactions involving the GS spacer, extension elements and amino acid. In the GS spacer, extension and amino acid elements can be�ü used reactive functional groups, which its properties are electrophilic, nucleophilic, or free. Reactive functional groups include, but are not limited to, a carboxy, a hydroxyl, a pair-nitrophenylarsonic, isothiocyanate and leaving groups, such as O-mesyl, O-tosyl, -Cl, -Br, -I or maleimide.

For example, the charged substituent such as sulfonate (-SO-3) or ammonium, may increase water solubility of the reagent and facilitate the binding reaction of the linker reagent with the antibody or group of medicines, or to facilitate the binding reaction of Ab-L (intermediate antibody-linker) with D, or D-L (intermediate drug-linker) with Ab, depending on the method of synthesis used to obtain the ADC.

Linker reagents

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

Conjugates of the antibody-drug can also be obtained using the following linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4 - vinylsulfonic)benzoate), and including 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 from Pierce Biotechnology, Inc., ThermoScientific, Rockford, IL, and other suppliers of reagents. Bis-maleimide reagents allow attaching thiol group of the antibody with built in his cysteine residues by thiol-containing group of the medicinal product, label, or linker intermediate compound, sequentially or simultaneously. Other functional groups, except maleimide that are reactive towards the thiol group of the antibody with built in his cysteine residues, a group of the medicinal product, label, or linker intermediate include connection todatetime, bromoacetamide, vinylpyridine, disulfide, pyridyldithio, isocyanate and isothiocyanate.

Suitable linker reagent� can also be obtained via other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO), or synthesized in accordance with methods described in Toki et al. (2002) J. Org. Chem. 67: 1866-1872; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7: 180 to 186; US 6214345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.

Lengthening the elements of the formula (IIIa) can be embedded into a linker by reaction of the following linker reagents with the N-end amino acid element:

where n is an integer in the range 1-10 and T is-H or-SO3Na;

where n is an integer in the range 0-3;

and

Extension elements can be embedded into a linker by reaction of the following bifunctional reagents with the N-end amino acid element:

where X represents Br or I.

Lengthening the elements of the formula can also be embedded in the linker by reaction of the following bifunctional reagents with the N-end amino acid element:

Illustrative dipeptide linker reagent valine-citrulline (val-cit or vc) having maleimide extension piece and pair-aminobenzeneboronic (PAB) �aboutwebsite spacer, has the structure:

Illustrative dipeptide linker reagent phe-lys(Mtr is mono-4-methoxytrityl) having maleimide the extension piece and smoothshapes GS spacer element PAB, can be obtained according to Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60, and he has the structure:

Obtaining conjugates antibodies against CD79b with built in his cysteine residues and drug

The ADC of formula I can be obtained in several ways, using the reaction conditions and reagents of organic chemistry, known to specialists in this area, including: (1) the reaction of the group of the cysteines in the antibody with built in his cysteine residues from the linker reagent with the formation of intermediate compounds antibody-linker Ab-L, via a covalent bond, followed by reaction with an activated group of the drug D; and (2) reaction of a nucleophilic group in the group of the drug with a linker reagent with the formation of the intermediate drug-linker D-L, via a covalent bond, followed by reaction with a group of cysteines in the antibody with built in his cysteine residues. To obtain conjugates antibody-drug of formula I conjugation methods (1) and (2) can be used with different antibodies�and with the built-in cysteine residues, groups drugs and linkers.

Thiol group of cysteine antibodies are nucleophilic and capable of entering into reaction with the formation of covalent bonds with electrophilic groups on linker reagents and intermediate compounds, the drug-linker, including: (i) active esters such as NHS esters, HOBt esters, halogenfree and acid halides; (ii) alkyl - and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimide group, and (iv) disulfides, including peredelnye disulfides, by sulphide exchange. Nucleophilic groups on a group of drugs include, but are not limited to: an amino group, Tilney, hydroxyl group, group, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazines, able to react with the formation of covalent bonds with electrophilic groups on linker groups and of the linker reagents.

Reactivity of the antibody for conjugation with linker reagents can be enhanced by treatment with a reducing agent such as DTT (dithiothreitol, the reagent Cleland) or TCEP (hydrochloride Tris(2-carboxyethyl)phosphine; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA), followed by re-oxidation with the formation of megapo�echnik and vnutrisemejnyh disulfide bonds (example 5). For example, intact monoclonal antibodies with the built-in cysteine residues (thio-Mab), expressed in CHO cells, recover by approximately 50-fold molar excess of TCEP for 3 hrs at 37°C to restore the disulfide bonds in cysteine adducts which may form between the newly integrated cysteine residues and the cysteine present in the culture medium. Restored thio-Mab was diluted and placed in a column HiTrap S in 10 mm sodium acetate, pH 5, and eluted by PBS containing 0.3 M sodium chloride. Disulfide bonds between cysteine residues of the original Mab reinstall with dilute (200 nm) aqueous copper sulfate (CuSO4) at room temperature over night. Alternative dehydroascorbic acid (DHAA) is an effective oxidant to re-restore vnutrisemejnyh disulfide groups of the antibody with built in his cysteine residues after reductive cleavage of the cysteine adducts. You can also use other oxidizing agents, i.e., oxidizing agents, and the conditions of oxidation, which is known in this field. Also an effective oxidation by ambient air. At this stage, soft partial re-oxidation effectively formed vnutriserdechne disulfides with high precision� and stored thiol group-embedded cysteine residues. Approximately 10-fold excess of the intermediate drug-linker, such as MC-vc-PAB-MMAE, was added, mixed and allowed to stand for about one hour at room temperature to ensure the conjugation and formation of the conjugate antibodies against CD79b-drug. Compound for conjugation was filtered and placed in column (HiTrap S and suirable it to remove the excess of the intermediate drug-linker and other impurities.

Figure 16 presents the overall process of obtaining antibodies with built in his cysteine residues, expressed from cell culture, conjugation. When the cell culture medium contains cysteine can form disulfide adducts between the newly integrated amino acid cysteine and cysteine from the medium. These adducts of cysteine, represented as a circle in the illustrative thio-Mab (left) figure 16, it is necessary to restore to generate antibodies with the built-in cysteine residues, reactive for conjugation. Adducts of cysteine, probably together with various megamachine disulfide bonds, is subjected to reductive cleavage with obtaining reduced forms of the antibody with reducing agents such as TCEP. Megamachine disulfide bonds IU�do paired cysteine residues are re-formed under conditions of partial oxidation with copper sulfate, DHAA, or under the influence of ambient oxygen. Newly built, constructed and unpaired cysteine residues remain available for reaction with linker reagents or intermediate compounds, the drug-linker with the formation of conjugates of the antibody according to the invention. Thio-Mab, expressed in cell lines of mammals, lead to conjugate outside Cys adduct with a built-Cys through education connection-S-S-. Thus, purified thio-Mab treated by the processes of recovery and re-oxidation as described in example 5 to obtain a reactive thio-Mab. These thio-Mab used in the conjugate containing maleimide cytotoxic drugs, fluorophores and other markers.

10. Immunoliposome

Antibodies against CD79b, described herein, also can be produced in the form of immunoliposome. "Liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactants that are suitable for drug delivery to a mammal. Components of liposomes usually form a two-layer structure, similar to the placement of the lipids of biological membranes. Liposomes containing the antibody, prepared by methods known in this field, such as described � Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); U.S. patent No. 4485045 and 4544545; and WO97/38731 published October 23, 1997. Liposomes with enhanced circulation time are described in U.S. patent No. 5013556.

Especially suitable liposomes can be obtained by the method of reversed-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and a derivative of phosphatidylethanolamine with PEG (PEG-PE). Liposomes are extruded through filters with defined pore size to obtain liposomes with the desired diameter. Fab'-fragments of the antibodies of the present invention can be konjugierte with liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via the reaction of disulfide exchange. In the liposome contains optional chemotherapeutic agent. Cm. Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989).

B. Specific methods of obtaining antibodies

1. Screening antibodies against CD79b with the required properties

Methods for obtaining antibodies that bind to CD79b polypeptides described above. If desirable, it is possible to select antibodies with certain biological characteristics.

The effects of antibodies against CD79b according to the invention on the inhibition of growth can be assessed by methods known in this field, e.g., using cells which Express CD79b polypeptide either endogenously or pokeransikte CD79b gene. For example, the corresponding cell line tumors and cells, transfetsirovannyh CD79b, you can handle a monoclonal antibody against the cage according to the invention at various concentrations for a few days (e.g., 2-7 days) and painted with crystal violet or MTT or analyzed by some other colorimetric method of analysis. Another way to define proliferation represents the comparison of capture3H-thymidine-treated cells in the presence and absence of antibodies against the cage according to the invention. After treatment with antibody, the cells were collected and determine the amount of radioactivity of the obtained DNA in a scintillation counter. Appropriate positive controls include treatment of the selected cell line growth inhibitory antibody against whom it is known that it inhibits the growth of this cell line. The inhibition of growth of tumor cellsin vivocan be defined in various ways known in this field. Cell tumors can be a cell that sverkhekspressiya CD79b polypeptide. Antibodies against CD79b inhibits cell proliferation expressing tumor cells CD79bin vitroorin vivoby about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and �still more preferably by about 50-100% or 70-100%, in one embodiment of the implementation, when the antibody concentration from about 0.5 to 30 μg/ml. the Inhibition of growth can be determined by the concentration of the antibody is from about 0.5 to 30 μg/ml or about 0.5 nm to 200 nm in cell culture, where the growth inhibition is determined 1-10 days after exposure of tumor cells to the antibody. The antibody is growth inhibitoryin vivoif the introduction of antibodies against CD79b in an amount of from about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size and decrease the proliferation of tumor cells in a range of approximately from 5 days to 3 months after the first administration of the antibody, preferably within about 5 to 30 days.

For selection of antibodies against CD79b, which cause cell death, it is possible to evaluate the loss of membrane integrity, as indicated, for example, by means of the engagement of propidium iodide (PI), trypan blue or 7AAD, relative to control. Analysis of capture PI can be carried out in the absence of complement and immune effector cells. Expressing CD79b polypeptide tumor cells were incubated with medium alone or with medium containing of the appropriate monoclonal antibody against CD79b (e.g., at a concentration of 10 μg/ml). Cells were incubated for 3 days. After each treatment the cells were washed � divided into aliquots served by the grid in 35-mm tubes 12×75 (1 ml per tube, 3 tubes in the treated group) for removal of cell clumps. Then in a test-tube add PI (10 μg/ml). Samples can be analyzed using a FACSCAN flow cytometerTMand software FACSCONVERTTMCellQuest (Becton Dickinson). Antibodies against CD79b, which lead to a statistically significant level of cell death, determined to seize the PI, it is possible to select as inducing cell death antibodies against CD79b.

For the screening of antibodies that bind to the epitope on CD79b polypeptide associated interest antibody, it is possible to carry out a common analysis of cross-blocking, such as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This analysis can be used to detect the presence of antibody binding to the same site or epitope is associated with known antibodies against CD79b according to this invention. Alternatively, or additionally, it is possible to carry out mapping of epitopes of ways known in this field.For example, to identify the contact residues in the sequence of an antibody can be made of a mutation, for example, alanine scanning. The mutant antibody is initially tested for binding to a polyclonal antibody to achieve the desired folding. In another method, the peptides, sootvetstvuyuschim parts of the CD79b polypeptide, can be used in ways competitive analysis with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

2. Some methods of screening libraries

Antibodies against CD79b according to the invention can be obtained by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, in this area there are different ways to generate phage display libraries and screening these libraries against antibodies having the desired binding characteristics. Such methods are described, mainly, in Hoogenboom et al. (2001), Methods in Molecular Biology 178:1-37 (O'brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, in Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

Essentially, the synthetic clones of antibodies are subjected to selection through phage display libraries that contain phage, which shows the different fragments of the variable regions (Fv) of the antibody, fused with the protein shell of the phage. Such phage library sorting using affinity chromatography against the desired antigen. Clones expressing Fv-fragments, is able to communicate with the desired antigen, adsorbed on the antigen and, thus, they are separated from non-binding clones in the library. Then linking clones eluted from the antigen, and with their�holding can be further improved through additional cycles of adsorption/elution of the antigen. By developing a suitable method of screening with antigen for selection of interest phage clone with the subsequent construction of a clone of intact antibodies against CD79b using the Fv sequences from the phage of interest clone and appropriate sequences of the constant region (Fc), described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3, can be obtained from any of antibodies against CD79b according to the invention.

In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one from the light (VL) and heavy (VH) chains, both of which are present three hypervariable loops (HVR) or complementarity determining region (CDR). The variable domains can be functionally displayed on phage, either as single-chain Fv fragments (scFv), in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they both are fused with a constant domain and interact ecovalence, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, phage clones encoding the scFv and phage clones encoding the Fab, jointly referred to as "fagbemi Fv clones" or "Fv clones".

The repertoire of VH genes and VL separately to clone polymerase chain reaction (PCR) and subjected to random recombination in phage libraries, which then conduct the search for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need of constructing a hybrid. Alternatively, you can clone the naive repertoire to ensure a single source of human antibodies to a wide range of improper and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). In conclusion, libraries naive repertoire can also be obtained synthetically by cloning are not subjected to rearrangeable segments V-genes from stem cells, and the use of primers for PCR containing random sequence to encode the highly variable CDR3 regions and to conduct the rearrangementin vitroas described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992).

In certain embodiments, using filamentous phages for exposing fragments of antibodies by fusion of minor protein shell pIII. Fragments of antibodies can be exposed in the form of single-chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain movable polypeptide spacer, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII, and each�I secreted into the periplasm of a bacterial host cell, going where the structure of the Fab protein shell, which is exhibited on the surface of phage by displacing some of the coat proteins of wild-type, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

Typically, nucleic acids encoding fragments of the antibody gene, derived from immune cells derived from humans or animals. If library is desirable, is shifted toward the clones directed against CD79b, immunize the individual through CD79b to ensure antibody response, and isolated spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBL) to construct the library. In a preferred embodiment of the implementation, you get a library of gene fragments of antibodies, is shifted toward the clones directed against CD79b, by ensuring the response of antibodies against CD79b in transgenic mouse having a functional set of genes of human immunoglobulins (and lacking a functional endogenous system products antibodies) so that immunization through CD79b leads to the emergence of B-cells producing antibodies against CD79b. The creation of producing human antibodies from transgenic mice are described below.

An additional increase in the content of populations of cells reactive against CD79b, can be ensured with the use�Institute of economy and management of a suitable screening method for the isolation of B-cells, expressing specific to CD79b membrane-bound antibody, e.g., by separation of cells using affinity chromatography with CD79b or adsorption of cells to labeled fluorochromes CD79b with subsequent fluorescence-activated sorting of cells (FACS).

Alternative use of spleen cells and/or B-cells or other PBL from an unimmunized donor provides a better representation of the possible antibody repertoire, and also allows the construction of libraries of antibodies using any animal (human or non-human), in which CD79b is not antigenic. For libraries, including the construction of antibody genesin vitrocollect the stem cells of an individual to obtain nucleic acids encoding is not subject to rearrangeable segments of antibody genes. Interest immune cells can be obtained from various species such as human, mouse, rat, species of lagomorphs, wolf, canine, feline, swine, cattle, horses and birds, etc.

Nucleic acid encoding the variable gene segments (including segments of VH and VL) was isolated from representing the interest of cells and amplificateur. In case subjected rearrangeable libraries of genes VH and VL, the desired DNA can be obtained by isolating genomic DNA or mRNA and� lymphocytes with subsequent polymerase chain reaction (PCR) with primers coinciding with the 5'- and 3'-ends subjected rearrangeable genes VH and VL, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby obtaining a varied repertoire of V-genes for expression. V-genes can be amplificatoare from cDNA and genomic DNA using reverse primers at the 5'end of the exon encoding the Mature V-domain and direct primers, located in the J-segment as described in Orlandi et al. (1989) and Ward et al., Nature, 341: 544-546 (1989). However, for amplification from cDNA, back primers can also be located in the leading exon as described in Jones et al., Biotechnol., 9: 88-89 (1991), and direct primers can be located in the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity, the primers can include degeneracy, as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, a variety of libraries to maximize the use of PCR primers designed for each family of V-genes for the amplification of all available locations VH and VL that are present in the sample nucleic acid of immune cells, for example, as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucl Acids Res., 21: 4491-4498 (1993). To clone the amplified DNA into expression vectors, in primer for PCR can be made of rare restriction sites � as a label on one end, as described in Orlandi et al. (1989), or by further amplification by PCR with labeled primer as described in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires subjected to the synthetic rearrangeable V-genes can be obtainedin vitrosegments V-genes. Most of the segments of the VH genes were cloned and tsakonian (as described in Tomlinson et al., J. Mol. Biol, 227: 776-798 (1992)), and mapped (described in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the loop H1 and H2) can be used to obtain various repertoires of VH-genes using primers for PCR, coding H3 loop with a diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). You can also obtain a repertoire of VH, where all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Segments are Vκ and Vλ were cloned and tsakonian (described in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to obtain synthetic light chain repertoires. Synthetic repertoires of V-genes, on the basis of a number of assemblies VH and VL, and the lengths L3 and H3, encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V-gene segments V-gene germ line can be rearrangeablein vito in accordance with the methods of Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992).

Repertoires of fragments of antibodies can be designed by combining a repertoire of VH genes and VL in different ways. Each repertoire can be created in separate vectors, and the vectors are subjected to recombinationin vitrofor example , as described in Hogrefe et al., Gene, 128: 119-126 (1993), orin vivoby combinatorial infection, e.g., by using the loxP system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). In recombination approachin vivoused double-stranded structure of the Fab-fragments to overcome the size limitations of the library defined by the efficiency of transformationE. coli. Naive repertoires of VH and VL cloned separately, one into the plasmid, and the other into a phage vector. Then combine two libraries by infection with phage containing fahmida bacteria so that each cell contains a different combination, and the library size is limited only by the number of cells present (about 1012clones). Both vectors contain the signals of recombinationin vivoso the genes of VH and VL recombine into a single replicon together and packaged into phage virions. These huge libraries provide large numbers of diverse antibodies with high affinity (Kd-1approximately 10-8M).

Alternative repertoires can CLO�ing sequentially in a single vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or combined by PCR and then cloned, for example as described in Clackson et al., Nature, 352: 624-628 (1991). Assembly by PCR can also be used to link the DNA of VH and VL DNA that encodes a movable peptide spacer, with the formation of a repertoire of single-chain Fv (scFv). Another way is to use the "build through PCR in cells" for combining genes of VH and VL in lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).

The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity (Kd-1approximately 106up to 107M-1), but affinity maturation can also simulatein vitroby means of constructing and re-selection from secondary libraries as described in Winter et al. (1994), above. For example, mutations can be made randomlyin vitrousing a polymerase with reduced accuracy (described by Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). In addition, affinity maturation can be performed by introducing mutations randomly in one or more CDRs, for example using PCR with primers having seq�tea sequence, spanning the CDR of interest, in selected individual Fv clones and screening for clones with the highest affinity. In WO 9607754 (published March 14, 1996) describes a method of inducing mutagenesis in a complementarity determining region light chain immunoglobulin to create a library of light chain genes. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring variants of the V-domains, obtained from unimmunized donors and screened for the highest affinity in several rounds of re-shuffling circuits, as described in Marks et al., Biotechnol., 10: 779-783 (1992). This method allows the production of antibodies and fragments of antibodies with an affinity of approximately 10-9M or less.

Screening libraries can be performed in various ways known in this field. For example, CD79b can be used for coating the wells of adsorption plates, to Express on the cell host, which are fixed on the adsorption plates or used in sorting cells, or konjugierte with Biotin for capture coated with streptavidin beads, or used in any way panning of phage display libraries.

Samples for phage display are contacted with immobilized CD79b in the�circumstances, suitable for binding at least part of the phage particles with the adsorbent. Usually choose the conditions, including pH, ionic strength, temperature, etc., simulating physiological conditions. Phages associated with the solid phase, washed, and then eluted with acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or through competition with CD79b antigen, for example in a way similar to the way competition with antigen in Clackson et al., Nature, 352: 624-628 (1991). In a single round of selection, the phage can be enriched in 20-1000 times. Moreover, the enriched phage can be grown in bacterial culture and subjected to subsequent rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and the ability to meet a large number of fragments of antibodies on a single phage with the antigen. Antibodies with fast dissociation kinetics (and a weak binding affinity) can be delayed by using a short washing, polyvalent phage display and high-density coating antigen on a solid phase. High density not only stabilizes the phage in multivalent interactions, but also promotes the re-binding of the phage, which dissociatives. Selection of antibodies with low kinetics of dissociati� (high affinity binding) can be achieved using a long wash and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and low density coating antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

Selection between fagbemi antibodies with different affinity to CD79b can even be used in affinity, which differs a bit. However, random mutation of a selected antibody (e.g., as occurs in some methods of affinity maturation) may lead to the emergence of many mutants, most of which binds to the antigen, and few of which have a higher affinity. When you restrict CD79b, a rare high-affinity phage could be competitive eliminated. To save all mutants with higher affinity, phages can be incubated with excess biotinylating CD79b, but with biotinylating CD79b in a concentration of lower molarity than the estimated molar affinity constant for CD79b. Then binding with high affinity phages can catch coated with streptavidin paramagnetic beads. Such "equilibrium capture" allows the selection of antibodies according to their affinity of binding, with a sensitivity that allows the selection of mutant clones with affinity, greater than only two times, from a large excess of phages with lower affinity. Also m�are encouraged to modify the terms and conditions used for washing phages associated with the solid phase, to differentiate on the basis of the kinetics of dissociation.

Clones directed against CD79b, can be selected on the basis of activity. In certain embodiments, the invention relates to antibodies against CD79b that bind to living cells that naturally Express CD79b. In one embodiment of implementation, the invention relates to antibodies against CD79b, which block the binding between the ligand and CD79b CD79b, but does not inhibit binding between the ligand and CD79b second protein. The Fv clones corresponding to such antibodies against CD79b, can be subjected to selection by (1) selection of clones directed against CD79b, ragovoy library, as described above, and optional reproduction of the selected population of phage clones by growing population in a suitable bacterial host; (2) breeding CD79b and a second protein against which blocking is desirable and non-blocking activity, respectively; (3) adsorption of phage clones directed against CD79b, immobilized CD79b; (4) the use of an excess of the second protein for the elution of any undesirable clones which recognise CD79b binding determinants which overlap or are shared with the binding determinants of the second protein; and (5) elution clone�in, which remain adsorbed after stage (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection processes described herein, one or more times.

DNA encoding educated from hybridomas monoclonal antibodies or clones Fv phage display according to the invention, are easily identified and sequenced using conventional methods (e.g. by using oligonucleotide primers designed for specific amplification of interest coding sections of the heavy and light chains from hybridomas or DNA-matrix phage). After DNA extraction can be placed in expressing vectors, which are then transferout in the host cell, such as cells ofE. coli, COS cells monkeys, cells of the Chinese hamster ovary (CHO) or myeloma cells that otherwise do not produce the protein immunoglobulin providing the synthesis of the desired monoclonal antibodies in the recombinant cell host. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding the constant parts of the heavy chain and/or Le�coy chains (e.g., the appropriate DNA sequence can be obtained in accordance with Kabat et al., above) with the formation of clones encoding full-length or incomplete heavy and/or light chains. It will be understood that for this purpose you can use the constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region can be obtained from the person or from any animal species. The Fv clone formed from the DNA of the variable domain of one type of animal (such as man), and then merged with the DNA of a constant region of another species with the formation of the coding sequence(s) "hybrid" full-size heavy chain and/or light chain that is included in the definition of "chimeric" and "hybrid" antibody as used herein. In certain embodiments, the Fv clone formed from the DNA of variable regions of human is merged with the DNA of a constant region of a human with the formation of the coding sequence(s) for full-or part-heavy and/or light chains.

DNA encoding the antibody against CD79b, originating from the hybridomas, it is also possible to modify, for example, by replacing the coding sequence of the constant domains of the heavy and light chains is homologous mouse sequences derived from clone hybridomas (e.g., as in �the p Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding derived from hybridomas or Fv-clone antibody or fragment can be further modified by covalent binding to the coding sequence of the immunoglobulin whole coding sequence non-immunoglobulin polypeptide, or part thereof. Thus, "chimeric" or "hybrid" antibodies that have the binding specificity originating from Fv-clone or clone the hybridomas antibodies according to the invention.

C. mediated antibody-dependent enzyme Pro-drug therapy (ADEPT)

The antibodies of the present invention can also be used in ADEPT by conjugation of an antibody to activating the prodrug by the enzyme which converts a prodrug (e.g., peptidyl chemotherapeutic agent, see WO81/01145) to an active drug against malignant tumors. See, for example, WO 88/07378 and U.S. patent No. 4975278.

Enzyme component immunoconjugate suitable for ADEPT includes any enzyme capable of acting on a prodrug in such a way as to transform it into the more active cytotoxic form.

Enzymes that are suitable in the method according to this invention include, but are not limited to, alkaline phosphatase, suitable for converting phosphate-containing prodrug in free� drug; the arylsulfatase suitable for converting sulfate-containing prodrug into the free drug; sitoindosides suitable for converting non-toxic 5-fertilizin in the drug against malignant tumors, 5-fluorouracil; proteases, such as proteaseserratiathermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins B and L), which are suitable for converting peptide-containing prodrug into free drugs; D-alanismorissette suitable for the conversion of the prodrug, which contain substituents in the form of D-amino acids; carbohydrate cleaving enzymes such as β-galactosidase and neuraminidase, which are suitable for converting glycosylated prodrug into the free drug; β-lactamase that is suitable for the conversion of medicines, converted into a derivative with β-lactams, available drug; and penicillinases, such as amidase penicillin V or amidase penicillin G, suitable for turning of medicines transformed into derived their nitrogen of the amino groups by phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Alternative antibodies with enzymatic activity, also known in this area as "AB�ima", can be used for the conversion of the prodrug of the invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Conjugates of the antibody-Abim can be obtained as described herein for delivery of abzyme in the population of tumor cells.

Enzymes according to this invention can covalently bind with antibodies against CD79b ways known in this field such as the application heterobifunctional reagents for crosslinking discussed above. Alternative fusion proteins containing at least the antigen-binding region of the antibody according to the invention is associated with at least a functionally active portion of an enzyme of the invention, can be designed using the methods of recombinant DNA, are well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984).

D. Antibodies against CD79b

In addition to antibodies against CD79b, described herein, provides that you can obtain versions of the antibodies against CD79b. Variants of antibodies against CD79b can be obtained by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Specialists in this field will understand that amino acid changes may alter post-translational processes of the antibody CD79b, for example, they can change the number or position of glycosylation sites or changing the characteristics of the anchor on the membrane.

Changes of antibodies against CD79b described herein can be performed, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set out, for example, in U.S. patent No. 5364934. Change can be a substitution, deletion or insertion of one or more codons encoding the antibody or peptide that lead to changes in amino acid sequence compared to the native antibody or polypeptide. Not necessarily the change is a replacement of at least one amino acid by another amino acid in one or more domains of antibodies against CD79b. Information about which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence antibodies against CD79b with sequence homologous known protein molecules and minimizing the number of amino acid sequence substitutions in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid by another amino acid having similar structural and/or chemical properties, so�th as replacement of leucine by serine, i.e., they may represent conservative amino acid substitutions. The size of insertions, deletions or substitutions may not necessarily be in the range of from about 1 to 5 amino acids. The allowable change is possible to determine the systematic conduct of insertions, deletions or substitutions of amino acids in the sequence and testing the obtained variants in relation to the activity shown by full-length or Mature native sequence.

This document provides fragments of antibodies against CD79b. Such fragments may be truncated from the N-end or C-end, or they may be deprived of internal residues, for example, compared with full-sized native antibody or protein. Such fragments are deprived of amino acid residues that are not essential for a desired biological activity of antibodies against CD79b.

Fragments of antibodies against CD79b can be obtained by any of the many conventional methods. Desired peptide fragments can be chemically synthesized. An alternative approach involves obtaining the antibody or polypeptide fragments by enzymatic cleavage, for example by treatment of a protein enzyme, which is known that it breaks down proteins in the areas defined by particular amino acid residues, or by splitting DNA suitable� the enzymes and secretions of the required fragment. Another suitable method involves the isolation and amplification of a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). As 5' and 3'primers in the PCR using oligonucleotides that define the ends of a DNA fragment. Preferably, fragments of antibodies against CD79b have at least one type of biological and/or immunological activity, coinciding with the activity of native antibodies against CD79b, described in this document.

In specific embodiments of interest the conservative substitutions provided in table 6 under the heading of "Preferred substitutions". If such substitutions result in a change in biological activity, then make more substantial changes, indicated in table 6 illustrative substitutions", or as described below in relation to classes of amino acids, and conduct screening products.

Table 6
The original balanceIllustrative 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
A His (H)asn; gln; lys; argarg
Ile (I)leu; val; met; ala; phe; norleucineleu
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
Pr (P) alaala

Ser (S)thrthr
Thr (T)serser
Trp (W)tyr; phetyr
Tyr (Y)trp; phe; thr; serphe
Val (V)ile; leu; met; phe; ala; norleucineleu

Significant modification of a function or immunological properties of antibodies against CD79b spend, 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, such as the conformation in the form of a layer or spiral, (b) the charge or hydrophobicity of the molecule at the site of the target, or (c) the volume of the side chain. Naturally occurring residues can be divided into groups based on common properties of their side chains:

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

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

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Changes can be carried out using methods known in this field, such as mediated by oligonucleotide (site-directed) mutagenesis, alanine scanning, and mutagenesis through PCR. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene. 34:315 (1985)), mutagenesis by restriction-selection (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known methods can be performed on the cloned DNA to obtain DNA variant antibodies against CD79b.

To identify one or more amino acids along a contiguous sequence using the analysis of scanning amino acids. To the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid in this group because it eliminates the side-chain addition to beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science. 244: 1081-1085 (1989)]. Also alanine is usually preferred as it is with�Oh the most common amino acid. In addition, it is often found both in depth and in exposed positions [Creighton, The Proteins, (W. H. Freeman & Co., N. Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not lead to adequate amounts of variant, you can use solernou amino acid.

Any cysteine residue not involved in maintaining the proper conformation of antibodies against CD79b, also can be replaced, mainly serine, increasing the stability of the molecule to oxidation and inhibiting aberrant cross-linking. On the contrary, to improve stability, in such a molecule, you can make cysteine bond(s) (particularly where the antibody is an antibody fragment such as an Fv fragment).

Especially preferred variant type with substitution includes replacement of one or more residues of the hypervariable portion of the source antibody (e.g., humanized antibody or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the original antibody from which they are derived. A convenient method of producing such variants with substitutions involves affinity maturation using phage display. In summary, several hypervariable regions of the plot (e.g., 6-7 areas) is subjected to mutation with�the teachings of all possible amino acid substitutions at each site. Thus obtained antibody variants exhibit as monovalent antibodies of the particles of filamentous phages as molecules, merged with the product of the gene III of M13 and Packed in every part. Then exposed on the phage variants is subjected to screening in relation to their biological activity (e.g. binding affinity) as described herein. For identification of hypervariable regions of the plot is a candidate for modification, can be performed alanine scanning mutagenesis, identifying the remains of the hypervariable area, participating largely in the binding to the antigen. Alternative or additionally, it may be appropriate to analyze the crystal structure of the complex antigen-antibody to identify contact points between the antibody and CD79b polypeptide. Such contact residues and neighboring residues are candidates for replacement in accordance with the methods described herein. After receipt of such options, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

The nucleic acid molecules encoding amino acid sequence variants of antibodies against CD79b, get R�slishnimi ways, known in this field. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring variants of the amino acid sequence) or oligonucleotide-mediated (or site-directed) mutagenesis, mutagenesis by PCR and cassette mutagenesis of the sequence previously obtained variant or non-variant version of the antibody against CD79b.

E. Modification of antibodies against CD79b

In the scope of this invention incorporated covalently modified antibodies against CD79b. One type of covalent modification includes reacting the specified amino acid residues of antibodies against CD79b with forming an organic derivative of a substance that can react with specific side chains or the N - or C-terminal residues of such antibodies against CD79b. Appropriate is the conversion of bifunctional substances, for example, for the crosslinking of antibodies against CD79b with water-insoluble matrix of the substrate or surface for use in the purification of antibodies against CD79b, and Vice versa. Commonly used cross-binding substances include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, esters of N-hydroxysuccinimide, for example, esters with 4-azidoaniline acid, homobifunctional� - to-date complex imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester), bifunctional maleimide, such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propionamide.

Other modifications include desalinatio glutaminergic and asparaginyl residues relevant to glutamine and aspartyl residues, respectively, hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups merilnyh and 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 Amidala any C-terminal carboxyl group.

Another type of covalent modification of antibodies against CD79b involves modifying the native glycosylation pattern of the antibody or polypeptide. "Changing the native glycosylation pattern" is defined as the removal of one or more carbohydrate groups found in native sequence antibodies against CD79b (either by removal of the primary glycosylation site, either through the elimination of the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in native posledovatelnosti.html against CD79b. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the structure and proportions of the various carbohydrate present groups.

Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked glycosylation refers to the accession of carbohydrate groups to the side chain of the asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are sequences that are recognized for enzymatic joining of carbohydrate groups to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the accession of one of the sugars constituting the N-acetylgalactosamine, galactose or xylose, to hydroxynicotinate, usually, to serina or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.

Addition of glycosylation sites to the antibody against CD79b convenient to carry out, changing amino acid sequence to contain one or more of the above Tripeptide sequences (for the�of Astrov N-linked glycosylation). Also the change can be performed by adding to the sequence or replacement by one or more residues of serine or threonine in the sequence of the original antibody against CD79b. Amino acid sequence antibodies against CD79b optional can be modified through changes at the DNA level, particularly by mutating the DNA encoding the sequence antibodies against CD79b, in a pre-selected basis to obtain codons, which will broadcast the required amino acids.

Another way of increasing the number of carbohydrate groups on the antibody against CD79b is a chemical or enzymatic joining of glycosides to the polypeptide. Such methods are described in this area, for example, in WO 87/05330, published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. pp. 259-306 (1981).

Removal of carbohydrate groups present on the antibody against CD79b, can be performed chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical methods of deglycosylation known in this field and are described, for example, Hakimuddin, et al., Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al., Anal. Biochem. 118:131 (1981). Enzymatic cleavage of carbohydrate groups on the polypeptides can be performed � many endo - and ectoparasites, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of antibodies against CD79b involves the binding of an antibody to one of the many non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene manner indicated in U.S. patents No.. 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337. The antibody can also be enclosed in microcapsules obtained, for example, by means of coacervation or interfacial polymerization (for example, the microcapsules on the basis of hydroxymethylcellulose or gelatin and microcapsules based on poly (methyl methacrylate), respectively), in colloidal systems drug delivery (for example, liposomes, microspheres based on albumin, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in Remington's Pharmaceutical Science, 16th edition, Oslo, A., Ed., (1980).

Antibodies against CD79b the present invention can also be modified in such a way as to formed a chimeric molecule containing an antibody against CD79b, merged with another a heterologous polypeptide or amino acid sequence.

In one embodiment, the implementation of such a chimeric molecule contains antibodies against CD79b, which are attached to a polypeptide tag, which provides an epitope to which can be selectively contacted by the antibody against the tag. The epitope-tag, as the rules�, placed on the N - or C-end of antibodies against CD79b. Detection of the presence of such epitope tagged forms of antibodies against CD79b can be carried out using antibodies against the polypeptide tags. Also, provision of the epitope tag enables easy purification of antibodies against CD79b affinity purification methods using antibodies against the tag or another type of affinity matrix that binds to the epitope-tag. Different polypeptides-markers and corresponding antibodies are well known in this field. Their examples include labels polyhistidine (poly-his) or poly-histidine-glycine (poly-his-gly); a polypeptide tag flu HA and antibody to it 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; label c-myc and antibodies thereto 8F9, 3C7, 6E10, G4, B7 and 9E10 [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and a tag in the form of glycoprotein D (gD) of herpes simplex virus and antibody to it [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other peptide tags include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the peptide-KT3 epitope [Martin et al., Science, 255:192-194 (1992)]; the peptide-epitope of α-tubulin [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the peptide tag protein gene 10 T7 [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

In an alternative embodiment of the chimeric molecule can contain antibodies against CD79b, fused with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (macieabney as "immunoadhesin"), such a fusion could be to the Fc-region of IgG molecules. The merger of Ig molecules preferably include the substitution of a soluble (deleted or inactivated transmembrane domain) form antibodies against CD79b is at least one variable segment in the Ig molecule. In a particularly preferred embodiment of the fused molecule of the immunoglobulin comprises a hinge region, CH2and CH3or hinge region, CH1-, CH2- and CH3-areas of the IgG1 molecule. To obtain fused immunoglobulin molecules also see U.S. patent No. 5428130, issued June 27, 1995.

F. antibodies against CD79b

The following description relates mainly to the receipt of antibodies against CD79b by culturing cells transformed or transfected with a vector containing nucleic acid encoding the antibody against CD79b. Of course, stipulates that to obtain such antibodies against CD79b you can use alternative methods that are well known in this field. For example, the appropriate amino acid sequence, or part thereof, can be obtained by direct peptide synthesis using solid-phase methods [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. The synthesis of proteinsin vitrocan be performed with use�Itanium manual methods or by using machines. Automated synthesis can be performed, for example, using Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. The various parts of antibodies against CD79b can be chemically synthesized separately and combined using chemical or enzymatic methods of obtaining the required antibodies against CD79b.

1. The isolation of the DNA encoding the antibody against CD79bDNA encoding the antibody against CD79b, can be obtained from a cDNA library derived from tissue allegedly possessing mRNA antibodies against CD79b, and expressing it on beyond the detection level. Thus, DNA antibodies against CD79b person can be a convenient way to get from a cDNA library derived from human tissue. The gene encoding the antibody against CD79b, also can be obtained from a genomic library or by known methods of synthesis (e.g., automated synthesis of nucleic acids).

Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or to encode them protein. Screening cDNA or genomic libraries of a particular probe can be carried out using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor aboratory Press, 1989). An alternative method for isolating the gene encoding the antibody against CD79b is the application of PCR technology [Sambrook et al., above; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

Methods of screening cDNA libraries are well known in this field. Sequences of oligonucleotides selected as probes should be of sufficient length and must be unique enough to minimize false-positive results. Preferably the oligonucleotide is labeled, so it can be identified by hybridization to DNA in the library being screened. Methods of labeling are well known in this area and include the use of radioactive labels, such as labeled32P ATP, biotinylation or tagging enzyme. The hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., above.

Sequences identified in such methods of screening 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. The sequence identity (at either the amino acids or nucleotides) in certain parts of the molecule, or for a full-sized placenta�coherence can be determined using the methods known in this area and as described in this document.

Nucleic acid having a sequence encoding protein, you can get certain screening cDNA libraries or genomic libraries using the established amino acid sequence described herein for the first time, and, if necessary, using conventional methods of lengthening primers, as described in Sambrook et al., above, for detection of precursor and intermediate forms during the processing of mRNA that may not be subjected to reverse transcription into cDNA.

2. Selection and transformation of host cells

The host cell transferout or transforming expressing or cloning vectors described herein for the production of antibodies against CD79b and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplification of genes encoding the desired sequences. Culturing conditions, such as environment, temperature, pH, etc., a qualified person may be selected without undue experimentation. As a rule, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Smbrook et al., above.

Methods transfection of eukaryotic cells and transformation of prokaryotic cells, which refers to the introduction of DNA into the cell, so that the DNA is replicated, either extrachromosomal or chromosomal integration, known to the person skilled in the art, for example, mediated CaCl2, CaPO4, a liposome method, and electroporation. Depending on the host cell, transformation is carried out using standard methods appropriate to such cells. Treatment with calcium using calcium chloride as described in Sambrook et al., above, or electroporation is generally used for prokaryotes. For transformation of certain plant cells using infection byAgrobacterium tumefaciensas described in Shaw et al., Gene. 23:315 (1983) and WO 89/05859, published 29 June 1989. For mammalian cells without such cell walls, you can use the method of deposition of calcium phosphate by Graham and van der Eb, Virology, 52:456-457 (1978). The main aspects of transpency systems host cells of mammals is described in U.S. patent No. 4399216. Transformation of yeast as a rule, is carried out in accordance with the method of Van Solingen et al., J. Bact, 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, you can also use other methods of introducing DNA into cells, e.g., by microinjection into the nucleus, electro�Orazio, merge protolayer bacteria with intact cells, or polycations, e.g., polybrene, polyamidine. For various ways of transforming mammalian cells, see 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 herein, cells include prokaryotes, yeast or higher eukaryotes.

a. Prokaryotic cells-owners

Suitable prokaryotes include but are not limited to eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such asE. coli. Different strains ofE. coliare publicly available, such asE. coliK12 strain MM294 (ATCC 31446);E. coliX1776 (ATCC 31537);E. colistrain W3110 (ATCC 27325) and K5 772 (ATCC 53635). Other suitable prokaryotic cells-owners, include Enterobacteriaceae, such asEscherichiafor example,E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonellafor example,Salmonellatyphimurium,Serratiafor example,Serratia marcescansandShigellaandBacillisuch asB. subtilisandB. Bacillus licheniformis(for example,B. Bacillus licheniformis41P described in DD 266710, published 12 April 1989),Pseudomonas,such asP. Aeruginosa, Rhizobia, Vitreoscilla, ParacoccusandStreptomyces. These examples are illustrative, but not limiting. Strain W3110 is one particularly preferred host and�and parent, since it is a common strain-host for fermentation of recombinant DNA product. Preferably, the host secretes 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; the number of the deposited sample of ATCC No. 27325) can be modified to achieve a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts includeE. coliW3110 strain 1A2, which has the complete genotype tonA;E. coliW3110 strain 9E4, which has the complete genotype oftonA ptr3;E. coliW3110 strain 27C7 (ATCC 55244), which has the complete genotype oftonA ptr3 phoA E15(argF-lac)169 degP ampT kanr;E. coliW3110 strain 37D6, which has the complete genotype oftonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvGkanr;E. coliW3110 strain 40B4, which is strain 37D6 with a deletion mutation degP without resistance to kanamycin;E. coliW3110 strain 33D3 having genotype W3110 ΔfhuAtonA)ptr3lacIqlacL8ΔompTΔ (nmpc-fepE)degP41kanR(U.S. patent No. 5639635) and a strain ofE. colihaving mutant periplasmic protease described in U.S. patent No. 4946783 issued 7 August 1990. Also fit other strains and derivatives thereof, such asE. coli294 (ATCC 31446),E. coliB,E. coliλ1776 (ATCC 31537), andE. coliRV308 (ATCC 31608). �behold these examples are illustrative, but not limiting. The methods for constructing derivatives of any of the above mentioned bacteria having defined genotypes are known in this field and described, for example, Bass et al., Proteins, 8: 309-314 (1990). As a rule, you must select the appropriate bacteria, given the capacity for replication of the replicon in the cells of Bactria. For example, species ofE. coli,SerratiaorSalmonellacan appropriately be used as hosts as to provide replicon use a well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. As a rule, the host should secrete minimal amounts of proteolytic enzymes, and may be desirable to add additional cell culture of protease inhibitors. Alternative suitable methods of cloningin vitrofor example , PCR or other polymerase reactions of nucleic acids.

Full-size antibody, the antibody fragments and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and effector function Fc is not required, such as when therapeutic antibody anywhereman with a cytotoxic agent (e.g. a toxin) and immunoconjugate shows effectiveness against the destruction of tumor cells. Full-size antibodies have a longer half-life in the shelter�current. Products in theE. coliis faster and more economical. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U. S. 5648237 (Carter et. al), U. S. 5789199 (Joly et al) and U.S. 5840523 (Simmons et al.) in which is described the site of translation initiation (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein as references. After expression, the antibody is recovered from the cell massE. coliin the soluble fraction, and it can be cleaned, for example by column with protein A or G, depending on the isotype. Final cleaning can be performed similarly to the process of purification of the antibody, a murine, e.g., in CHO cells.

b. Eukaryotic cells-owners

In addition to prokaryotes, suitable hosts for cloning and expression vectors encoding antibody against CD79b are eukaryotic microbes such as filamentous fungi or yeast. Often use a lower eukaryotic microorganism-hostSaccharomyces cerevisiae. Other owners includeSchizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [1981]; EP 139383, published 2 may 1985); the hosts ofKluyveromyces(U.S. patent No. 4943529; Fleer et al., Bio/Technology, 9:968-975 (1991)), such asK. 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 5600), K. drosophilarum(ATCC 36906; Van den Berg et al., Bio/Technology, 8: 135 (1990)),K. thermotoleransandK. marxianus;yarrowia(EP 402226);Pichia pastoris(EP 183070; Sreekrishna et al., J. Basic Microbiol. 28:265-278 [1988]);Candida;Trichoderma reesia(EP 244234);Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomycessuch asSchwanniomyces occidentalis(EP 394538 published 31 October 1990); and filamentous fungi such asNeurospora, Penicillium, Tolypocladium(WO 91/00357, published 10 January 1991), and hosts theAspergillussuch asA. 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]) andA. niger(Kelly and Hynes, EMBO J. 4:475-479 [1985]). Suitable for this methylotrophy yeast and they include, but are not limited to, yeast capable of growing on methanol selected from the genera consisting ofHansenula, Candida, Kloeckera, Pichia, Saccharomyces, TorulopsisandRhodotorula. A list of specific species that are examples of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Appropriate cell hosts for the expression of glycosylated anti-CD79b antibody are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as the culture of cotton, corn, potato, soybean, Petunia, tomato and tobacco. It was identified a large number of baculovirus strains, variants and the corresponding�following allowed the host cell, such asSpodoptera frugiperda(caterpillar),Aedes aegypti(mosquito),Aedes albopictus(mosquito),Drosophila melanogaster(fruit fly) andBombyx mori. Available a variety of viral strains for transfection, for example, a variant L-1Autographa californicaNPV and the strain Bm-5Bombyx moriNPV, and such viruses may be used as the virus of the present invention, particularly for transfection of cellsSpodoptera frugiperda.

However, of most interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become an accepted way. Examples of suitable cell lines of mammals are line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); line kidney of a human embryo (293 cells or 293 cells, sublimirovanny 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 77:4216 (1980)); the cells of mouse Sertoli (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); cell carcinoma human cervical (HELA, ATCC CCL 2); cells dog kidney (MDCK, ATCC CCL 34); cells rat liver Buffalo (BRL 3A, ATCC CRL 1442); lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); breast tumor mouse (MMT 060562, ATCC CCL51); cell TRI (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (192)); the cells, MRC 5; FS4 cells; and human hepatoma (Hep G2).

The host cell is transformed as described above expressing or cloning vectors for production of antibodies against CD79b and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplification of genes encoding the desired sequences.

3. The choice and use of a replicable vector

For recombinant production of an antibody according to the invention nucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody, easily allocate and sequeiros using conventional methods (e.g., by using oligonucleotide probes that are capable of specific contact with the genes encoding the heavy and light chain antibodies). A variety of vectors. The choice of vector depends, in part, on the host cell to be used. As a rule, preferred the host cell are either prokaryotic or eukaryotic (typically mammalian) origin.

For example, a vector can be represented in the form of plasmids, Comedy, viral particle, or phage. Corresponding follower�spine nucleic acid can be inserted into the vector in a variety of ways. Typically, the DNA inserted into an appropriate site(s) for the restriction enzyme using methods known in this field. Vector components generally include, but are not limited to, one or more signal sequences, origin of replication, one or more marker genes, enhancer element, a promoter and termination sequence transcription. In the design of suitable vectors containing one or more of these components, use the standard methods of ligation, which is known to a qualified specialist.

CD79b can be produced by recombinant methods not only directly, but also as a polypeptide fused with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-end of the Mature protein or polypeptide. Typically, the signal sequence may be a component of the vector, or it may be part of the DNA that encodes the antibody against CD79b, which are inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penicillinase, lpp, or thermostable�wow enterotoxin II. In the case of secretion in yeast, the signal sequence may be, for example, a leader sequence of yeast invertase, leader sequence of alpha-factor (including the leader sequence of α-factorSaccharomycesandKluyveromyces,the last of which is described in U.S. patent No. 5010182) or leader sequence of acid phosphatase, leader sequence of the glucoamylaseC. albicans(EP 362179 published 4 April 1990), or the signal sequence described in WO 90/13646 published 15 November 1990. When overexpressed in mammalian cells, for directing secretion of the protein can be used signal sequence mammals, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leader sequence.

a. Prokaryotic cells-owners

Polynucleotide sequences encoding polypeptide components of the antibody according to the invention, can be obtained using standard recombinant methods. The desired polynucleotide sequences can be identified and sequenced from antibody-producing cells, such as cells of hybridomas. Alternative polynucleotides can be synthesized using the device for the synthesis of nucleotides �whether PCR methods. After receipt, the sequence encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. For the purposes of the present invention can use a variety of vectors are available and known in this field. The selection of an appropriate vector depends mainly on the size of nucleic acids that are to be embedded in the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotide, or both of them) and its compatibility with specific host cell in which it resides.

Generally, with regard to those owners use of plasmid vectors containing replicon and sequence control from species compatible with the host cell. As expressing and cloning vectors contain a nucleic acid sequence that enables replication of the vector in one or more selected cells of the host, as well as marker sequences which are capable of providing phenotypic selectivity of transformed cells. Such sequences are well known for many bacteria, droge�th and viruses. Origin of replication from the plasmid pBR322, which contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet) and, thus, provides a simple means of identifying transformed cells is suitable for most gram-negative bacteria, plasmid 2µ origin is suitable for yeast, and various viral oridzhiny (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells. pBR322, its derivatives, or other microorganisms or plasmids bacteriophage may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies, are described in detail in Carter et al., U.S. patent No. 5648237.

In addition, with regard to those owners, as the transformed vectors can be used phage vectors containing replicon and sequence of controls that are compatible with the microorganism-host. For example, when producing recombinant vector can be used bacteriophage, such as λGEM.TM.-11 that can be used to transform susceptible to it host cells, such asE. coliLE392.

Expressing the vector according to the invention may contain two or more pairs of PR�motor-cistron, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence, located above (5') cistron that modulates its expression. Prokaryotic promoters typically fall into one of two classes, inducible and constitutive. The inducible promoter is a promoter that initiates increased levels of transcription of cistron under its control, in response to a change in the cultivation conditions, for example on the presence or absence of nutrients or temperature changes.

It is well known a large number of promoters recognized by a variety of potential cells-hosts. The selected promoter can functionally associate with castronno DNA encoding the light or heavy chain, by removing the promoter from the DNA, which is its source, through cleavage by a restriction enzyme and embedding the selected promoter sequence into the vector according to the invention. For the direction of amplification and/or expression of target genes can be used as the native promoter sequence and many heterologous promoters. In some embodiments, use of heterologous promoters, as they permit greater transcription and higher o�d a murine gene-targets compared to the native promoter of the polypeptide target.

Well known promoters recognized by a variety of potential cells-hosts. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, promoter of the system β-galactosi and lactose [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], the promoter system, the alkaline phosphatase, the tryptophan (trp) promoter system [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 trc. Promoters for use in bacterial systems also contain a sequence Shine-Dalgarno (S. D.), functionally linked to DNA that encodes the antibody against CD79b. However, also other suitable promoters that are functional in bacteria (such as other known bacterial or phage promoters). Their nucleotide sequences are published, thereby allowing a qualified professional to functionally ligitamate them with cisternae, code required light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adapters to provide any required restriction sites.

In one aspect of the invention, each cistron in the recombinant vector contains a component representing a secretory signal sequence that directs the movement expressed�s polypeptides across the membrane. Typically, the signal sequence may be a component of the vector, or it may be part of the DNA of the polypeptide target, which is inserted into the vector. The signal sequence selected for the purposes of this invention must be a sequence that is recognized by the host cell, and subjected to processing (i.e. cleaved by a signal peptidase) in it. For prokaryotic host cells that do not recognize and do not carry out processing of the signal sequences native to the heterologous polypeptides, the signal sequence is replaced prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII), LamB, PhoE, PeIB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistronic expressing systems represent the signal sequence STII or their variants.

In another aspect, the production of immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and, thus, it does not require the presence of secretory signal sequences in each cistron. In this regard, light and heavy chains of the immunoglobulins expresser�are, curl up and going with the formation of a functional immunoglobulin in the cytoplasm. Certain strains-owners (for example, strains trxB-E. coli) provide conditions in the cytoplasm, which contribute to the formation of disulfide bonds, thereby ensuring proper folding and Assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention relates to expressing the system in which the quantitative relationship expressed polypeptide components can be modulated to maximize the yield of secreted and properly folded antibody according to the invention. Such a modulation is carried out at least partially simultaneously by modulating the efficiency of translation of the polypeptide components.

One way of modulating the efficiency of translation is described in Simmons et al., U.S. patent No. 5840523. It uses versions of the site of translation initiation (TIR) in castrone. For this TIR, you can create a series of variants of the amino acid sequence or nucleic acid sequences with a range of efficiency broadcast, thereby providing convenient tools to correct this factor for the desired level of expression of a particular circuit. Options TIR can be obtained by the conventional methods of mutagenesis, which when�ADAT to changes in the codons, which can change the amino acid sequence, are preferred although silent changes in the nucleotide sequence. Changes TIR can include, for example, changing the number or placement of sequences Shine-Dalgarno, together with changes in the signal sequence. One method of producing a mutant signal sequences is to obtain "Bank of codons in the early coding sequence that do not alter the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be done by changing the third nucleotide position of each codon; in addition, some amino acids, such as leucine, serine and arginine, have multiple first and second positions, which can complicate the receiving Bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

Preferably, get a set of vectors with a range of efficiency TIR for each cistron in them. This limited set provides a comparison of the expression levels of each circuit as well as the yield of the desired product antibodies at various combinations of the effectiveness of TIR. The effectiveness of TIR can be determined by quantifying the level of expression of reporter gene, as described in detail in Simmons t al., U.S. patent No. 5840523. Based on the comparison of the effectiveness of the broadcast, select the required individual TIR to be combined in the construction of expression vectors according to the invention.

b. Eukaryotic cells-owners

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

(1) Component - signal sequence

Vector for use in eukaryotic the host cell may also contain a signal sequence or other polypeptide having site for specific cleavage at the N-end of the Mature protein or of interest polypeptide. Selected heterologous signal sequence is preferably a sequence that is recognized and converted (i.e., cleaved by a signal peptidase) in the host cell. For expression in mammalian cells, the available 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 preceding section be ligated in frame sitiveni� with DNA encoding the antibody.

(2) the origin of replication

As a rule, for expressing vectors mammals component, representing the origin of replication is not required. For example, as a rule, you can use the SV40 origin, because it contains the early promoter.

(3) Component - selective gene marker

Expressing and cloning vectors usually contain a gene for selection, also known as selective marker. Typical genes for breeding encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complementary defects of auxotrophs or (c) provide essential nutrients not available from complex media, e.g. the gene encoding D-alaninate forBacilli.

In one example, the schema selection using a drug to stop the growth of the host cell. Cells that are successfully transformed with a heterologous gene produce a protein which confers resistance to the drug and, thus, survive mode selection. Examples of such dominant species selection is the use of drugs neomycin, mycophenolic acid and hygromycin.

Examples of suitable selective markers for mammalian cells�itausa are markers which enable the identification of cells capable of capturing the nucleic acid encoding the antibody against CD79b, such as DHFR genes, timedancing, metallothionein-I and-II, preferably genes metallothionein primates, adenosine deaminase, emitintermediate etc. a Suitable host cell, when using wild-type DHFR is a CHO cell line with a deficit of DHFR activity (e.g., ATCC CRL-9096), obtained and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with the gene for DHFR selection, first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, the host cell (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibody, protein DHFR wild-type and the other a selective marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing cells on media containing a substance for selection by a selective marker such as aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

Suitable gene for selection for use in yeast is the genetrp1, in the yeast plasmid YRp7 [Stichcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. Genetrp1 is a selective marker for a mutant strain of yeast, which lacks the ability to grow in the absence of tryptophan, for example ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85: 12 (1977)].

(4) Promoter component

Expressing and cloning vectors usually contain a promoter functionally linked to a nucleic acid sequence that encodes an antibody against CD79b to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site of initiation of transcription. Another sequence, which is from 70 to 80 basis point above the start of transcription of many genes, represents an area of CNCAAT, where N can be any nucleotide. At the 3'-end of most eukaryotic genes is the sequence AATAAA, which may represent a signal to add a poly-A tail to the 3'-end of the coding sequence. All of these sequences can be embedded into eukaryotic expression vectors.

Examples of suitable promoter sequences for use in yeast hosts include PR�motors 3-fosfogliceratkinazy [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, pyruvate kinase complex, triazolopyrimidine, phosphoglucomutase and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of controlled growing conditions transcription, represent the promoter sites of alcohol dehydrogenase 2, sociogram C, acid phosphatase, enzymes of the degradation associated with the metabolism of nitrogen, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Furthermore, the suitable vectors and promoters for use for expression in yeast is described in EP 73657.

Transcription antibodies against CD79b with vectors in the cells of the host mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, smallpox birds (UK 2211504 published 5 July 1989), adenovirus (such as adenovirus 2), human papilloma virus of cattle, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis-B and most preferably, the virus of monkeys� 40 (SV40), heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, heat shock promoters, provided such promoters are compatible with the systems of host cells.

Early and late promoters of SV40 virus usually get as an SV40 restriction fragment that also contains the origin of replication of SV40 virus. Pretani the promoter of the human cytomegalovirus is usually produced as a HindIII restriction fragment E. the System for expression of DNA in hosts belonging to mammals, with the use of human papilloma virus of cattle in the vector as described in U.S. patent No. 4419446. Modification of this system is described in U.S. patent No. 4601978. Cm. also Reyes et al., Nature 297:598-601 (1982) on the cDNA expression of β-interferon in cells of mice under the control of the promoter timedancing from herpes simplex virus. Alternative as a promoter, you can use the long terminal repeat of the rous sarcoma virus.

(5) Component - enhancer

Transcription of DNA encoding the antibody against CD79b, in higher eukaryotes can be increased by embedding in vector enhancer sequence. Enhancers are CIS-regulatory elements of DNA, usually about from 10 to 300 BP that act on a promoter, increases� its transcription. There are many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). However, as a rule, use enhancer of virus eukaryotic cells. Their examples include the SV40 enhancer on the late side of the replication origin (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyoma on the late side of the replication origin, and adenovirus enhancers. Cm. also Yaniv, Nature 297: 17-18 (1982) on the enhancer for activation of eukaryotic promoters. The enhancer may be attached to the vector to the encoding antibodies against CD79b sequence in position 5' or 3', but preferably it is located in the site located in the 5'direction from the promoter.

(6) Component for transcription termination

Expression vectors used in eukaryotic cells-the masters (in yeast cells, fungi, insects, plants, animals, humans, or containing core cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are usually located at the 5' and, occasionally 3'-noncoding sites of eukaryotic or viral DNA or cDNA. These parcels contain nucleotide fragments are transcribed as polyadenylated� fragments in the untranslated section of the mRNA, the encoding antibodies against CD79b. One suitable component for the termination of transcription is the site of polyadenylation bovine growth hormone. Cm. WO94/11026 and the described expressing vector.

Other methods, vectors, and the host cell suitable for adaptation to the synthesis of antibodies against CD79b in the culture of recombinant cells of vertebrates are described in 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

The host cell used for the production of antibodies against CD79b according to the invention, can be cultured in a variety of environments.

a. Prokaryotic cells-owners

Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in this field, and they are suitable for culturing a selected host cells. Examples of suitable media include broth, Luria (LB) together with the necessary nutrients. In some embodiments, the medium also contains a means for selection, selected on the basis of constructs expressing vector for selective growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium of cells expressing the gene of resistance to ampicillin.

Also, in addition to a source� carbon, nitrogen and inorganic phosphate, you can add any necessary additives in appropriate concentrations, are added separately or in admixture with another Supplement or medium such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic cells-the hosts are cultivated under suitable temperatures. For the growth ofE. colifor example , the preferred temperature is in the range from about 20°C. to about 39°C, more preferably from about 25°C to about 37°C, preferably at approximately 30°C. the pH of the medium can be any value in the range from about 5 to about 9, depending mainly on the host organism. ForE. colithe pH is preferably from about 6.8 to about 7,4, and more preferably about 7.0.

If in expressing the vector according to the invention using an inducible promoter, protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the invention, for controlling transcription of the polypeptides using the PhoA promoter. Thus, for the induction of transformed� cells are cultivated in an environment with limited phosphate content. Preferably, Wednesday with a limited content of phosphate is an environment C. R. A. P (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). You can use many other inducing substances, in accordance with vector design, as is well known in this field.

In one embodiment, the implementation of a murine polypeptides of the present invention are secreted into the periplasm of the host cell and from it they are. Protein expression usually involves the destruction of a microorganism, usually by methods such as osmotic shock, sonication or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. In addition, proteins can be cleaned, for example, affinity chromatography with a resin. Alternative proteins can be transported into the culture medium and can be isolated from it. Cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification of proteins produced. The expressed polypeptides can be further identified and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

In one aspect of the invention, the antibody production is carried out in large quantities by the process� fermentation. For production of recombinant proteins are available in various large-scale fermentation methods with nourishment. Large-scale fermentation has a capacity of at least 1000 liters, preferably from about 1,000 to 100,000 liters. In these fermenters are used agitators for the distribution of oxygen and nutrients, especially glucose (the preferred source of carbon/energy). Small scale fermentation refers mainly to the fermentation in the fermenter, which has a capacity of not more than approximately 100 liters, and it can be in the range from about 1 liter to about 100 liters.

During the fermentation process, induction of protein expression, usually begin after the cells grow in suitable conditions to a desired density, e.g., OD550approximately 180-220, at the stage when the cells are in early stationary phase. You can use different inductors in accordance with the design of the vector, as known in the field and as described above. Before induction, the cells can be grown for more than short periods of time. The cells are usually induced for about 12-50 hours, although you can use a longer or shorter induction.

For improving yield and quality�ETS polypeptides according to the invention, you can modify various fermentation conditions. For example, to improve the proper Assembly and folding of secreted antibody polypeptides, can be used for more vectors, sverkhekspressiya proteins-chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (peptideprophet-CIS,TRANS-isomerase with chaperone activity) for cotransformation prokaryotic host cells. It has been shown that proteins-chaperones facilitate proper folding and solubility heterologic 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 and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39: 199-210.

To minimize proteolysis of expressed heterologous proteins (especially proteins that are sensitive to proteolysis), for the present invention it is possible to use certain strains-owners with a deficiency of proteolytic enzymes. For example, strains of host cells can be modified to ensure genetic mutation(s) in the genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and combinations thereof. Available some strains ofE. coliwith deficiency of proteases and these are described, for example, Joly et a. (1998), above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment of the implementation, as host cells in the expression system of the invention used strains ofE. coliwith deficiency of proteolytic enzymes and transformed with plasmids, sverkhekspressiya one or more proteins-chaperones.

b. Eukaryotic cells-owners

For the cultivation of host cells are suitable commercially available medium, such as medium ham's F10 (Sigma), minimal maintenance medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Wednesday Needle, modified by way of Dulbecco, ((DMEM), Sigma). In addition, host cells as the culture medium, you can use any of the media described in 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; WO9003430; WO 87/00195; or U.S. patent Re. 30985. Any of these media can be supplemented if necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as a drug GENTAMYCIN™), trace elements (defined as inorganic compounds usually represented in the final to�ncentrated micromolar range), and glucose or an equivalent energy source. You can also add any other additional substances in relevant concentrations, which are known to specialists in this field. Culturing conditions, such as temperature, pH, etc., are conditions that have used selected for expression host cells, and they will be understood by a person skilled in this field.

5. Detection of amplification/expression gene

Amplification and/or expression of the gene can be directly determined in the sample, for example, by conventional southern blotting, nazaren blotting to quantify the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or hybridizationin situusing appropriately labeled probe based on the sequences presented herein. Alternatively, antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes DNA-RNA or DNA duplexes-protein. The antibodies in turn can be labeled, and analysis can be carried out where the duplex is bound to the surface, so that upon the formation of duplex on the surface, it was possible to detect the presence of antibodies associated with the duplex.

Alternative gene can determine immunological STRs�Obama, such as immunohistochemical staining of cells or tissue sections and analysis of cell culture or body fluids, to quantify directly the expression of gene product. Antibody suitable for immunohistochemical staining and/or analysis of sample fluids may be either monoclonal or polyclonal, and can be obtained from any mammal. Conveniently, the antibodies suitable for the method according to the present invention, it was possible to obtain against CD79b polypeptide with native sequence, or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA CD79b and coding specific antibody epitope.

6. Purification of antibodies against CD79b

Forms antibodies against CD79b may be recovered from culture medium or from lysates of the host cells. In the case associated with the membrane form, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells used for expression of antibodies against CD79b, you can destroy various physical or chemical means, such as cycles of freezing and thawing, exposure to ultrasound, mechanical destruction or SR�of DSTV for cell lysis.

It may be desirable purification of antibodies against CD79b from recombinant proteins or polypeptides of the cells. The following methods illustrate suitable methods for cleaning up: fractionation on an ion-exchange column; precipitation with ethanol; reversed-phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; column with protein a-separate to remove contaminants such as IgG; and chelating metals column for labeled binding epitope forms antibodies against CD79b. You can use various methods of protein purification, and such methods are known in this field and are described, for example, Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Selected stage(s) of cleaning will depend, for example, from the nature of the process of obtaining and produced specific antibodies against CD79b.

When using recombinant methods, the antibody may are produced intracellularly, in the periplasmic space, or directly secretariats on Wednesday. If such antibodies are produced intracellularly, in the first stage, debris in the form of particles, either host cells or lysed fragments, is removed, e.g. by centrifugation or �ultrafiltration. In Carter et al., Bio/Technology 10:163-167 (1992) described a method for isolating antibodies which are secreted into the periplasmic spaceE. coli. In brief, cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl (PMSF) over about 30 min, the Cell debris can be removed by centrifugation. When the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available filter for concentrating the protein, for example, the element for ultrafiltration Amicon or Millipore Pellicon. To any of the preceding stages can include a protease inhibitor such as PMSF to inhibit proteolysis, and to prevent the unnecessary growth of contaminating organisms can add antibiotics.

The composition of the antibodies obtained from cells can be purified using, for example, chromatography with hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification method. 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 antibodies, which are based on heavy chains γ1, γ2, or γ4 (Lindmark et al, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which is attached an affine ligand, is most often agarose, but other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene allow for faster flow rates and shorter processing time than the flow rate and the treatment time, which can be achieved with agarose. When the antibody contains CH3-domain, suitable for cleaning resin Bakerbond ABXTM(J. T. Baker, Phillipsburg, NJ). Also available other methods of protein purification such as fractionation on an ion-exchange column, precipitation with ethanol, reversed-phase HPLC, chromatography on silica, chromatography on heparin-SEPHAROSETM, chromatography on anyone - or cation-exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate, depending on subject selection antibody.

After the preliminary stage(s) of purification, a mixture containing interest is an antibody and impurities, can be subjected to hydrophobic interaction chromatography at low pH using a buffer for elution at a pH between about 2.5 to 4.5, preferably providemore low concentrations of salt (e.g., from about 0-0,25 M salt).

G. Pharmaceutical formulations

Conjugate antibody-drug (ADC) according to the invention can be entered by any method suitable for the condition targeted by the treatment. ADC, typically administered parenterally, i.e., by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

For the treatment of these malignant tumors, in one embodiment, the implementation, the conjugate antibody-drug administered by intravenous infusion. Dosage, administered by infusion, is in the range from about 1 μg/m2to about 10000 μg/m2per dose, generally one dose per week just with one, two, three or four doses. Alternative dosage is in the range from about 1 μg/m2to about 1000 μg/m2from about 1 μg/m2to about 800 μg/m2from about 1 μg/m2to about 600 mg/m2from about 1 μg/m2to about 400 μg/m2from about 10 μg/m2to about 500 μg/m2from about 10 μg/m2to about 300 mg/m2from about 10 μg/m2to about 200 μg/m2and from about 1 μg/m2up to approximately 200 m�g/m 2. To alleviate or ameliorate symptoms of the disease the dose can be entered once a day, once a week, several times a week, but less than once a day, several times a month but less than once a day, several times a month but less than once a week, once a month or periodically. The introduction can be continued during any of these intervals until remission of the tumor or symptoms of lymphoma, leukemia exposed to treatment. The introduction can be continued after remission or mitigation of symptoms, where such remission or mitigation shall be extended by such continued administration.

The invention also relates to a method of alleviating an autoimmune disease, comprising administering to a patient suffering from an autoimmune disease, a therapeutically effective amount of the conjugate is a humanized antibody 2F2-medicament according to any one of the preceding embodiments. In preferred embodiments, the antibody is administered intravenously or subcutaneously. Conjugate the antibody-drug is administered intravenously at a dosage in the range from about 1 μg/m2to about 100 mg/m2per dose and in a specific embodiment, the implementation, the dosage is 1 mg/m2to about 500 μg/m2. For softening or weakening of the si�of Tomov disease dose you can enter once a day, once a week, several times a week, but less than once a day, several times a month but less than once a day, several times a month but less than once a week, once a month or periodically. The introduction can be continued during any of these intervals to alleviate or ameliorate symptoms of autoimmune disease susceptible to treatment. Introduction to achieve a softening or weakening of symptoms, where such a softening or weakening of the extended by such continued administration.

The invention also relates to a method of treatment of B-cell disorders, comprising administering to a patient suffering from B-cell violation, such as B-cell-proliferative disorder (including, but not limited to, lymphoma and leukemia) or an autoimmune disease, a therapeutically effective amount of a humanized antibody 2F2 according to any one of the preceding embodiments, wherein the antibody is not conjugated with a cytotoxic molecule or capable of detection molecule. The antibody is typically administered in the range of dosages from about 1 μg/m2to about 1000 mg/m2.

In one aspect, the invention also relates to pharmaceutical compositions containing at least one antibody against CD79b to invent�of and/or at least one immunoconjugate and/or at least one conjugate is an antibody against CD79b-medicament according to the invention. In some embodiments, the pharmaceutical composition contains (1) an antibody according to the invention and/or its immunoconjugate, and (2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains (1) an antibody according to the invention and/or its immunoconjugate, and optionally (2) at least one additional drug. Additional drugs include, but are not limited to, drugs, described below. ADC, as a rule, can be administered parenterally, i.e., by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

Therapeutic compositions containing antibodies against CD79b or immunoconjugate against CD79b used in accordance with the present invention are prepared for storage by mixing the antibody or immunoconjugate having a desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers such as acetate, Tris, phosphate, citrate and other organic acids; antioxidant�Dante, including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; hexamethonium chloride; benzalkonium chloride, chloride benzathine; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptides; 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; chelating agents such as EDTA; changing the correct tonicity agents 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). Pharmaceutical compositions to be used for the introduction ofin vivoas a rule , are sterile. This is easily accomplished by filtration through sterile filtration membranes.

The compositions presented in the present document, �also may contain more than one active connection, if required for particular indications, which is directed to the treatment, preferably compounds with complementary types of activities that will not adversely effect each other. For example, in addition to antibodies against CD79b, it may be desirable inclusion in one additional antibody, e.g. a second antibody against CD79b, which binds to a different epitope on CD79b polypeptide, or an antibody to some other target such as a growth factor that affects the growth of malignant tumors. Alternative or additionally, the composition may further contain a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent and/or cardioprotector. Such molecules, respectively, are present in combination in amounts that are effective for the goal.

The active ingredients can also be enclosed in microcapsules obtained, for example, by means of coacervation or interfacial polymerization, for example, the microcapsules on the basis of hydroxymethylcellulose or gelatin and microcapsules based on poly (methyl methacrylate), respectively, in colloidal systems drug delivery (for example, liposomes, microspheres based on albumin, microemulsions, nanoparticles and nanocapsules), or in microa�Ulzii. Such methods are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

You can get drugs with a slow release. Suitable examples of drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are presented in the form of shaped articles, e.g. films or microcapsules. Examples of matrices with a slow release include polyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactic acid called PLA (U.S. patent No. 3773919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, degradiruem ethylene vinyl acetate, degradiruete copolymers of lactic acid-glycolic acid such as the LUPRON DEPOT®(injectable microspheres composed of a copolymer of lactic acid and glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid provide the 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 time, they may be subject to denaturation or to form aggregates under the influence of humidity at 37°C, which leads to loss of bio�logicheskoi activity and possible changes in immunogenicity. Depending on the involved mechanism it is possible to develop rational strategies for stabilization. For example, if you discovered that the mechanism of aggregation is the intermolecular formation of S-S communication through thio-disulfide interchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer compositions of matrices.

The antibody may be manufactured in any suitable form for delivery to the tissue/cell-target. For example, antibodies can be produced in the form of immunoliposome. "Liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactants that are suitable for drug delivery to a mammal. Components of liposomes usually form a two-layer structure, similar to the placement of the lipids of biological membranes. Liposomes containing the antibody, prepared by methods known in this field, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); U.S. patent No. 4485045 and 4544545; and WO97/38731 published October 23, 1997. Liposomes with enhanced circulation time are described in U.S. patent No. 5013556.

Especially suitable liposomes can be obtained way� reversed-phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and a derivative of phosphatidylethanolamine with PEG (PEG-PE). Liposomes are extruded through filters with defined pore size to obtain liposomes with the desired diameter. Fab'-fragments of the antibodies of the present invention can be konjugierte with liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via the reaction of disulfide exchange. In the liposome contains optional chemotherapeutic agent. Cm. Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989).

Pharmaceutical compositions to be used for the introduction ofin vivoas a rule , are sterile. This is easily accomplished by filtration through sterile filtration membranes.

H. Treatment with antibodies against CD79b

To determine CD79b expression in malignant tumors of various tests available for detection. In one embodiment, the implementation, sverkhekspressiya CD79b polypeptide can be analyzed by immunohistochemistry (IHC). Immersed in paraffin tissue sections from biopsy specimens of tumors can be subjected to IHC analysis and agree with the criteria of staining intensity of protein CD79b as follows:

Indicator 0 - no staining is observed or observed membrane staining in less than 10% of tumor cells.

Figure 1+ - detected faint/barely perceptible membrane staining in more than 10% SDA�olewig cells. Cells are stained only in part of their membrane.

Indicator 2+ - watch from weak to moderate complete membrane staining in more than 10% of tumor cells.

Indicator 3+ - see moderate to strong complete membrane staining in more than 10% of tumor cells.

Tumors with indicators of expression of CD79b polypeptide, components 0 or 1+, can be described as not sverkhekspressiya CD79b, and the tumors with 2+ or 3+ can be described as sverkhekspressiya CD79b.

Alternative or additionally, to determine the degree of sverkhekspressiya (if available), CD79b in the tumor can be performed the FISH analyses, such as the INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois) on formalin fixed immersed in paraffin tumor tissue.

Sverkhekspressiya or amplification CD79b can be assessed with the use of analysis for the detection ofin vivofor example , by administering a molecule (such as an antibody) which binds the molecule to be detected, and is labeled amenable to detection label (e.g. a radioactive isotope or a fluorescent label) and externally scanning the patient in relation to localization of the label.

As described above, antibodies against CD79b according to the invention have various non-therapeutic applications. Antibodies against CD79b the present invention can �to emanate to determine the stage of expressing CD79b polypeptide of malignant tumors (e.g., if radioitalia). Antibodies are also useful for purification or immunoprecipitation of CD79b polypeptide from cells, for detection and quantification of the CD79b polypeptidein vitrofor example , in an ELISA or a Western blot, to kill and eliminate expressing CD79b cells from a population of mixed cells as a stage in the purification of other cells.

Currently, depending on the stage of a malignant tumor, the treatment of a malignant tumor involves one or a combination of the following therapies: surgery to remove tissue malignant tumor, radiation therapy and chemotherapy. Therapy with antibody against CD79b may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy, and in metastatic disease where radiation therapy has limited applicability. Aimed at the tumor antibodies against CD79b according to the invention is suitable for the attenuation of expressing CD79b tumors at initial diagnosis of the disease or during an exacerbation. For therapeutic applications, antibodies against CD79b can be used separately, or in combination therapy with, for example, hormones, antiangiogenic agents or radioactively labeled compounds, or with surgery, cryotherapy, and/or radiation therapy. The treatment� antibody against CD79b can be performed along with other forms of conventional therapy, or sequentially with conventional therapy, either before or after conventional treatment. For the treatment of malignant tumors, particularly in patients with good risk, use of chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (paclitaxel), estramustine and mitoxantrone. In the present method of the invention for the treatment or mitigation of a malignant tumor, the patient with malignant tumor, you can enter the CD79b antibody against together with the introduction of one or more of the aforementioned chemotherapeutic agents. In particular, it provides combination therapy with paclitaxel and modified derivatives (see, for example, EP0600517). Antibodies against CD79b you can enter with a therapeutically effective dose of chemotherapeutic agents. In another embodiment, the implementation, the antibody against CD79b administered together with chemotherapy to enhance the activity and efficacy of chemotherapeutic agents, e.g., paclitaxel. In Physicians' Desk Reference (PDR) describes the dosage of these funds that can be used for the treatment of various malignant tumors. The dosing regimen and dosages of these aforementioned chemotherapeutic agents that are therapeutically effective will depend on the specific malignant tumors exposed to treatment, the degree of ill�tion and other factors, known qualified therapist, and they may be determined by the physician.

In one particular variant of implementation, the patient is administered with a conjugate containing an antibody against CD79b, anywhereman with a cytotoxic agent. Preferably, immunoconjugate associated with protein CD79b, internalized cell, resulting in increased therapeutic efficacy immunoconjugate for the destruction of malignant cells with which it is associated. In a preferred embodiment of implementation, the cytotoxic agent targeted to a nucleic acid in a malignant cell or hurts her. Examples of such cytotoxic funds described above and include maytansinoid, calicheamicin, ribonuclease and DNA endonuclease.

Antibodies against CD79b or their conjugates with toxins administered to the patient is a person, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a certain period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, vnutricinovialnoe, intrathecal, oral, local or inhalation methods. Preferred is intravenous or subcutaneous administration of the antibody.

With the introduction of antibodies against CD79b mo�but to combine other treatment regimens. The combined introduction includes co-administration using separate formulations or a single pharmaceutical composition, and sequential introduction in any order, preferably where there is a time period while both (or all) active ingredient simultaneously exert their biological activities. Preferably, such combination therapy leads to a synergistic therapeutic effect.

Also, it may be desirable that the combined introduction of antibodies or antibodies against CD79b, with the introduction of an antibody directed against another tumor antigen associated with a particular malignancy.

In another embodiment of implementation, methods of medical treatment of the present invention involve the combined introduction of antibodies against CD79b (or antibodies) and one or more chemotherapeutic agents or inhibiting the growth of funds, including co-administration of cocktails of different chemotherapeutic agents, or other cytotoxic funds(the funds), or other medicinal products(tools) that also inhibit tumor growth. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxypaclitaxel (such as paclitax�l and docetaxel) and/or anthracycline antibiotics. Drugs and dosing schemes for such chemotherapeutic agents can be used in accordance with the manufacturer's instructions or as empirically determines a qualified technician. Drugs and dosing schemes for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, MD (1992). The antibody can be combined with anti-hormonal compound; e.g., connection with the antiestrogen such as tamoxifen; with antiprogesterone such as onapristone (see, EP 616812); or an anti-androgen such as flutamide, in dosages known for such molecules. When a malignant tumor to be treated, is an androgen-dependent tumor, the patient is first subjected to anti-androgen therapy and, after the tumor becomes androgen-independent, the patient can introduce antibodies against CD79b (and optionally other means as described herein).

Sometimes, also useful is the co-administration to the patient of cardio protector (to prevent or reduce myocardial dysfunction associated with therapy) or one or more cytokines. In addition to the above therapeutic regimens, the patient can be subjected to surgical removal of malignant cells and/or radiation therapy (e.g., radiation by external beam �whether therapy with radioactive means such as an antibody), prior to, simultaneously or after antibody therapy. Suitable dosages for any of the above jointly administered funds are those which are currently in use, and can be reduced due to the combined action (synergy) funds and antibodies against CD79b.

The composition of the antibody according to the invention can be produced, dosing and introduce ways consistent with "Good medical practice". Factors considered in this context include the specific violation is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the violation, the region of the delivery means, the method of administration, schedule of administration, and other factors known to physicians. The antibody should not be made, however, it is not necessarily manufactured, with one or more of the funds currently used for the prevention or treatment of the disorders. An effective amount of such other funds depends on the number of antibodies according to the invention present in the composition, the type of disorder or treatment, and other factors discussed above. They are commonly used in the same dosages and with the same routes of administration as described herein above or in the amount of from about 1 to 99% from that described above only� dosages.

For the prevention or treatment of disease, the dosage and method of administration can be selected by the physician according to known criteria. The appropriate dosage of the antibody may depend on the type of disease targeted by the treatment, as defined above, the severity and course of the disease, a preventive or therapeutic purposes, the introduction of molecules, previous treatment, clinical history of the patient and response to the antibody and from the decision of the attending physician. The antibody is suitably administered to the patient at one time or over a series of injections. Preferably, the antibody is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, the original intended dosage for administration to the patient is from about 1 μg/kg to about 50 mg/kg of body weight (e.g., about 0.1-15 mg/kg/dose) of antibody, for example, or through one or more separate administrations, or by continuous infusion. The dosing scheme may include the introduction of an initial loading dose of approximately 4 mg/kg, with subsequent maintenance dose antibodies against CD79b approximately 2 mg/kg every week. However, there may be other suitable feeding patterns. A typical daily dosage may range from about 1 μg/kg to 10 mg/kg or more, depending on the factors mentioned above. In the case of multiple injections over several days or more, depending on the state, the introduction is repeated until the occurrence of a desired suppression of disease symptoms. Monitoring of the progress of this therapy is easily carried out using conventional methods and analysis, and based on the criteria known to the physician and other qualified professionals.

In addition to the introduction of the protein of the antibody to the patient, the present application provides for the introduction of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression "the introduction of a therapeutically effective amount of an antibody". See, for example, WO96/07321 published March 14, 1996, regarding the application of gene therapy to generate intracellular antibodies.

There are two main approaches for introducing nucleic acid (optionally contained in a vector) into the cells of the patient;in vivoandex vivo. Deliveryin vivo, the nucleic acid is injected directly to the individual, and usually the area where you want the antibody. For the treatment ofex vivothe cells of the individual is removed, these cells are injected nucleic acid, and the modified cells back to the individual, either directly or, for example, encapsulated in the form of porous membranes, which� implanted the patient (see, for example, U.S. patent No. 4892538 and 5283187). There are many ways of introducing nucleic acids into viable cells. The methods vary depending on, carry Lee nucleic acid in cultured cells ofin vitroor carry themin vivoin the cells of the intended host. Ways suitable for the transfer of nucleic acid into mammalian cellsin vitroinclude the use of liposomes, electroporation, microinjection, cell fusion, method with DEAE-dextran and deposition of calcium phosphate. A widely used vector for gene deliveryex vivois a retrovirus.

Preferred in the present methods of transferring nucleic acidsin vivoinclude transfection with viral vectors (such as adenovirus, herpes simplex virus I, or adeno-associated virus) and systems based on lipids (suitable lipids for mediated lipid transfer gene are, for example, DOTMA, DOPE and DC-Chol). To review the currently known protocols of marking the genes and gene therapy, see Anderson et al., Science 256:808-813 (1992). Also see WO 93/25673 and references cited therein.

Antibodies against CD79b according to the invention can be presented in various forms covered by the definition of "antibody" herein. Thus, the antibodies include full-length or intact antibody, frag�patients under stood antibodies, antibody with a native sequence and variant amino acids, humanized, or chimeric antibody fused, immunoconjugate and their functional fragments. In the fusion of the antibody sequence of the antibody is fused with a heterologous polypeptide sequence. Antibodies can be modified in the Fc-region for providing the desired effector functions. As discussed in more detail in the sections of the present document, a “naked” antibody with the appropriate Fc regions associated on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by involvement of complement in complement dependent cytotoxicity, or some other mechanism. Alternative when it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, you can use some other Fc-region.

In one embodiment of the antibody competes for binding or binds essentially the same epitope as the antibody of the invention. Also provides antibodies having the biological characteristics of the presented antibodies against CD79b according to the invention, specifically including targeting tumorin vivoand any inhibition tion�the stripes cell proliferation or cytotoxic characteristics.

Methods of obtaining the above-mentioned antibodies discussed in more detail in this document.

These antibodies against CD79b suitable for targeting expressing CD79b tumor or mitigate one or more symptoms of a malignant tumour in a mammal. Such a malignant tumor includes, but is not limited to, hematopoietic malignant tumors or tumor-related blood malignancies such as lymphoma, leukemia, myeloma or lymphoid malignancies, and malignant tumors of the spleen and malignant tumors of the lymph nodes. More specific examples of such associated with B-cell malignancies include, for example, well-differentiated, intermediate or low-grade lymphomas (including B cell lymphomas such as B-cell lymphoma-associated lymphoid tissue of the mucous membranes, and nehodgkinski lymphoma, b-cell lymphoma mantle zone, Burkitt lymphoma, malcolmfeijten lymphoma, lymphoma marginal zone cells, diffuse large cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma and T-cell lymphomas) and leukemias (including secondary leukemia, chronic lymphocytic leukemia, such as B cell leukemia (CD5+ B lymphocytes), myeloid leukemia, such as acute mie�kidny leukemia, chronic myeloid leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia and myelodysplasia), and other hematological and/or associated with B-cells or T-cells of a malignant tumor. Malignant tumors encompass metastatic malignant tumors of any of the above types. The antibody is able to bind with at least a part of malignant cells that Express the CD79b polypeptide in the mammal. In a preferred embodiment of the implementation, the antibody is effective to destroy or destruction of expressing CD79b tumor cells or inhibiting the growth of such tumor cells,in vitroorin vivowhen binding to CD79b polypeptide in the cell. Such an antibody includes a naked antibody against CD79b (not anywhereman with any tool). Simple antibodies that have cytotoxic or cell growth inhibitory properties can further be combined with a cytotoxic agent to ensure that they were more effective in destroying tumors. Cytotoxic properties can be given to the CD79b antibody against, for example, by conjugation of antibody with a cytotoxic agent, with the formation of immunoconjugate, as described herein. Cytotoxic agent or growth inhibitory agent, the assumption�plant is a low-molecular compound. Preferred are toxins, such as calicheamicin or maytansinoid and its analogs or derivatives.

The invention relates to compositions containing antibodies against CD79b according to the invention and a carrier. For the purposes of treatment of malignant tumors, the compositions can be administered to the patient in need of such treatment, where the composition may contain one or more antibodies against CD79b present as immunoconjugate or “naked” antibodies. In the next version of the implementation, the compositions may contain these antibodies in combination with other drugs, such as cytotoxic or inhibiting the growth of funds, including chemotherapeutic agents. The invention also relates to compositions containing antibodies against CD79b according to the invention and a carrier. In one embodiment, the implementation, the composition is a therapeutic composition comprising a pharmaceutically acceptable carrier.

Another aspect of the invention relates to isolated nucleic acids encoding antibodies against CD79b. Provides for nucleic acids encoding both the H-and L-chains, and especially the remains of the hypervariable region, the chains that encode the antibody with a native sequence, and variants, modifications and humanized versions of the antibody.

The invention also relates to FPIC�BAM, suitable for the treatment of expressing CD79b polypeptide malignant tumor or mitigate one or more symptoms of a malignant tumour in a mammal, comprising administering to the mammal a therapeutically effective amount of antibodies against CD79b. Therapeutic antibody compositions can introduce short-term (acute) or long-term, or interrupted, in accordance with the discretion of the physician. Also provides methods for inhibiting the growth and destruction of expressing CD79b polypeptide of the cell.

The invention also relates to kits and articles containing at least one antibody against CD79b. Kits containing antibodies against CD79b, applicable, for example, for analyses of cell killing CD79b, for purification or immunoprecipitation of CD79b polypeptide from cells. For example, for isolation and purification CD79b, the kit may contain antibodies against CD79b associated with the pellets (e.g., pellets from sepharose). Can be provided kits, which contain antibodies for detection and quantification CD79bin vitrofor example , in an ELISA or a Western blot. This antibody is suitable for detection, may be provided with a label such as a fluorescent or radioactive label.

I. Treatment of the conjugate antibody-drug

It is envisaged that conjuga�s antibody-drug (ADC) according to the present invention can be used to treat various diseases or disorders, for example, characterized by sverkhekspressiya tumor antigen. Illustrative of the condition or hyperproliferative disorders include benign or malignant tumors; leukemias and lymphoid malignant tumors. Other conditions include neuronal, glial, astroglial, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocele, inflammatory, angiogenic and immunologic, including autoimmune, disorders.

Connection ADC which are identified in the animal models in cellular assays can be further tested with the tumor higher primates and in clinical trials in humans. Clinical trials in humans may be designed to test the effectiveness of monoclonal antibodies against CD79b or immunoconjugate according to the invention in patients suffering from B-cell-proliferative violation, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone. A clinical trial may be designed� to assess the effectiveness of the ADC in combination with known regimens, such as radiation therapy and/or chemotherapy involving chemotherapeutic and/or cytotoxic funds.

Typically, the disease or disorder to be treated, is a hyperproliferative disease such as B-cell-proliferative violation and/or B-cell malignant tumor. Examples of malignant tumor to be treated according to this document include, but are not limited to, B-cell-proliferative violation selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

A malignant tumor can contain expressing CD79b cells, so that the ADC of the present invention is able to bind with malignant cells. To determine CD79b expression in malignant tumors are available in a variety of diagnostic/prognostic methods of analysis. In one embodiment, the implementation, sverkhekspressiya CD79b can be analyzed by IHC. Immersed in the paraffin-embedded tissue sections of biopsy of the tumor can be subjected to IHC analysis and Apasov�VAT with the criteria of staining intensity of protein CD79b, associated with the degree of staining and the share of the investigated tumor cells.

For the prevention or treatment of disease, the appropriate dosage of the ADC depends on the type of disease to be treated, as defined above, the severity and course of the disease, a preventive or therapeutic purposes, the introduction of molecules, previous treatment, clinical history of the patient and response to the antibody and from the decision of the attending physician. The molecule is suitably administered to the patient at one time or over a series of injections. Depending on the type and severity of the disease, the original intended dosage for administration to the patient is from about 1 μg/kg to 15 mg/kg of body weight (e.g., 0.1 to 20 mg/kg) molecules, for example, or through one or more separate administrations, or by continuous infusion. A typical daily dosage may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Illustrative dosage ADC to be administered to a patient, is in the range from approximately 0.1 to approximately 10 mg/kg of body weight of the patient.

In the case of multiple injections over several days or more, depending on the state, the introduction is repeated until the occurrence of a desired suppression of disease symptoms. Illustrative of sh�mA dosage includes the introduction of the initial loading dose, approximately 4 mg/kg, with subsequent weekly maintenance dose of approximately 2 mg/kg antibody against ErbB2. May be suitable other dosing schemes. Monitoring of the progress of this therapy is easily carried out using conventional methods and analyses.

J. Combination therapy

Conjugate antibody-drug (ADC) according to the invention can be combined in a pharmaceutical combination composition or scheme of dosing as combination therapy, with a second compound having properties against malignant tumors. The second compound of the pharmaceutical composition or combined feeding patterns preferably has activities, complementing the activities of the ADC in combination, so that they do not have any adverse effect on each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent and/or cardioprotector. Such molecules are suitably present in combination in amounts that are effective for the intended purpose. Pharmaceutical composition containing ADC according to the invention, may also have a therapeutically effective amount of chemotherapeutic agents, such �AK formation inhibitor of tubulin, the topoisomerase inhibitor or a DNA binding agent.

In one aspect, the first compound is a ADC against CD79b the present invention, and the second compound is an antibody against CD20 (or “naked” antibody or ADC). In one embodiment, the implementation of the second compound is an antibody against CD20 rituximab (Rituxan®) or 2H7 (Genentech, Inc., South San Francisco, CA). Other antibodies suitable for combination immunotherapy by ADC against CD79b according to the invention, include, but are not limited to, antibody against VEGF (e.g., Avastin®).

Other treatment regimens can be combined with treatment against malignant tumors, identified in accordance with this invention, including, but not limited to, radiation therapy and/or bone marrow transplantation and peripheral blood, and/or cytotoxic agent, chemotherapeutic agent, or growth inhibitory agent. In one of these embodiments, the chemotherapeutic agent is a means or combination of means, such as cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (OncovinTM), prednisolone, CHOP, CVP, or COP, or immunotherapeutic agents such as an antibody against CD20 (e.g., Rituxan®) or an antibody against VEGF (e.g., Avastin®).

Kombinirovanno� therapy can be performed as a simultaneous or sequential circuits. The sequential introduction, the combination can be entered in two or more injections. The combined introduction includes co-administration using separate formulations or a single pharmaceutical composition, and sequential introduction in any order, preferably where there is a time period while both (or all) active ingredient simultaneously exert their biological activities.

In one embodiment of implementation, the treatment by ADC combo involves the introduction of anti-malignant tumor identified herein, and one or more chemotherapeutic agents or inhibiting the growth of funds, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Drugs and dosing schemes such chemotherapeutic agents can be used in accordance with the manufacturer's instructions, or as may empirically determine a qualified technician. Drugs and dosing schemes for such chemotherapy are also described in Chemotherapy Service", (1992) Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.

Suitable dosages for any of the above jointly administered funds represent dosiro�and, currently used and can be reduced through the combined action (synergy) of the newly identified funds and other chemotherapeutic agents or treatments.

Combination therapy may provide "synergy" and "synergistic", i.e. the effect achieved by joint application of the active ingredients, exceeds the sum of effects due to the use of compounds separately. The synergistic effect can be achieved when the active ingredients are: (1) jointly produced and administered or delivered simultaneously in a combined unit dosage composition; (2) delivered serially or in parallel as separate formulations; or (3) apply with any other scheme. Upon delivery by means of alternate therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. Typically, in the course of the alternate therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, as in combination therapy, effective dosages of two or more active ingredients are administered together.

K. Products and kits

Another embodiment of the this invention relates to a product containing materials, suitable �La treatment, prevention and/or diagnosis of expressing CD79b malignant tumors. The product includes a container and a label on the container or liner in the package attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. the Containers can be made of many materials such as glass or plastic. The container contains a composition which is effective for treatment, prevention or diagnosis of a malignant condition, and may have a sterile access port (for example, the container may be a bag of intravenous solution or vial having a stopper penetrable needle for subcutaneous injection). At least one active substance in the composition is an antibody against CD79b according to the invention. On the label or the liner in the packaging indicates that the composition is used to treat malignant tumors. In addition, the label or the liner in the package contains information on the introduction of the composition of the antibody to the patient with malignant tumor. Moreover, the product may further contain a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose solution. In addition, it may include other materials desirable from to�marcheschi and user point of view, including other buffers, diluents, filters, needles, syringes.

Also provides kits that are suitable for different purposes, such as analysis of the destruction of expressing CD79b cells, for purification or immunoprecipitation of CD79b polypeptide from cells. For isolation and purification of CD79b polypeptide, the kit can contain an antibody against CD79b associated with the pellets (e.g., pellets from sepharose). Can be provided kits, which contain antibodies for detection and quantification of the CD79b polypeptidein vitrofor example , in an ELISA or a Western blot. As in the case of products, the kit contains a container and the label on the container or liner in the package attached to the container. The container contains a composition comprising at least one antibody against CD79b according to the invention. May include additional containers, which contain, e.g., diluents and buffers, control antibodies. On the label or the liner in the package may be provided with the description of the composition, and instructions for intended use ofin vitroor detection.

L. the Use of CD79b polypeptides

This invention relates to methods of screening compounds to identify compounds that mimic the CD79b polypeptide (agonists) or prevent the effect of CD79b polypeptide (antagonists). I�Tana screening tests for suspected drugs-antagonists for identification of compounds which bind or form a complex with CD79b polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, including e.g., inhibiting the expression of CD79b polypeptide from cells. Such screening assays will include assays suitable for high throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drugs candidates.

Tests can be performed in a variety of formats, including analyses of protein-protein binding, biochemical screening assays, immunoassays and cell-based assays, which are well characterized in the field.

All assays for antagonists are common in that they require contacting the drug candidate with a CD79b polypeptide encoded by a nucleic acid identified herein under conditions and for a time period sufficient to ensure the interaction of these two components.

In binding assays, the interaction is binding and the complex formed can distinguish or discriminate detection in the reaction mixture. In a particular variant of implementation, the CD79b polypeptide encoded by GE�Ohm, identified herein or the drug candidate is immobilized on a solid phase, for example, the microplate for titration, by covalent or non-covalent joining. Non-covalent attachment, as a rule, is carried out by coating a solid solution phase of a CD79b polypeptide and drying. Alternative immobilized antibody, e.g. a monoclonal antibody, specific to the CD79b polypeptide, subject to immobilization, can be used to anchor it to a solid surface. The analysis is carried out by adding neimmunizirovannah component that can be labeled amenable to the detection of the label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is completed, unreacted components are removed, e.g. by washing, and carry out the detection of complexes anchored on the solid surface. When the original neemalirovannym component has a measurable detection label, the detection of label immobilized on the surface indicates that there was a formation of the complex. When the original neemalirovannym component does not have a label, the detection of complex formation can be performed, for example, using a labeled antibody specific binding immobiliza�tion complex.

If a connection-candidate interacts with a specific CD79b polypeptide encoded by the gene identified herein, but is not associated with him, its interaction with that polypeptide can be analyzed well-known methods of detecting protein-protein interactions. Such assays include traditional approaches, such as cross-linking, coimmunoprecipitate and co-purification through gradients or chromatographic columns. In addition, monitoring protein-protein interactions 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)) as described Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many activators of transcription, such as yeast GAL4, consist of two physically separated modular domains, one acting as the DNA-binding domain and the other domain functions as activation of transcription. Yeast ekspresowa system described in the above publications (usually called "twohybrid system") uses this property, and it uses two hybrid protein, one protein target is merged with the DNA binding domain of GAL4, and the other of which activates proteins-candidates are merged with the domain Akti�ation. The expression of reporter gene GAL1-lacZ under the control of a GAL4-activated promoter depends on the restoration of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKERTMto identify protein-protein interactions between two specific proteins using twohybrid method is commercially available from Clontech. This system can also be extended to map protein domains involved in specific interactions of proteins, as well as to determine the position of amino acid residues that are important for these interactions.

Compounds that inhibit the interaction of a gene encoding a CD79b polypeptide identified herein and other intra - or extracellular components can be tested as follows: usually receive a reaction mixture containing the product of the gene and the intra - or extracellular component under conditions and for a time period that allows interaction and binding of the two products. To test the ability of the compounds of the candidate to inhibit binding, the reaction is carried out in the absence and in the presence of the tested compounds. In addition, in the third reaction mixture can�about to add placebo she served as positive control. Monitoring the binding (complex formation) between the test compound and the intra - or extracellular component present in the mixture, is carried out as described herein above. The formation of the complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

For analysis of antagonists, the CD79b polypeptide can be added to a cell along with the compound to be screened for a particular activity and the ability of compounds to inhibit interest activity in the presence of CD79b polypeptide indicates that the compound is an antagonist of a CD79b polypeptide. Alternatively, the detection of antagonists can be performed by combining the CD79b polypeptide and a potential antagonist with membrane-bound receptors CD79b polypeptide or recombinant receptors under appropriate conditions for a competitive analysis of binding. The CD79b polypeptide can be labeled, e.g., radioactive label, so the number of molecules of a CD79b polypeptide bound to the receptor, can be used to determine the effectiveness of the potential antagonist. The gene encoding the-counter�'or you can identify a variety of ways known to specialists in this field, for example, penninga ligand and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably use expressing cloning, where get polyadenylated RNA from cells that are responsible for the CD79b polypeptide and a cDNA library created from this RNA is divided into pools and used for transfection of COS cells or other cells that do not respond to the CD79b polypeptide. Transfetsirovannyh cells that are grown on glass slides, exposed to labeled CD79b polypeptide. The CD79b polypeptide can be labeled in various ways, including iodination or inclusion of a recognition site for a particular site-specific protein kinase. After fixation and incubation, the glass is subjected radioautographic analysis. Identify positive pools and get subpoly and re transferout them using an agreed process of obtaining subpool and re-screening, which ultimately leads to a single clone that encodes the intended receptor.

As an alternative approach for the identification of the receptor labeled with a CD79b polypeptide can be subjected photoaffinity binding with cell membrane or drugs extracts that Express molecule recepto�and. Cross stitched material separated by PAGE and show on x-ray film. The labeled complex containing the receptor can be cut, divided into peptide fragments, and subjected to protein microsequencing. Amino acid sequence obtained by mikroekonomia, can be used to design a set of degenerate oligonucleotide probes for screening the cDNA library to identify the gene encoding the presumed receptor.

In another analysis of antagonists, mammalian cells or a membrane preparation expressing the receptor, incubate with labelled CD79b polypeptide in the presence of the connection candidate. Then you can measure the ability of the compound to enhance or block this interaction.

More specific examples of potential antagonists include an oligonucleotide that binds to immunoglobulin, merged with a CD79b polypeptide, and, in particular, antibodies including, but not limited to, poly - and monoclonal antibodies and fragments of antibodies, single-chain antibodies, antiidiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and fragments of antibodies. Alternatively, a potential antagonist may be highly related protein, for example, a mutant of form� CD79b polypeptide, which recognizes the receptor but has no effect, thus, competitive inhibiting effect of CD79b polypeptide.

Antibody that is specific binds CD79b polypeptide identified herein, as well as other molecules identified by the screening assays described herein above, can be introduced for the treatment of various disorders, including malignant tumor, in the form of pharmaceutical compositions.

If the CD79b polypeptide is intracellular and as inhibitors using whole antibodies, are preferred internalities antibodies. But also to deliver the antibody or fragment of the antibody into the cells, you can use lipofectin or liposomes. When using fragments of antibodies, preferred is the smallest inhibitory fragment that binds to a specific binding domain of the target protein. For example, on the basis of sequences of the variable regions of the antibody, can be designed molecules peptides that retain the ability to bind a protein sequence as the target. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The composition described herein may also contain more than one active �connection if necessary, to specific evidence, which is directed to the treatment, preferably, the connection forms a complementary activity that will not adversely impact on each other. Alternative or additionally, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

M. Derivatives of antibodies

The antibodies of the present invention can be further modified to contain additional non-protein groups that are famous in this area and easily accessible. Preferably, groups that are suitable for deriving antibodies, are water-soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, copolymer ethylene/maleic anhydride, polyaminoamide (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polietilen�icol, probabilistical the homopolymers, copolymers of polypropylene oxide/ethylene oxide, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Propionaldehyde of polyethylene glycol may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number of polymers related to the antibody may vary, and if it is more than one polymer, they may be the same or different molecules. Typically, the number and/or type of polymers used to obtain the derivative, can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, the use of derived antibodies in therapy in certain conditions, etc.

N. Method of screening

Another embodiment of the present invention relates to a method for determining the presence of CD79b polypeptide in a sample suspected to contain a CD79b polypeptide, where the method includes the impact on the sample conjugate antibody-drug that binds to a CD79b polypeptide and determining binding of the conjugate antibody-drug with the CD79b polypeptide in the sample, where the presence of such an a� binding indicates the presence of CD79b polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) presumably expressing CD79b polypeptide. Conjugate antibody-drug, used in the method, optionally, you can mark beyond detection label to associate with a firm pad or similar.

Another embodiment of the present invention relates to a method for diagnosing the presence of a tumor in a mammal, where the method includes (a) contacting the test sample containing tissue cells obtained from the mammal with the conjugate antibody-drug that binds to a CD79b polypeptide, and (b) detection of complex formation between cojugation antibody-drug and CD79b polypeptide in the sample, where the formation of the complex indicates the presence of tumor in the mammal. Optionally, the conjugate antibody-drug, is labelled amenable to detection label associated with the solid substrate or similar, and/or the test sample of tissue cells obtained from the individual, assumed to be of a malignant tumor.

IV. Additional uses of antibodies against CD79b and immunoconjugates

A. Diagnostic methods and methods of detection

In one aspect, antibodies against C79b and immunoconjugate according to the invention are suitable for detecting the presence of CD79b in a biological sample. The term "detection" as used herein includes qualitative and / or quantitative detection. In certain embodiments, the biological sample comprises a cell or tissue. In certain embodiments, such tissues include normal and/or cancerous tissues that Express CD79b at higher levels relative to other tissues, for example, B-cells and/or associated with B-cells of the tissues.

In one aspect, the invention relates to a method of detecting the presence of CD79b in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an antibody against CD79b under conditions that allow the binding of an antibody against with CD79b CD79b, and detection of complex formation between the antibody against and CD79b CD79b.

In one aspect, the invention relates to a method for diagnosing disorders associated with increased expression of CD79b. In certain embodiments, the method comprises contacting the test cells with an antibody against CD79b; determining the level of expression (either quantitatively or qualitatively) CD79b the test cell by detecting binding of antibodies against CD79b with CD79b; and comparing the level of expression of CD79b of the test cell with the level of expression of CD79b a control cell (e.g., a normal cell of origin Taiwan brand�Dasha of the same fabric, that of the test cell, or a cell that expresses CD79b at levels comparable to those for normal cells), where a higher level of expression of CD79b the test cell compared to the control cell indicates the presence of disorders associated with increased expression of CD79b. In certain embodiments, the test cell is obtained from the individual with an alleged violation, associated with increased expression of CD79b. In certain embodiments, the offense is a cell-proliferative violation, such as a malignant tumor or a tumor.

Illustrative of cell-proliferative disorders that can be diagnosed using an antibody of the invention include B-cell trait and/or B-cell-proliferative violation, including, but not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In certain embodiments, a method of diagnosis or detection, such as the methods described above, includes� detection of binding of antibodies against CD79b with CD79b, expressed on the cell surface or in a membrane preparation obtained from cells expressing CD79b on the surface. In certain embodiments, the method comprises contacting the cells with an antibody against CD79b under conditions that allow the binding of an antibody against with CD79b CD79b, and detection of complex formation between the antibody against and CD79b CD79b on the surface of the cell. Illustrative analysis for the detection of binding antibodies against CD79b with CD79b expressed on the cell surface, is an analysis of "FACS".

For detection of binding of antibodies against CD79b with CD79b you can use some other ways. Such methods include, but are not limited to, analyses of binding to the antigen, which are well known in this field, such as Western blots, radioimmunoassay tests, ELISA (ELISA), immune "sandwich"assays, analysis of immunoprecipitation, fluorescent immunoassays, immunoassays to protein A and immunohistochemistry (IHC).

In certain embodiments, antibodies against CD79b are marked. Labels include, but are not limited to, a label or group that is subjected to detection directly (such as fluorescent, chromophoric, electronmobility, chemiluminescent and radioactive labels), as well as groups such as f�renty or ligands, which is subjected to indirect detection, for example, by enzymatic reaction or molecular interaction. Illustrative labels include, but are not limited to, radioisotopes32P,14C,125I,3H and131I, fluorophores such as rare earth chelates metals or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, 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, oxidase saccharides, e.g., glucose oxidase, galactosidase and glucose-6-phosphatedehydrogenase, heterocyclic oxidase, such as uricase and xanthine oxidase associated with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microbiocides, Biotin/avidin, spin labels, bacteriophobia labels, stable free radicals, etc.

In certain embodiments, antibodies against CD79b is immobilized on an insoluble matrix. Immobilization involves separating antibodies against CD79b from any CD79b, which remains free in solution. It is carried out, or providing the insolubility antibodies against CD79b before analysis, for example, put�m adsorption on water-insoluble matrix or surface (Bennich et al., U. S. 3720760), or by covalent coupling (for example, using cross-linkage with glutaraldehyde), or providing the insolubility antibodies against CD79b after the formation of a complex between the antibody against and CD79b CD79b, for example, by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be done using immunoconjugate according to the invention is antibodies against CD79b, or in addition thereto.

B. Methods of treatment

The antibody or immunoconjugate according to the invention can be used, for example, in the treatment methodsin vitro,ex vivoandin vivo. In one aspect, the invention relates to methods of inhibiting the growth or proliferation of cells, orin vivoorin vitroand the method includes the impact on cell antibodies against CD79b or immunoconjugate under conditions that allow binding immunoconjugate with CD79b. "Inhibition of cell growth or proliferation" means a reduction of cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and includes the induction of cell death. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a B-cell. In certain embodiments, the cell is a combintion of�th xenograft, for example, as illustrated herein.

In one aspect, the antibody or immunoconjugate according to the invention is used for the treatment or prophylaxis of B-cell-proliferative disorders. In certain embodiments, cell-proliferative violation associated with increased expression and/or activity CD79b. For example, in certain embodiments, B-cell-proliferative violation associated with increased expression of CD79b on the surface of B-cells. In certain embodiments, B-cell-proliferative infringement is a tumor or a malignant tumor. Examples of B-cell-proliferative disorders to be treated by the antibodies or immunoconjugate according to the invention, include, but are not limited to, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

In one aspect, the invention relates to methods of treating B-cell-proliferative disorders, comprising administering to the individual an effective amount of antibodies against CD79b or immunology�Gata. In certain embodiments, a method of treating B-cell-proliferative disorders comprises administering to the individual an effective amount of a pharmaceutical composition containing the antibody against CD79b or immunoconjugate against CD79b and optionally at least one additional drug, such as medicines below. In certain embodiments, a method of treating cell-proliferative disorders comprises administering to the individual an effective amount of a pharmaceutical composition comprising 1) immunoconjugate containing antibodies against CD79b and a cytotoxic agent; and optionally, 2) at least one additional drug, such as a vehicle, is provided below.

In one aspect, at least some of the antibodies or immunoconjugates according to the invention can bind to CD79b species different from man. Thus, antibodies or immunoconjugate according to the invention can be used to associate CD79b, for example, in a cell culture containing CD79b, human or other mammal having a CD79b, which antibody or immunoconjugate according to the invention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus monkey and macaque-rhesus, pig or mouse). In one embodiment, the implementation, the antibody or immune�conjugate against CD79b can be used for targeting CD79b on B cells by contacting the antibody or immunoconjugate with CD79b with the formation of a complex of the antibody or immunoconjugate-antigen, so the conjugated cytotoxin immunoconjugate penetrates the cell. In one embodiment, the implementation, CD79b CD79b is a person.

In one embodiment, the implementation, the antibody or immunoconjugate against CD79b can be used in the method of CD79b binding of an individual suffering from a violation associated with increased expression and/or activity CD79b, wherein the method comprises administering to the individual the antibody or immunoconjugate, so that was a CD79b binding of the individual. In one embodiment, the implementation of the associated antibody or immunoconjugate internalized in B-cell that expresses CD79b. In one embodiment, the implementation, CD79b CD79b is a person, and the individual is man. Alternative individual can be a mammal expressing CD79b, which binds CD79b antibody against. In addition, the individual may be a mammal that was introduced CD79b (e.g., by introducing CD79b or expression of the transgene encoding CD79b).

The antibody or immunoconjugate against CD79b you can enter to man for therapeutic purposes. Moreover, the antibody or immunoconjugate against CD79b you can enter non-human mammal, which CD79b is expressed with which the antibody cross-reacts (e.g., a Primate, pig, rat or mouse) for veterinary purposes or in ka�ETS animal model for human disease. Regarding the latter, such animal models for can be useful for evaluating therapeutic efficacy of antibodies or immunoconjugates according to the invention (e.g., testing of dosages and intervals of administration).

Antibodies or immunoconjugate according to the invention can be applied either alone or in combination with other compositions in a therapy. For example, the antibody or immunoconjugate according to the invention can be jointly introduce at least one additional pharmaceutical agent and/or adjuvant. In certain embodiments, the additional drug is a cytotoxic agent, chemotherapeutic agent, or growth inhibitory agent. In one of these embodiments, the chemotherapeutic agent is a means or combination of means, such as cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (OncovinTM), prednisolone, CHOP, CVP, or COP, or immunotherapeutic agents such as an antibody against CD20 (e.g., Rituxan®) or an antibody against VEGF (e.g., Avastin®), where combination therapy is suitable for treatment of malignant tumors and/or B-cell disorders such as B-cell-proliferative disorders, including lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, and recurrent�resivour NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

These types of combination therapies listed above encompass combined administration (where two or more drugs included in one or in separate formulations), and separate introduction, in which case the introduction of antibodies or immunoconjugate according to the invention can occur prior to, simultaneously and/or after the administration of additional medications 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 drug or adjuvant) can be entered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal introduction, and, if it is desirable for the local introduction, introduction to the lesion. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate suitably administered by pulse infusion, particularly with declining doses of anti�ate or immunoconjugate. Dosing can be performed by any suitable method, for example, by injection, such as intravenous or subcutaneous injections, depending in part, on whether the introduction of short-term or long-term.

Antibodies or immunoconjugate according to the invention can be produced, dosing and introduce ways consistent with "Good medical practice". Factors considered in this context include the specific violation is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the violation, the region of the delivery means, the method of administration, schedule of administration, and other factors known to physicians. The antibody or immunoconjugate should not be made, however they are not necessarily made with one or more tools, currently used for the prevention or treatment of the disorders. An effective amount of such other funds depends on the number of antibodies or immunoconjugate present in the composition, the type of disorder or treatment, and other factors discussed above. They are commonly used in the same dosages and with the same routes of administration as described herein above or in the amount of from about 1 to 99% of the dosages described herein, or in any d�Sarovka and with it in any way that is empirically/clinically determined, as appropriate.

For the prevention or treatment of disease, the dosage and method of administration of the antibody or immunoconjugate according to the invention (when used alone or in combination with one or more additional drugs, such as chemotherapeutic agents) may depend on the type of disease targeted by the treatment, the type of antibody or immunoconjugate, the severity and course of the disease, where the antibody or immunoconjugate administered for the prevention or treatment, previous treatment, clinical history of the patient and response to the antibody or immunoconjugate, and from the decision of the attending physician. The antibody or immunoconjugate suitably administered to the patient at one time or over a series of injections. Depending on the type and severity of the disease, the original intended dosage for administration to the patient is from about 1 μg/kg to about 100 mg/kg (e.g. 0.1 mg/kg to 20 mg/kg) of antibody or immunoconjugate, for example, or through one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Typical daily doziruemoy to vary from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. In the case of multiple injections over several days or more, depending on the state, the introduction is repeated until the occurrence of a desired suppression of disease symptoms. One exemplary dosage of the antibody or immunoconjugate may be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, the patient can enter one or more doses comprising about 0.5 mg/kg, 2.0 mg/kg 4,0 mg/kg or 10 mg/kg (or any combination) of the antibody or immunoconjugate. Such doses can be entered periodically, for example every week or every three weeks (e.g., so that the patient receives from about two to about twelve, or e.g. about six doses of the antibody or immunoconjugate). You can enter an initial higher loading dose, and then one or more lower doses. Illustrative dosing scheme may include the introduction of an initial loading dose of approximately 4 mg/kg, with subsequent maintenance dose of antibody is about 2 mg/kg every week. However, there may be other suitable feeding patterns. Monitoring of the progress of this therapy is easily carried out using conventional methods and analyses.

C. activity Assays

Antibodies against CD79b � immunoconjugate according to the invention can be characterized for their physical/chemical properties and/or biological activities by various assays known in this field.

1. Analyses activity

In one aspect, provides assays for the identification of antibodies against CD79b or immunoconjugate having biological activity. Biological activity may include, for example, the ability to inhibit the growth or proliferation of cells (for example, the activity "destruction of cells"), or the ability to induce cell death, including programmed cell death (apoptosis). Antibodies or immunoconjugate having such biological activity ofin vivoand/orin vitroalso foreseen.

In certain embodiments, antibodies against CD79b or immunoconjugate tested with respect to its ability to inhibit the growth or proliferation ofin vitro. Tests of inhibition of growth or cell proliferation are well known in this field. In certain assays cell proliferation, illustrated by analyses of cell killing", as described in this document, measure cell viability. One such analysis is the fluorescent analysis of cell viability CellTiter-GloTMwhich is commercially available from Promega (Madison, WI). In this analysis, determine the number of viable cells in culture based on quantification the presence of ATP, which is recognized�Ohm 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 that enables automated high-performance screening (HTS). Cm. Cree et al. (1995) Anticancer Drugs 6:398-404. Method of analysis involves adding the single reagent (reagent CellTiter-Glo®) directly to cultured cells. This leads to lysis of cells and the formation of fluorescent signal in the reaction of luciferase. The fluorescent signal is proportional to the number the presence of ATP, which is directly proportional to the number of living cells present in culture. Data can be registered in the luminometer or device for imaging with CCD camera. The output luminescence expressed as relative light units (RLU).

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

In one aspect, a CD79b antibody against tested with respect to its ability to induce cell deathin vitro. Analyses of the induction of cell death good� known in this field. In some embodiments, such assays measure, for example, loss of membrane integrity, demonstrated by the capture of propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology, 17: 1-11) or 7AAD. In the illustrative analysis of capture PI, the cells are cultured in a modified way the Needle of Dulbecco (D-MEM):Ham's F-12 (50:50) supplemented with 10% inactivated by heating FBS (Hyclone) and 2 mm L-glutamine. Thus, the analysis carried out in the absence of complement and immune effector cells. Cells are seeded at a density of 3×106a Cup Cup in 100×20 mm and allow to attach overnight. The medium is removed and replaced with fresh medium alone or medium containing various concentrations of antibody or immunoconjugate. Cells were incubated for 3 days. After treatment, the monolayers were washed by PBS and ukreplyayut by treatment with trypsin. Then the cells are centrifuged at 1200 rpm.min. for 5 minutes at 4°C, the pellet resuspended in 3 ml cold Ca2+-binding buffer (10 mm Hepes, pH of 7.4, 140 mm NaCl, 2.5 mm CaCl2) and divided into aliquots in sealed 35 mm mesh tubes 12×75 mm (1 ml per tube, 3 tubes in the treated group) for removal of cell pieces. Then in a test-tube add PI (10 μg/ml). Samples analyzed using a FACSCAN flow cytometerTMand software FACSCONVERTT CellQuest (Becton Dickinson). Thus, to identify antibodies or immunoconjugate that induce statistically significant levels of cell death as determined by the capture of PI.

In one aspect, the antibody or immunoconjugate against CD79b tested for their ability to induce apoptosis (programmed cell death)in vitro. Illustrative analysis of antibodies or immunoconjugates that induce apoptosis, is an analysis of the binding of annexin. In the illustrative analysis of the binding of annexin, cells are cultivated and sown in cups as described in the previous paragraph. The medium is removed and replaced with fresh medium alone or medium containing 0.001 to 10 μg/ml antibody or immunoconjugate. After incubation for three days, the monolayers washed with PBS and ukreplyayut by treatment with trypsin. Then the cells are centrifuged, was resuspended in Ca2+-binding buffer, and divided into aliquots in test tubes, as discussed in the previous paragraph. Then in a test-tube add labeled annexin (e.g. annexin V-FITC) (1 μg/ml). Samples analyzed using a FACSCAN flow cytometerTMand software FACSCONVERTTMCellQuest (BD Biosciences). Thus, to identify antibodies or immunoconjugate that induce statistically significant levels of binding of annexin otnositelnaya. Another illustrative analysis of antibodies or immunoconjugates that induce apoptosis, is a colorimetric analysis with ELISA histone DNA for the detection of vnutriregionaljnoj degradation of genomic DNA. Such analysis can be carried out using, for example, a kit for detection of cell death by ELISA (Roche, Palo Alto, CA).

Cells for use in any of the above testsin vitroinclude cells or cell lines that naturally Express CD79b or modified methods engineering to expressively CD79b. Such cells include tumor cells that sverkhekspressiya CD79b relative to normal cells originating from the same cloth. Such cells also include cell lines (including tumor cell lines) that Express CD79b, and a cell line that normally does not Express CD79b, but which were transfected by a nucleic acid that encodes CD79b.

In one aspect, a CD79b antibody against his or immunoconjugate tested for their ability to inhibit the growth or proliferation of cellsin vivo. In certain embodiments, antibodies against CD79b or immunoconjugate tested for their ability to inhibit the growth of tumorsin vivo. For this test it is possible to use the model system�s in vivosuch as a xenograft model. In the illustrative system compared to xenograft, tumor cells of a human is administered a suitable immunodeficient animal, non-human, e.g., a SCID mouse. The antibody or immunoconjugate according to the invention is administered to an animal. Measure the ability of antibodies or immunoconjugate to inhibit or reduce tumor growth. In certain embodiments of the above systems with the xenograft, tumor cells are tumor cells from the patient-person. Such cells are suitable for models with the xenograft include cell lines of leukemia and lymphoma rights, which include, but are not limited to, cells of BJAB-luc (EBV-negative cell line of Burkitt lymphoma, transfetsirovannyh reporter gene luciferase), 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 certain embodiments, the cells of human tumors injected a suitable immunodeficient animal, not human, by subcutaneous injection or by transplantation into a suitable area, such as the fat body of the mammary gland.

2. Analyses of binding and each�e methods of analysis

In one aspect, a CD79b antibody against tested with respect to its antigen-binding activity. For example, in certain embodiments, antibodies against CD79b tested against its ability to bind to CD79b expressed on the cell surface. For this kind of testing can be used FACS analysis.

In one aspect, you can use competitive analysis to identify a monoclonal antibody that competes with antibody against 2F2 mouse and/or humanized antibody 2F2.D7 for binding to CD79b. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by antibody 2F2 mouse and/or humanized antibody 2F2.D7. Illustrative competitive assays include, but are not limited to, conventional analyses, such as analyses are presented in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed description of illustrative ways of mapping the epitope to which the antibody binds, provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). Two antibodies consider communicating with one epitope if each blocks the binding of the other by 50% or more.

Illustrative competitive analysis, immobilized D79b incubated in solution, containing the first labeled antibody that binds to CD79b (e.g., antibody 2F2 mouse and/or a humanized antibody 2F2.D7) and a second unlabeled antibody that is subjected to testing with respect to its ability to compete with the first antibody for binding to CD79b. The second antibody may be present in the supernatant of hybridomas. As a control, immobilized CD79b incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the binding of the first antibody to CD79b, the excess is not bound antibodies are removed, and measure the amount of label associated with immobilized CD79b. If the amount of label associated with immobilized CD79b, is essentially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to CD79b. In certain embodiments, immobilized CD79b is present on the cell surface or in a membrane preparation obtained from cells expressing CD79b on the surface.

In one aspect, the purified antibodies against CD79b can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, the analysis of amino acids, gel-filtration in adenocarinoma �word, high performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain cleavage.

In one embodiment of implementation, the invention relates to altered antibody that possesses some but not all effector functions, which make it a desirable candidate for many applications where is important the half-life of the antibody isin vivobut also certain effector functions (such as complement-dependent cytotoxicity and ADCC) are unnecessary or harmful. In certain embodiments, Fc measured activity of the antibody in order to ensure that stored only the required properties. Can perform analysis of cytotoxicityin vitroand/orin vivoto confirm the reduction/elimination activity against CDC and/or ADCC. For example, it is possible to conduct analyses of the binding of Fc-receptor (FcR) to ensure that the antibody lacks FcγR binding (thus, likely devoid of activity against ADCC), but retains the ability to bind FcRn. Basic cells for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is presented in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An example of the analysis ofin vitroto assess the act�activity, of interest of the molecule in relation to the ADCC are described in U.S. patents No. 5500362 or 5821337. Suitable for these assays effector cells include peripheral mononuclear blood cells (PBMC) and natural killer (NK) cells. Alternative or additionally, the activity of interest molecules against ADCC is possible to estimatein vivofor example , in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998). It is also possible to conduct analyses of the binding of C1q to confirm that the antibody is unable to bind C1q and, thus, lacks CDC activity. To assess complement activation, it is possible to analyze the CDC, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Definition of binding FcRn and excretion/time-lifein vivoalso can be carried out using methods known in this field.

The examples below are given for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

All patent and literature references cited in this description are incorporated herein as references in full.

EXAMPLES

Commercially available reagents referred to in the examples were used according to the manufacturer's instructions, unless otherwise indicated. Antibodies the operation of the examples include commercially available antibodies. The source of those cells identified in the following examples and throughout the description under registration numbers ATCC is American Type Culture Collection, Manassas, VA.

EXAMPLE 1: Obtaining humanized antibodies against CD79b

Rooms residues correspond to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Use one-letter abbreviations of amino acids. The degeneracy of the DNA represented using the IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y=C/T).

Chimeric antibody 2F2 (denoted herein as "ch2F2) were obtained as previously described in the application U.S. No. 11/462336, filed August 3, 2006.

A. Humanized antibodies against CD79b with a transplanted sequence

Received a humanized antibody against CD79b. VL - and VH domains of antibodies 2F2 mouse (mu2F2) aligned domain with a consensus VL Kappa I (huKI) and consensual domain, the VH subgroup III (huIII). To embed transplanted HVR used frame acceptor VH region, which differs from the consensus VH domain subgroup III person at 3 positions: R71A, N73T and L78A (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992)). Hypervariable region of the antibody 2F2 mouse (mu2F2) embedded in acceptor consensus frame region h�possibility of obtaining directly 2F2-graft HVR (denoted herein as "2F2-graft sequence" or"humanized" antibody with a transplanted sequence 2F2" or "hu2F2 with a transplanted sequence"). In the VL domain the following plots were transplanted in the consensus acceptor sequence person: position 24-34 (L1), 50-56 (L2) and 89-97 (L3) (figure 7). In the VH domain was transplanted provisions 26-35 (H1), 49-65 (H2) and 93-102 (H3) (figure 8A and 8B). Also available in et al. (Also available in et al., J. Mol. Biol, 262: 732-745 (1996)) have analyzed the crystal structure of the complex of antibody and antigen, and found that the provisions of 49, 93 and 94 of the heavy chain are part of the contact area, and thus they are included in the definition HVR-H2 and HVR-H3 in the humanization of antibodies.

The option of directly transplanted sequence (2F2-graft sequence) were obtained by mutagenesis Kunkel, both as Fab, exposed on the phage, and as IgG, using a separate oligonucleotide for each hypervariable region. Correct clones was assessed by DNA sequencing.

B. Variants of humanized antibodies against CD79b with a transplanted sequence

Variants of antibodies against CD79b with a transplanted sequence that included mutational diversity in hypervariable regions "humanized" antibodies with a transplanted sequence 2F2, created using phage libraries. Variants of antibodies against CD79b with a transplanted sequence options included on many points in HVR figure 9).

C. Phage selection

For ragovoy breeding, huCD79becd(2 ág/ml) immobilizovana in PBS on the microplates for titration MaxiSorp (Nunc) overnight at 4°C. the plates were blocked for at least 1 h in blocking of funds on the basis of casein (Pierce). The phage were collected from the culture supernatant and suspended in PBS containing 0.5% BSA and 0.05% Tween 20 (PBSBT). After adding ragovoy library and ragovoy selection for 2 h, the wells for micrometrology were thoroughly washed with PBS containing 0.05% Tween 20 (PBST) for removal of non-bound phage, the bound phage were suirable by incubating the wells with 100 mm HCl for 30 min. Stiffness of selection can be increased during successive rounds of selection by increasing the number of PBST washes or by incubation with soluble huCD79becdover extended periods of time before elution.

Elyuirovaniya phage frustrated 1M Tris, pH 8, and were amplified using cells XLl-Blue and phage-assistant M13/KO7 and grown over night at 37°C in 2YT, 50 μg/ml of carbenicillin. The titers of phage, - eluted with the target of the wells were compared with the titers of phage obtained for non-target wells, to evaluate the increase of its content.

D. the Products are Fab and IgG production

For protein expression of Fab in order to measure the affinity, �the projector for phage display built in a stop codon between the heavy chain and g3. Clones of transformed cells inE. coli34B8 and were grown in complete medium C. R. A. P. at 30°C (Presta et al. Cancer Res. 57: 4593-4599 (1997)). The cells were collected by centrifugation, suspended in PBS, 100 µm PMSF, 100 µm benzamidine, 2.4 mm EDTA and destroyed with the use of microfluidizer. Fab was purified by affinity chromatography with protein G.

For the purposes of screening initially receive the IgG variants in 293 cells. Vectors encoding the VL and VH (25 µg) were transfusional in 293 cells using the FuGene system. 500 µl of FuGene was mixed c with 4.5 ml of DMEM containing no FBS and incubated at room temperature for 5 min. To the mixture was added each circuit (25 µg) and incubated at room temperature for 20 min, and then transferred into the vial for transfection overnight at 37°C in5% CO2. The following day, the medium containing the mixture for transfection, was removed and replaced with 23 ml of medium PS04 with 0.1 ml/l trace elements and 10 mg/l insulin. Cells were incubated for additional 5 days, after which the medium was collected at 1000 Rev./min. for 5 min and subjected to sterile filtration using a 0.22-µm filter with low protein binding. Samples can be stored at 4°Cafteradd 2.5 ml of 0.1% PMSF for every 125 ml of medium.

E. Determination of affinity (Biacore analysis)

To determine the affinity of the variants of "humanized" antibodies 2F2 s p�residenoe sequence, the extracellular domain of human CD79b(huCD79becd) expressively in CHO cells alone or in the form fused with Fc (huCD79becd-Fc) and purified by the conventional methods. In addition, the synthesized peptide of 16 amino acids (ARSEDRYRNPKGSACK) (SEQ ID NO:78), which contains the epitope for 2F2, in a common manner.

Karakterizacija epitope for antibody 2F2 (labeled as "test peptide" in figure 14) has been previously described in the application U.S. No. 11/462336, filed August 3, 2006. The epitope for antibody 2F2 is located in the extracellular region peptide, distal to the transmembrane domain, and he is represented in full-length and truncated forms of human CD79b (Cragg, Blood, 100(9): 3068-76 (2002)), described for normal and malignant B-cells (Hashimoto, S. et al., Mol. Immunol, 32(9): 651-9 (1995); Alfarano et al., Blood, 93(7): 2327-35 (1999)). The truncated form of CD79b devoid of the entire extracellular Ig-like domain (extracellular Ig-like domain that is not present in playerwindow truncated form of CD79b, enclosed in a frame figure 14).

Binding of Fab and IgG variants ch2F2, "humanized" antibodies with a transplanted sequence 2F2 (hu2F2 with a transplanted sequence) or option 7, "humanized" antibodies with a transplanted sequence 2F2 (hu2F2.D7) with immobilized huCD79becdor CD79b-Fc or a peptide of 16 amino acids containing the epitope for 2F2, measured by CEMS�restage plasmon resonance. The definition of affinity were performed by surface plasmon resonance using a BIAcoreTM-2000. Antigen, huCD79becdor huCD79b-Fc immobilizovana (approximately 50 - 200 RU) in 10 mm sodium acetate, pH of 4.8 on a sensor chip CM5. In the experiments in which we measured the binding with the peptide of 16 amino acids (ARSEDRYRNPKGSACK) (SEQ ID NO:78), containing the epitope (amino acids 1-11 of SEQ ID NO:78) for 2F2, biotinylating peptide caught (approximately 20 EN) coated with streptavidin sensor chip. Cleared version of "humanized" antibodies with a transplanted sequence 2F2 (as Fab or IgG) (2-fold serial dilution of 0.5 to 1000 nm in PBST) were injected at a flow rate of 30 μl/min. Each sample was analyzed at the Association for 4 minutes and dissociation for 10 minutes. After each injection, the chip was regenerated using 10 mm glycine pH of 1.7.

Binding response was corrected by subtracting the values of the control flow cell from the values of the flow cell options "humanized" antibodies with a transplanted sequence 2F2 (Fab or IgG). For analysis of the kinetics of the model used the Langmuir 1:1 for simultaneous alignment of konand koff.

F. Analysis of binding (FACS Analysis)

To further determine binding of variant 7 "humanized" an�of Itala with a transplanted sequence 2F2 (hu2F2.D7), obtained from the libraries of the SR, with BJAB cells, the binding of labeled antibodies hu2F2.D7 (IgG) BJAB cells were analyzed using FACS analysis.

For FACS analysis, monoclonal antibodies ch2F2 and 2F2.D7 was labeled with a set of Zenon® Alexa Fluor® 488 Human IgG Labeling Kit (Invitrogen, Carlsbad, California) according to the manufacturer's instructions. BJAB cells (1×106in a volume of 100 µl) were stained with 1 µg of each labeled antibodies: hIgG1 isotype, ch2F2 or 2F2.D7.

G. Determination of affinity (analysis of Scatchard)

To further determine binding of IgG variants variant 7 "humanized" antibodies with a transplanted sequence 2F2 (hu2F2.D7) having substitutions in the HVR-L3, analyzed the binding of iodinated antibodies against CD79b (with the same epitope as that of ch2F2) BJAB cells expressing CD79b of human rights and CD79b Javanese macaque, using competition with unlabeled ch2F2 and analyzed Scatchard.

For analysis of Scatchard, 0.5 nm labeled with I125antibodies against CD79b person with the same epitope as that of ch2F2, or 0.5 nm labeled with I125option 7, "humanized" antibodies with a transplanted sequence 2F2 (hu2F2.D7) were subjected to competition against unlabeled or ch2F2 hu2F2.D7, respectively, in the range from 50 to 0.02 nm (12 furlongs serial dilutions 1:2) in the presence transfetsirovannyh line BJAB stably expressing CD79b Evans�wow macaque and endogenous human CD79b. After incubation for four hours at 4°C, cells were washed and conducted the count in the cell precipitate with a gamma counter (1470 WIZARD Automatic Gamma Counter; Perkin Elmer, Walthem, MA). All points were obtained in triplicate and counted for 10 minutes. To calculate the Kd used the average CPM using the New Ligand (Genentech, South San Francisco, CA).

Results and discussion

A. the creation of humanized antibodies against CD79b

Acceptor of a frame region of a person used to produce humanized antibodies against CD79b contains consensual domain VL Kappa I person and the option of a consensual domain, the VH subgroup III person. Variant VH domain had 3 substitutions relative to the consensus sequence of a person: R71A, N73T and L78A. VL - and VH domains 2F2 mouse (mu2F2) aligned with the domains of the Kappa I and subgroup III of human rights; identify each HVR, and then transplanted into the acceptor frame region of a person to receive a transplanted HVR, which could be exposed as an Fab on phage (figures 7 and 8).

Phage, exposure transplanted sequence 2F2 in the form of Fab, not contacted with immobilized huCD79becd(data not shown). In addition, transplanted sequence 2F2 in the form of Fab is not connected huCD79becdand transplanted the sequence 2F2 in the form ab or Ig did not associate huCD79b ecd-Fc (figure 10, NB = no binding), in the determination by Biacore analysis.

1. Recovery CDR

Identified options "humanized" antibodies with a transplanted sequence 2F2 who were able to communicate with immobilized huCD79becdthe following modifications of the sequence.

In libraries, containing many changes, observed only change sequences aimed at HVR in L3, and they are presented in the figure 9 (L3 mutations: W89F and Y96F (mutation 2F2.D7) (SEQ ID NO:18).

Selected clones were transformed into the form Fab for FACS analysis and in the form of IgG for further analysis Biacore and Scatchard.

a. The definition of affinity (Biacore analysis)

As shown on figure 10, showing Biacore analysis, this approach of recovery CDR identified sequence changes in HVR-L3 (hu2F2.D7) that restores affinity "humanized" antibodies with a transplanted sequence 2F2. Analysis of surface plasmon resonance showed that the changes in L3 (hu2F2.D7) had similar affinity (figure 10) with ch2F2 when linking with immobilized huCD79becdor a peptide of from 16 amino acids (SEQ ID NO:78), containing the epitope (amino acids 1-11 of SEQ ID NO:78) for 2F2, as determined by Biacore analysis.

b. The definition of affinity (analysis of Scatchard)

As assessed by analysis of Scatchard, atopobium recovery CDR identified sequence changes, which increase the affinity of "humanized" antibodies with a transplanted sequence 2F2. Specifically, analysis of cell binding showed that the affinity and ch2F2 option 7 "humanized" antibodies with a transplanted sequence 2F2 (hu2F2.D7) (converted to form IgG) against the binding of BJAB cells stably expressing CD79b Javanese macaque and endogenous human CD79b, had Kd values, the components of 1 nm (ch2F2; Kd=0,99±0,23 nm) and 2 nm (hu2F2.D7; Kd=2,0±0,53 nm), respectively (data not shown), in determining the analysis of Scatchard.

c. Definition of binding (FACS analysis)

As assessed by FACS analysis, this approach of recovery CDR identified sequence changes, which increased the binding of "humanized" antibodies with a transplanted sequence 2F2 (hu2F2 with a transplanted sequence) with BJAB cells (data not shown). Specifically, FACS analysis of monoclonal hu2F2.D7 (IgG), identified from phage libraries, with BJAB cells showed binding options hu2F2.D7 with BJAB cells (data not shown).

B. Discussion of immunization antibodies 2F2

Since transplant 6 HVR 2F2 mouse (defined as positions 24-34 (L1), 50-56 (L2), 89-97 (L3), 26-35 (H1), 49-65 (H2) and 93-102 (H3)) in a consensus VL Kappa I and VH subgroup III (containing A71, T73 and A78), used the recovery of CR to identify changes in HVR 1-6, which increase the affinity of binding. Changes HVR sequences identified in figure 10, has led to humanized variants 2F2 with affinity similar to that of ch2F2.

EXAMPLE 2: Obtaining conjugates antibodies against CD79b-drug (ADC)

To test the efficacy of IgG variants of "humanized" antibodies with a transplanted sequence 2F2, the options are "humanized" antibodies with a transplanted sequence 2F2 conjugated to drugs, such as DM1. Variants conjugated to DM1, include variants having changes in HVR-L3.

Medicines used to obtain conjugates antibody-drug (ADC), for antibodies against CD79b included maytansinoid DM1 and may include derivatives of dolastatin, monomethylaniline E (MMAE) and monomethylaniline F (MMAF). (Cm. US2005/0276812; US 2005/0238649; Doronina et al., Bioconjug. Chem., 17:114-123 (2006)), DM1, MMAE and MMAF are inhibitors of mitosis, which are at least 100 times more cytotoxic than inhibitors of mitosis on the basis of the Vinca alkaloids used in chemotherapy treatment of NHL (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), all of which are incorporated herein as references in full). Linkers suitable for the ADC, before�provide a BMPEO, SPP or SMCC (also referred to herein as "MCC") for DM1 or MC, or MC-vc-PAB for MMAE and MMAF. In the case of DM1, the antibodies associated with tigraphy DM1 and through the ε-amino group of lysine using the linker reagent SMCC. Alternatively, in the case of DM1, the antibodies can be linked to DM1 through the ε-amino group of lysine using the linker SPP. SPP (N-Succinimidyl-4-(2'-pyridyldithio)pentanoate) reacts with the Epsilon-amino group of lysine residues, leaving a reactive 2-pyridylsulfonyl the linker to the protein. In the case of the SPP linker, when reacted with the free sulfhydryl (e.g., DM1), Peregrina group is displaced, leaving DM1 associated recoverable through a disulfide bond. DM1, linked via a linker SPP is released in reducing conditions (i.e., for example, in cells), and DM1, linked via the SMCC linker is resistant to cleavage in reducing conditions. In addition, ADC SMCC-DM1 induce toxicity in cells, if the ADC internalized and sent to the lysosome causing the release of lysine-Nε-DM1, which is an effective antimitotic agent inside the cell, and when he is released from the cell, lysine-Nε-DM1 is non-toxic (Erickson et al., Cancer Res., 66: 4426-4433 (2006)). In the case of MMAE and MMAF, the antibodies can be linked to MMAE or MMAF through the cysteine by maleimidomethyl-valine-citrulline-(c)-p-aminobenzonitrile (MC-vc-PAB). In the case of MMAF, the antibodies alternative can be linked to MMAF through the cysteine by maleimidomethyl (MC) linker. The linker MC-vc-PAB is amenable to cleavage by intracellular proteases, such as cathepsin B, and the cleavage releases the free drug (Doronina et al., Nat. Biotechnol, 21: 778-784 (2003)), while the linker MC is resistant to cleavage by intracellular proteases.

Conjugates of the antibody-drug (ADC) for antibodies against CD79b, using SMCC-DM1, received, similar to the method described in US2005/0276812. Purified antibodies against CD79b was subjected to replacement of the buffer solution containing 50 mm potassium phosphate and 2 mm EDTA, pH 7.0. SMCC (Pierce Biotechnology, Rockford, IL) was dissolved in dimethylacetamide (DMA) and added to the antibody solution with a final molar ratio of SMCC/Ab of 10:1. The reaction was allowed to proceed for three hours at room temperature with stirring. Then modified by SMCC antibody was purified on a column for desalting GE Healthcare HiTrap (G-25), equilibrated in 35 mm sodium citrate with 150 mm NaCl and 2 mm EDTA, pH 6.0. In the drug antibody and SMCC was added DM1, dissolved in DMA, up to a molar ratio of DM1 to antibody of 10:1. The reaction was allowed to proceed for 4-20 h at room temperature with stirring. The solution of the modified DM1 antibody was subjected to diaf�litraly with 20 volumes of PBS to remove unreacted DM1, was subjected to sterile filtration and stored at 4°C. typically, a process by which we reached the exit of the antibody, comprising 40-60%. The drug usually was >95% Monomeric estimated by gel filtration and scattering of laser radiation. Since DM1 had a maximum absorption at 252 nm, the amount of drug that is associated with the antibody, it was possible to determine a differential measurement of the absorption at 252 and 280 nm. Typically, the ratio of the drug and antibody was from 3 to 4.

Conjugates of the antibody-drug (ADC) for antibodies against CD79b described herein that use a linker SPP-DM1, can be obtained similarly to the method described in US 2005/0276812. Purified antibodies against CD79b was subjected to replacement of the buffer solution containing 50 mm potassium phosphate and 2 mm EDTA, pH 7.0. SPP (Immunogen) was dissolved in DMA and added to the antibody solution to obtain the final molar ratio SPP/Ab of approximately 10:1, with the exact ratio depends on the required load of the antibody drug. The ratio of 10:1, usually leads to the relation of the drug to the antibody of approximately 3-4. SPP was allowed to react for 3-4 hours at room temperature with stirring. Then the modified SPP antibody was purified on Kolo�ke for desalination GE Healthcare HiTrap (G-25), equilibrated in 35 mm sodium citrate with 150 mm NaCl and 2 mm EDTA, pH 6.0 or phosphate-buffered saline, pH 7,4. DM1 was dissolved in DMA and added to the SPP and the drug antibody to a molar ratio of DM1 to antibody of 10:1, which leads to a 3-4-KRATA molar excess relative to the available SPP linkers on the antibody. Reaction with DM1 was allowed to proceed for 4-20 h at room temperature with stirring. The solution of the modified DM1 antibody was subjected to diafiltration with 20 volumes of PBS to remove unreacted DM1, subjected to sterile filtration and stored at 4°C. typically, in this process was reached exit antibodies 40-60% or more. The drug usually was >95% Monomeric estimated by gel filtration and scattering of laser radiation. The amount of bound drug was determined by differential measurement of the absorption at 252 and 280 nm, as described for the preparation of conjugates SMCC-DM1 (described above).

Conjugates of the antibody-drug (ADC) for antibodies against CD79b described herein that use linkers to the drug MC-MMAF, MC-MMAE, MC-val-cit(vc)-PAB-MMAE or MC-val-cit(vc)-PAB-MMAF, can also be obtained analogously to the method described in US 2005/0238649. Purified antibodies against CD79b dissolved in 500 mm sodium borate and 500 mm sodium chloride at pH 8.0 and then treated with an excess of 100 mm Dimitri�Ola (DTT). After incubation at 37°C for approximately 30 minutes to replace the elution buffer through the resin Sephadex G25 and eluted in PBS with 1 mm DTPA. The value of the thiol/Ab checked by determining the concentration of the recovered antibodies from absorption of the solution at 280 nm and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the absorption at 412 nm. The recovered antibody was dissolved in PBS and cooled on ice. The linker medicines, for example, MC-val-cit (vc)-PAB-MMAE, in DMSO, dissolved in acetonitrile and water, and add to the cooled recovered antibody in PBS. After incubation for one hour, add an excess of maleimide to quench the reaction and closing any unreacted tylnej groups of the antibody. The reaction mixture is concentrated by ultrafiltration by centrifugation and the conjugate antibody-drug purified and subjected to desalting by elution through G25 resin in PBS, filtered through a 0.2-μm filters under sterile conditions, and frozen for storage.

Conjugates of the antibody-drug (using antibodies against CD79b described herein) was diluted to 2×10 μg/ml in the medium for analysis. Conjugates were associated with crosslinking the linker SMCC (for SPP for toxin maytansinoid DM1 you can use an alternative disulfide linker) (Cm. US 2005/0276812 and US 2005/0238649) in addition, conjugates can be linked to MC-valine-citrulline(vc)-PAB or MC for derivative dolastatin, toxin monomethylaniline E (MMAE) or toxin monomethylaniline F (MMAF) (see application U.S. No. 11/141344, filed may 31, 2005, and application U.S. No. 10/983340, filed November 5, 2004). Negative controls consisted of conjugates on the basis of HERCEPTIN® (trastuzumab) (antibody against HER2) (SMCC-DM1 or SPP-DM1 or MC-vc-MMAE or MC-vc-MMAF). Positive controls can include free form L-DM1, is equivalent to a load dose of the conjugate. The samples were shaken to ensure a homogeneous mixture before dilution.

Antibodies against CD79b for conjugation with drugs include chimeric antibodies 2F2 (described in example 1A) and antibodies, further described herein (see example 1), including hu2F2.D7.

EXAMPLE 3: Analysis of destruction of tumor cellsin vivo

A. Xenograft

To test the efficacy of IgG variants of "humanized" antibodies with a transplanted sequence 2F2, having changes in HVR-L3 (hu2F2.D7), option hu2F2.D7 conjugated to DM1 and analyzed the effect of the conjugated variant on tumors in mice.

Specifically, we examined the ability of antibodies to cause regression of tumors in multiple xenograft models, including cells RAMOS, BJAB cells (cell line of Burkitt lymphoma, which contain�it translocation t(2; 8)(p112;q24) (IGK-MYC), mutant p53 gene and is negative for the Epstein-Barr (EBV)) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), the cells Granta 519 (cell line lymphoma cells of the mantle zone that contains the translocation t(11;14)(q13;q32) (BCL1-IGH), which leads to sverkhekspressiya detected D1 (BCL1), contains deletions P16INK4B and P16INK4A and is positive for EBV) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), the cells U698M (B-lymphoblastic cell line transplantation; (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001) and DoHH2 cells (cell line follicular lymphoma that contains the translocation characteristic of follicular lymphoma t(14;18)(q32;q21), which leads to sverkhekspressiya Bcl-2 directed Ig heavy chain, contains the P16INK4A deletion, contains the translocation t(8;14)(q24;q32) (IGH-MYC) and an EBV negative) (Drexler, H. G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)).

To analyze the performance of options "humanized" antibodies with a transplanted sequence 2F2, female mice CB17 ICR SCID (aged 6-8 weeks from Charles Rivers Laboratories; Hollister, CA) were inoculable subcutaneously 2×107cells of BJAB-luciferase cells or Granta-519 via injection into the flanks of mice CB17 ICR SCID and xenotransplantation tumors were allowed to grow to a size of about 200 mm2. Day 0 refers to the day when the tumor size was autho�him 200 mm 2and when he introduced the first/or only dose administered funds if no other specific instructions below. Tumor size was calculated based on two dimensions, measured using calipers, and was expressed in mm3according to the formula: V=0.5 a×b2where a and b represent the long and short diameters of the tumor, respectively. Data obtained for each experimental group were expressed as mean values+SE. In groups of 10 mice underwent the introduction of a single intravenous (i.v.) dose, component between 50 and 210 μg associated with the antibody medicines/m2mouse (corresponding to ~1-4 mg/kg mouse), with options of "humanized" antibodies with a transplanted sequence 2F2 or control conjugates antibody-drug. Tumors were measured either one or two times a week during the experiment. Body weight of mice was measured either one or two times a week during the experiment. Mice were killed before reaching a tumor volume of 3000 mm3or when the tumors observed signs of impending ulceration. All protocols with animals were approved by the Institutional Animal Care and Use Committee (IACUC).

The linkers between the antibody and the toxin that was used was a simple thioether crosslinking linker SMCC to DM1. Additional linkers m�may include a disulfide linker SPP or simple thioether crosslinking linker SMCC to DM1 or MC or MC-valine-citrulline(vc)-PAB (or a dipeptide linker reagent valine-citrulline(vc))), having maleimide component and pair-aminobenzeneboronic (PAB) smoothshapes component for monomethylaniline E (MMAE) or monomethylaniline F (MMAF). Used toxins consisted of DM1. Additional toxins may include MMAE or MMAF.

Antibodies against CD79b for this experiment consisted of a chimeric antibody 2F2 (ch2F2), as described in the application U.S. No. 11/462336, filed August 3, 2006 (see example 1A), as well as options of the "humanized" antibodies with a transplanted sequence 2F2 described herein (see example 1). Additional antibodies may include antibodies 2F2, obtained from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006.

Negative controls consisted of conjugates on the basis of HERCEPTIN® (trastuzumab) (antibody against HER2) (SMCC-DM1).

B. Results

1. Xenograft BJAB-luciferase

Within 36 hours option 7 "humanized" antibodies with a transplanted sequence 2F2 (option hu2F2.D7) (converted in the form of IgG and chimeric antibodies against CD79b (ch2F2), anywhereman with DM1 (hu2F2.D7-SMCC-DM1 and ch2F2-SMCC-DM1, respectively), showed inhibition of tumor growth in SCID mice with tumors of BJAB-luciferase compared to negative control, HERCEPTIN® (trastuzumab)-SMCC-DM1 (antibody against HER2-SMCC-DM1). ADC was administered as a single dose (as indicated in table 7) n� 0 day for all ADC and controls. Specifically, antibodies hu2F2.D7-SMCC-DM1 (converted to form IgG) and ch2F2-SMCC-DM1 significantly inhibited tumor growth (figure 19). In addition, table 7 shows the number of mice out of total tested number showing PR = partial regression (where the tumor volume at any time after administration dropped below 50% of the tumor volume measured at 0 day) or CR = complete remission (where the tumor volume at any time after administration dropped to 0 mm3).

Table 7:
Xenograft BJAB-Luc (20 million cells/mouse) in mice SCID
The injected antibody (treatment)PRCRDose Ab
(mg/kg)
The dose of the drug-DM1
(µg/m2)
The ratio of the medicinal product (Drug/Ab)
The control antibody against HER2-SMCC-DM10/100/1021003,3
ch2F2-SMCC-DM10/101/102,3100 3
ch2F2-SMCC-DM10/100/101,2503
hu2F2.D7-SMCC-DM11/101/102,91002,3
hu2F2.D7-SMCC-DM11/100/101,5502,3

2. Xenograft Granta-519 (b-cell lymphoma mantle zone of man)

Within 14 days, option 7 "humanized" antibodies with a transplanted sequence 2F2 (option hu2F2.D7) (converted to form IgG) (hu2F2.D7-SMCC-DM1) showed inhibition of tumor growth in SCID mice with tumors Granta-519 compared to negative control, HERCEPTIN® (trastuzumab)-SMCC-DM1 (antibody against HER2-SMCC-DM1). ADC was administered in a single dose (as indicated in table 8) on 0 day for all ADC and controls. Specifically, antibodies hu2F2.D7-SMCC-DM1 (converted to form IgG) significantly inhibited tumor growth (figure 20A).

In addition, the introduction hu2F2.D7-SMCC-DM1 and control HERCEPTIN® (trastuzumab)-SMCC-DM1 (antibody against HER2-SMCC-DM1) did not lead to lower interest mass of bodies� mice (figure 20B). Moreover, table 8 shows the number of mice out of the total number of mice tested showing PR = partial regression (where the tumor volume at any time after administration dropped below 50% of the tumor volume measured at 0 day) or CR = complete remission (where the tumor volume at any time after administration dropped to 0 mm3).

Table 8:
Xenograft, Granta-519 (20 million cells/mouse) in mice SCID
The injected antibody (treatment)PRCRDose Ab
(mg/kg)
The dose of the drug-DM1
(µg/m2)
The ratio of the medicinal product (Drug/Ab)
The control antibody against HER2-SMCC-DM10/100/1042063,4
hu2F2.D7-SMCC-DM10/100/1041662,8

Because of the capacity of ADC "humanized" antibodies is consistent with a transplanted�STU 2F2 significantly inhibit tumor progression in xenograft, the CD79b molecules may be excellent targets for therapy of tumors in mammals, including associated with B-cell malignancies such as lymphomas (i.e. nehodgkinski lymphoma), leukemias (i.e. chronic lymphocytic leukemia) and other malignant tumors of hematopoietic cells. In addition, ADC "humanized" with a transplanted sequence 2F2 suitable for reducing the growth of tumorsin vivoin tumors, including associated with B-cell malignancies such as lymphomas (i.e. nehodgkinski lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and other malignant tumors of hematopoietic cells.

EXAMPLE 4: Colocalization antibodies against CD79b

For the determination of cases when "humanized" antibodies 2F2-graft and variants of the antibodies are delivered through internalization into the cell, can be used to examine colocalization antibodies against CD79b, internalized in B-cell lines, the cell lines Ramos. LAMP-1 is a marker of 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 the compartment MHC class II (MIIC), which is a late endosome/lysosome-like compartment. HLA-DM is a marker for MIIC.

The Ramos cells were incubated for 3 hours at 3°C with 1 µg/ml of "humanized" antibodies with a transplanted sequence 2F2 and variants of antibodies, with FcR blocking agent (Miltenyi) and 25 μg/ml Alexa647-transferrin (Molecular Probes) in complete do not contain carbonates medium (Gibco) in the presence of 10 μg/ml leupeptin (Roche) and 5 μm of pepstatin (Roche) to inhibit degradation lysosomes. Then the cells were washed twice, fixed with 3% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature, quenched with 50 mm NH4C1 (Sigma), provide their permeability with 0.4% saponin/2% FBS/1% BSA for 20 minutes and then incubated with 1 μg/ml antibodies against antibodies mouse Cy3 (Jackson Immunoresearch) for 20 minutes. Then the reaction block for 20 minutes through mouse IgG (Molecular Probes), followed by incubation for 30 minutes with signal amplifier Image-iT FX Signal Enhancer (Molecular Probes). In the end the cells were incubated with labeled with Zenon Alexa488 antibody against mouse LAMP1 (BD Pharmingen), a marker for lysosomes and MIIC (a lysosome-like compartment that is part of the cascade MHC class II), for 20 minutes, and then fixed with 3% PFA. Cells was resuspended in 20 µl of buffer with saponin and attached to the subject of glass, coated with poly-lysine (Sigma) before placing the coverslip containing DAPI VectaShield (Vector Laboratories). For immunofluorescence MIIC or lysosomes, the cells are fixed, their permeability and increase, as described above, and then stained jointly labeled with Zenon Alexa555-HLA-DM (BD Pharmingen and Alexa488-Lamp1 in the presence of excess mouse IgG according to the manufacturer's instructions (Molecular Probes).

Thus, colocalization "humanized" antibodies with a transplanted sequence 2F2 or variants of antibodies with MIIC or lysosomes B-cell lines as assessed by immunofluorescence, may indicate that the molecules are excellent agents for the treatment of tumors in mammals, including associated with B-cell malignancies such as lymphomas (i.e. non-Hodgkin's lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and other malignant tumors of hematopoietic cells.

EXAMPLE 5: antibodies against CD79b with the built-in cysteine residues

Antibodies against CD79b with the built-in cysteine residues was performed as described herein.

DNA encoding the antibody ch2F2 (light chain, SEQ ID NO:4, figure 4; and a heavy chain, SEQ ID NO:5, figure 5), can be subjected to mutagenesis by methods of modification of the light chain and heavy chain, as described in this document.

DNA encoding the antibody hu2F2.D7 (heavy chain (SEQ ID NO:90) and light chain (SEQ ID NO:89), figure 13) was subjected to mutagenesis by methods of modification of the heavy chain described herein. DNA encoding the antibody hu2F2.D7 (heavy chain (SEQ ID NO:90), figure 13), also can be subjected to mutagenesis by methods of modification of the Fc region of the heavy chain described herein.

With more inform� antibodies against CD79b with the built-in cysteine residues, DNA encoding light chain, was subjected to mutagenesis to replace cysteine valine at position 205 in Kabat light chain (position in the sequence 210, as shown in figure 18 (light chain SEQ ID NO:88 hu2F2.D7 thio-MAb). DNA encoding a heavy chain, was subjected to mutagenesis to cysteine replacement of alanine at position 118 in the EU heavy chain (position in sequence 118; Kabat number 114), as shown in figure 17 (heavy chain SEQ ID NO:85 hu2F2.D7 thio-MAb). Fc-region of antibodies against CD79b can be subjected to mutagenesis to replace cysteine, serine at position 400 in EU in the Fc-region of the heavy chain (position in the sequence 400; Kabat number 396), as shown in table 2-3.

A. antibodies against CD79b with the built-in cysteine residues for conjugation by restoring and re-oxidation

Full-size monoclonal antibodies against CD79b with the built-in cysteine residues (thio-Mab) was expressively in CHO cells and purified by affinity chromatography with protein A, followed by size exclusion chromatography. Purified antibody was diluted in 500 mm sodium borate and 500 mm sodium chloride at about pH 8.0 and restore approximately 50-100-fold molar excess of 1 mm TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA) for approximately 1-2 hours at 37°C. Restore�established thio-Mab was diluted and placed in a column HiTrap S in 10 mm sodium acetate, pH 5, and eluted by PBS containing 0.3 M sodium chloride. - Eluted restored thio-Mab treated with 2 mm dehydroascorbic acid (dhAA) at pH 7 for 3 hours, or 2 mm aqueous copper sulfate (CuSO4) at room temperature over night. It can also be effective oxidation by ambient air. The buffer is replaced by elution through the resin Sephadex G25 and eluted by PBS with 1 mm DTPA. The value of the thiol/Ab estimated by determining the concentration of the recovered antibodies from the absorption values at 280 nm in solution and the concentration of thiol in the reaction with DTNB (Aldrich, Milwaukee, WI) and determining the absorption at 412 nm.

EXAMPLE 6: Obtaining conjugates antibodies against CD79b with built in his cysteine residues and drugs by conjugation of antibodies against CD79b with the built-in cysteine residues and intermediates drug-linker

After the processes of recovery and re-oxidation of example 5, the CD79b antibody against with built in his cysteine residues was diluted in PBS (phosphate-buffered saline) and cooled on ice. About 1.5 molar equivalents of the intermediate drug auristatin-linker, such as MC-MMAE (maleimidomethyl-monomethylaniline E), MC-MMAF, MC-val-cit-PAB-MMAE or MC-val-cit-PAB-MMAF, with a reactive to thiol functional group�Noah group like maleimido, relatively built-cysteine residue on the antibody was dissolved in DMSO, is diluted in acetonitrile and water, and add to the cooled restored, re-oxidized antibody in PBS. After approximately one hour add the excess maleimide to quench the reaction and the closure of any unreacted tylnej groups of the antibody. The reaction mixture is concentrated by ultrafiltration by centrifugation and conjugate antibodies against CD79b with built in his cysteine residues and medicines cleanse and absoluut by elution through G25 resin in PBS, filtered through a 0.2-μm filters under sterile conditions, and frozen for storage.

Getting hu2F2.D7-HC(A118C) thio-MAb-BMPEO-DM1 can be performed as follows. The free cysteine on thio-MAb hu2F2.D7-HC(A118C) alter bis-multimediacenter BM(PEO)3 (Pierce Chemical), leaving an unreacted maleimido on the surface of the antibody. This is accomplished by dissolving BM(PEO)3 in a mixture of 50% ethanol/water to a concentration of 10 mm and adding a tenfold molar excess of BM(PEO)3 to a solution containing thio-MAb hu2F2.D7-HC(A118C) in phosphate-buffered saline at a concentration of approximately 1.6 mg/ml (10 micromol) and allowing it to react for 1 hour. Excess BM(PEO)3 is removed by gel filtration (HiTrap column, Pharmacia) in 30 mm citrate buffer, pH 6 with 150 mm NaCl. To intermediate with�the Association of the thio-MAb-BMPEO hu2F2.D7-HC(A18C) add approximately 10-fold molar excess of DM1, dissolved in dimethylacetamide (DMA). Dimethylformamide (DMF) can also be used for dissolution of the reagent of the group of medicines. The reaction mixture was allowed to react overnight before gel filtration or by dialysis in PBS to remove unreacted drug. Gel filtration on S200 columns in PBS is used to remove high molecular weight aggregates and providing purified thio-MAb-BMPEO-DM1 hu2F2.D7-HC(A118C).

Using the same Protocol, you can get a thio control hu-anti-HER2-HC(A118C)-BMPEO-DM1, thio control hu-anti-HER2-HC(A118C)-MC-MMAF, thio control hu-anti-HER2-HC(A118C)-MCvcPAB-MMAE and thio-control antibody against CD22-HC(A118C)-MC-MMAF.

C using the above method, you can get and test conjugates antibodies against CD79b with built in his cysteine residues and medicines (TDC), for example, but not limited to, the following:

1. thio-hu2F2.D7-HC(A118C)-MC-MMAF by conjugation of A118C thio-hu2F2.D7-HC(A118C) and MC-MMAF;

2. thio-hu2F2.D7-HC(A118C)-BMPEO-DM1 by conjugation of A118C thio-hu2F2.D7-HC(A118C) and BMPEO-DM1;

3. thio-hu2F2.D7-HC(A118C)-MCvcPAB-MMAE by conjugation of A118C thio-hu2F2.D7-HC(A118C) and MC-val-cit-PAB-MMAE;

4. thio-ch2F2-HC(A118C)-MC-MMAF by conjugation of thio-ch2F2-HC(A118C) and MC-MMAF; and

5. thio-ch2F2-LC(V205C)-MC-MMAF by conjugation of thio-ch2F2-LC(V205C) and MC-MMAF.

EXAMPLE 7: Karakterizacija affinity binding conjugates thio-MAb with built in his OST�worry-beads cysteine and drugs to cell surface antigen

The binding affinity of conjugates thio-hu2F2.D7-drug and conjugates thio-ch2F2-a drug with CD79b expressed on the cells of BJAB-luciferase determined by FACS analysis.

Briefly, approximately 1×106cells in 100 μl in contact with various amounts of 1.0 μg, 0.1 μg or 0.01 μg Ab per million cells of BJAB-luciferase) one of, but not limited to, the following conjugates thio-MAb against CD79b and drug or naked antibodies (unconjugated Ab as control): (1) thio-ch2F2-LC(V205C)-MC-MMAF or (2) thio-ch2F2-HC(A118C)-MC-MMAF; (3) thio-hu2F2.D7-HC(A118C)-MCvcPAB-MMAE, (4) thio-hu2F2.D7-HC(A118C)-BMPEO-DM1, or (5) thio-hu2F2.D7-HC(A118C)-MC-MMAF. As a secondary antibody used for detection anywhereman with PE antibody against mouse Ig (BD catalog# 555787).

Deccio antibodies against CD79b associated with the cell surface, is carried out using conjugated with PE mouse antibody against human Ig.

EXAMPLE 8: Analysis of reducing proliferation of cells ofin vitroby conjugates thio-Mab against CD79b and drug

The efficacy ofin vitroconjugates thio-MAb against CD79b-a drug (including, but not limited to thio-hu2F2.D7-HC(A118C)-MCMMAF, thio-hu2F2.D7-HC(A118C)-MCvcPAB-MMAE and thio-hu2F2.D7-HC(A118C)-BMPEO-DM1), is measured through the analysis of cell proliferation (e.g., in cells of BJAB-luciferase Granta-519, WSU-DLCL2). Fluorescent analysis of cell viability CellTiter-Glo® is a commercially available (Promega Corp., Madison, WI), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (US 5583024; US 5674713; US 5700670). In this analysis of cellular proliferation determine the number of viable cells in culture based on quantification the presence of ATP, the indicator of metabolically active cells (Crouch et al., J Immunol Metho., 160: 81-88 (1993); US 6602677). Analysis of CellTiter-Glo® is carried out in 96-well format, making it suitable for automated high-performance screening (HTS) (Cree et al., Anticancer Drugs 6:398-404 (1995)). Method homogeneous analysis involves adding the single reagent (reagent CellTiter-Glo®) directly to cells cultured in serum supplemented medium.

Uniform format "add-mix-measure" ensures lysis of cells and the formation of the luminescent signal proportional to the presence of ATP. The substrate, luciferin, beetles, oxidative decarboxylated recombinant Firefly luciferase with simultaneous conversion of ATP to AMP and the formation of photons. Viable cells expressed as relative luminescence units (RLU). The data can be detected using a luminometer or imaging device with CCD camera. The output luminescence expressed�t as relative light units (RLU). %RLU represents the normalized percentage of RLU compared to the control in the form of "not conjugate with medicine." Alternative photons from the luminescence can be counted in a scintillation counter in the presence of the scintillator. Light units can be expressed as CPS (number of pulses per second).

The efficiency of the conjugates thio-MAb-drug measured through analysis of cell proliferation using the following Protocol, adapted from fluorescent analysis of cell viability CellTiter Glo, Promega Corp. Technical bulletin TB288; Mendoza et al., Cancer Res., 62: 5485-5488 (2002)):

1. Aliquot 40 μl of cell culture containing approximately 3000 cells BJAB, Granta-519 or WSU-DLCL2 in the environment is applied into each well of 384-well opaque tablet.

2. In experimental wells in quadruplicate add TDC (conjugate thio-Mab-drug) (10 μl) to a final concentration of 10000, 3333, 1111, 370, 123, 41, 13,7, 4,6 or 1.5 ng/ml, with the control wells with no drug conjugate", which added to the medium separately, and incubated for 3 days.

3. The precipitate counterbalance to room temperature for about 30 minutes.

4. Add reagent CellTiter-Glo (50 µl).

5. The contents mixed for 2 minutes on an orbital device for shaking to induce lysis �notches.

6. Tablet incubated at room temperature for 10 minutes to stabilize luminescent signal.

7. The luminescence register and submit it to the charts as %RLU (relative luminescence units). Data from cells incubated with the medium containing no drug conjugate, is applied to a graph at a concentration of 0.51 ng/ml.

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

EXAMPLE 9: Analysis of inhibition of tumor growthin vivoby conjugates thio-Mab against CD79b and drug

In a similar study using the Protocol of the study compared to xenograft described in example 3 (see above), varying the drug conjugates and doses, are exploring the efficacy of conjugates thio-MAb and the drug in respect of a reduction of B-cell tumors in xenograft models, for example, the xenograft Granta-519 (b-cell lymphoma mantle zone), the xenograft DOHH2 (follicular lymphoma) xenograft WSU-DLCL2 (diffuse large cell lymphoma) or xenograft BJAB (Burkitt's lymphoma).

It is believed that the above written description enables the person skilled in the art to apply the invention in practice. The present invention is not limited to the� by volume of the deposited structure, since the deposited embodiment of the it is assumed as a single illustration of certain aspects of the invention, and any constructs that are functionally equivalent, apply to the scope of this invention. The deposited material described herein should not be construed as an admission that the written description contained herein is not sufficient to be allowed to practice any aspect of the invention, including the best method of its implementation, and it should not be interpreted as limiting the scope of the claims to the specific illustrations that it presents. Indeed, various modifications of the invention in addition to the modifications shown and described herein will become apparent to experts in the art from the above descriptions, and apply to the scope of the attached claims.

1. A humanized antibody against CD79b or antigen-binding fragment, comprising the following hypervariable region (HVR):
(i) HVR-L1 containing the sequence A1-A16, where A1-A16 is a KSSQSLLDSDGKTYLN (SEQ ID NO:23);
(ii) HVR-L2 containing a sequence B1-B7, where B1-B7 is a LVSKLDS (SEQ ID NO:24);
(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 before�provide a FQGTHFPFT (SEQ ID NO:25);
(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 is a GYTFTSYWMN (SEQ ID NO:31);
(v) HVR-H2 containing the sequence E1-E18, where E1-E18 is a GMIDPSDSETHYNHIFKD (SEQ ID NO:32); and
(vi) HVR-H3 containing the sequence F1-F6, where F1-F6 is a ARNLYL (SEQ ID NO:33).

2. The antibody or antigen-binding fragment of claim 1, wherein at least part of the sequence of frame area represents the consensus sequence of frame area of the person.

3. The antibody or antigen-binding fragment of claim 1 that binds to the same epitope as that of monoclonal antibody containing the variable domains of SEQ ID NO:10 and SEQ ID NO:14,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

4. The antibody or antigen-binding fragment of claim 1 that binds to the same epitope as the
(i) the Fab fragment of a monoclonal antibody produced by hybridoma with an access number ATCC PTA-7712; and/or
(ii) a chimeric antibody containing the variable domains of the antibody produced by hybridoma with an access number ATCC PTA-7712,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

5. The antibody or antigen-binding fragment according to claim 1, wherein the monovalent affinity of the antibody against human CD79b is
(a) by a mere�TSS is the same as the monovalent affinity of the mouse antibody containing the sequence of the variable light chain sequence and the heavy chain as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14);
(b) at least 1, 2 or 3 times greater than the monovalent affinity of the mouse antibody or chimeric antibody containing the variable light chain sequence and the heavy chain as indicated in Fig. 7 (SEQ ID NO:10) and Fig. 8A-B (SEQ ID NO:14); or
(c) at least 1, 2 or 3 times less than the monovalent affinity of the mouse antibody or chimeric antibody containing the variable light chain sequence and the heavy chain as indicated in Fig. 7 (SEQ ID NO:10) and Fig. 8A-B (SEQ ID NO:14);
where antibodies against CD79b binds to the same epitope as that of monoclonal antibody containing the variable domains of SEQ ID NO:10 and SEQ ID NO:14,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

6. The antibody or antigen-binding fragment of claim 1, wherein the affinity of the antibody in its bivalent form to human CD79b is
(a) essentially the same as the affinity of the mouse antibody in its bivalent form and comprising a sequence of variable region light chain and heavy chain, as shown in figure 7 (SEQ ID NO:10) and figures 8A-B (SEQ ID NO:14);
(b) at least 1, 2 or 3 times bol�more than the affinity of the mouse antibody or chimeric antibody in its bivalent form and containing the variable light chain sequence and the heavy chain as indicated in Fig. 7 (SEQ ID NO:10) and Fig. 8A-B (SEQ ID NO:14);
(c) 2.0 nm +/- 0,53; or
(d) more than 1.5 nm;
where antibodies against CD79b binds to the same epitope as that of monoclonal antibody containing the variable domains of SEQ ID NO:10 and SEQ ID NO:14,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

7. The antibody or antigen-binding fragment according to any one of claims.1, 5 and 6, where a humanized antibody, if it anywhereman with a cytotoxic agent inhibits the growth of tumor cells.

8. The antibody or antigen-binding fragment according to any one of claims.1, 5 and 6, containing a single Fab region linked to an Fc region.

9. The antibody or antigen-binding fragment according to claim 5 or 6, where the mouse antibody produced by hybrid cell line deposited with the ATCC under number PTA-7712.

10. The antibody or antigen-binding fragment according to claim 5 or 6, where the binding affinity is expressed as Kd value.

11. The antibody or antigen-binding fragment according to claim 5 or 6, where the binding affinity measured Biacore, ELISA or radioimmunoassay analysis.

12. The antibody or antigen-binding fragment according to claim 1 comprising sequentially�th consensus frame region κ subgroup 1 person.

13. The antibody or antigen-binding fragment according to claim 1, containing the consensus sequence of frame area of the subgroup III heavy chain.

14. The antibody or antigen-binding fragment according to claim 13, wherein the frame sequence field contains a substitution at position 71, 73 and/or 78.

15. The antibody or antigen-binding fragment according to claim 14, where the specified substitution is an R71A, N73T and/or L78A.

16. A humanized antibody against CD79b or antigen-binding fragment, which contains
(a) the variable domain of the heavy chain containing the sequence of the HVR1-HC, HVR2-HC and HVR3-HC presented on figure 13 (SEQ ID NO:31-33);
(b) variable domain light chain containing the sequence of the HVR1-LC, HVR2-LC and HVR3-LC presented on figure 13 (SEQ ID NO:23-25).

17. The antibody or antigen-binding fragment according to claim 16, where the variable region contains a sequence FR1-HC, FR2-HC, FR3-HC and/or FR4-HC shown in Fig. 13 (SEQ ID NO:27-30).

18. The antibody or antigen-binding fragment according to claim 16 and 17, where the antibody contains a sequence of CH1 and/or Fc, is presented in (SEQ ID NO:34 and/or 35).

19. The antibody or antigen-binding fragment according to claim 16, where the variable domain contains the sequence of FR1-LC, FR2-LC, FR3-LC and/or FR4-LC presented on figure 13 (SEQ ID NO:19-22).

20. The antibody or antigen-binding fragment that �vyzyvaetsya with CD79b, where the antibody contains:
(a) the variable domain of the heavy chain that is at least 90% identical to the amino acid sequence SEQ ID NO:16, and the variable domain light chain that is at least 90% identical to the amino acid sequence SEQ ID NO:12; or
(b) the variable domain of the heavy chain that is at least 90% identical to the amino acid sequence SEQ ID NO:14, and the variable domain light chain that is at least 90% identical to the amino acid sequence SEQ ID NO:10,
where the antibody binds to the same epitope as that of monoclonal antibody containing the variable domains of SEQ ID NO:10 and SEQ ID NO:14, and/or a Fab fragment of a monoclonal antibody produced by hybridoma with an access number ATCC PTA-7712, and/or a chimeric antibody containing the variable domains of the antibody produced by the hybridoma with an access number ATCC PTA-7712,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

21. A method of producing antibodies against CD79b, where the method comprises (a) cultivating a host cell selected from the group consisting of eukaryotic cell, and the cell SNO, in conditions suitable for expression of the polynucleotide encoding the antibody according to any one of claims.1-2, 16-20, and (b) isolation of antibody.

22. Antibodies against CD79b or antigen-binding fragment, soda�containing the variable region of the heavy chain with the sequence, shown in Fig.13 (SEQ ID NO:16), and variable region light chain with a sequence shown in Fig.13 (SEQ ID NO:12).

23. The antibody or antigen-binding fragment according to claim 22, where the antibody is monovalent and contains an Fc region.

24. The antibody or antigen-binding fragment according to claim 1 or 16, where the antibody contains
(a) variable region light chain that is at least 90% identical to the amino acid sequence SEQ ID NO:12; and/or
(b) the variable region of the heavy chain that is at least 90% identical to the amino acid sequence SEQ ID NO:16,
where the antibody binds to the same epitope as that of monoclonal antibody containing the variable region SEQ ID NO:10 and SEQ ID NO:14, and/or a Fab fragment of a monoclonal antibody produced by the hybridoma with the number of ATSS MOUTH-7712, and/or a chimeric antibody containing the variable region of the antibody produced by the hybridoma with the number of ATSS MOUTH-7712,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

25. The antibody or antigen-binding fragment according to claim 1 or 16, where the antibody contains
(a) variable region heavy chain containing one, two, three or four amino acid sequence of frame regions selected from SEQ ID NO:69, 70, 71 and 72; and/or
(b) variabeln� region light chain, containing one, two, three, or four amino acid sequence of frame regions selected from SEQ ID NO:65, 66, 67 and 68.

26. The antibody or antigen-binding fragment according to claim 1 or 16, where the antibody contains
(a) variable region heavy chain containing one, two, three or four amino acid sequence of frame regions selected from SEQ ID NO:69, 70, 71 and 72; and/or
(b) variable region light chain containing one, two, three or four amino acid sequence of frame regions that are at least 90% identical to the amino acid sequence selected from SEQ ID NO:65, 66, 67 and 68,
where the antibody binds to the same epitope as that of monoclonal antibody containing the variable region SEQ ID NO: 10 and SEQ ID NO:14, and/or a Fab fragment of a monoclonal antibody produced by the hybridoma with the number of ATSS MOUTH-7712, and/or a chimeric antibody containing the variable region of the antibody produced by the hybridoma with the number of ATSS MOUTH-7712,
where the epitope corresponds to amino acids 1-11 of the sequence SEQ ID NO:78, as determined by Biacore analysis.

27. Immunoconjugate for inhibiting b-cell proliferation containing the antibody according to any one of claims.1, 16, 17, 19 and 20, covalently attached to a cytotoxic agent or to the agent for inhibition of cell growth.

28. Immunoconjugate� according to claim 27, where the cytotoxic agent is selected from a toxin, chemotherapeutic agents, drug molecule, an antibiotic, a radioactive isotope and nucleotidase enzyme.

29. Immunoconjugate according to claim 28, where immunoconjugate has the formula I

where:
(a) Ab is an antibody according to any one of claims.1-2, 20 or 23;
(b) L is a linker;
(c) D is a drug molecule,
where p is equal to from about 1 to about 8.

30. Immunoconjugate according to claim 29, wherein 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-iodo-acetyl)aminobenzoate (SIAB).

31. Immunoconjugate according to claim 29, where D is selected from the group consisting of maytansinoid, auristatin and dolastatin.

32. Immunoconjugate according to claim 31, where D represents auristatin or dolastatin, and where D is a drug molecule of the formula DE or DF:


and where each R2and R6is bromide, each R3and R4represents isopropyl, R5represents-H, R7represents sec-butyl, each R8netavis�mo is selected from CH 3O-CH3OH 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-C(R8)2-C(R8)2-aryl; and
(d) the value of p is about 1 to 6,
where lekarstvennye molecule selected from MMAE and MMAF.

33. Immunoconjugate according to claim 29, which has the formula Ab-(L-ME)p, where L is a linker, and a value of p is from 2 to 5.

34. Immunoconjugate according to claim 29, which has the formula Ab-(L-MMAF)p, where L is a linker, and a value of p is from 2 to 5.

35. Immunoconjugate according to claim 33 or 34, wherein L contains a val-cit, MS, RABBI or MS-RAV.

36. Immunoconjugate according to claim 29, where D represents maytansinoid.

37. Immunoconjugate according to claim 36, where D is selected from DM1, DM3 and DM4.

38. Immunoconjugate according to claim 36, where the value p is 2-4 or 3-4.

39. Immunoconjugate according to claim 29, where immunoconjugate selected from the structures:





where Val is a valine, a Cit represents a citrulline.

40. Pharmaceutical composition for inhibiting b-cell proliferation, contains effective to�icesto immunoconjugate according to claim 29 and a pharmaceutically acceptable carrier.

41. The antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for use for inhibiting growth of cells that Express CD79b.

42. The antibody or immunoconjugate according to claim 41, where the specified antibody anywhereman with a cytotoxic agent.

43. The antibody or immunoconjugate according to claim 41, where the specified antibody anywhereman with a growth inhibitory agent.

44. The use of an antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 to obtain a medicine for inhibiting growth of cells that Express CD79b.

45. The use according to claim 44, where the specified antibody anywhereman with a cytotoxic agent.

46. The use according to claim 44, where the specified antibody anywhereman with a growth inhibitory agent.

47. The antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for use in the effective amount for treatment of an individual suffering from cancer.

48. The antibody or immunoconjugate according to claim 47, where the malignant tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

49. And�titulo or immunoconjugate according to claim 47, where the specified antibody anywhereman with a cytotoxic agent.

50. The antibody or immunoconjugate according to claim 47, where the specified antibody anywhereman with a growth inhibitory agent.

51. The use of an antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for obtaining a medicinal product for the treatment of an individual suffering from cancer.

52. The use according to claim 51, where the malignant tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

53. The use according to claim 51, where the specified antibody anywhereman with a cytotoxic agent.

54. The use according to claim 51, where the specified antibody anywhereman with a growth inhibitory agent.

55. The antibody according to any one of claims.1, 16, 17, 19 and 20 for use in an effective amount for the treatment of proliferative disease in the individual.

56. The antibody according to claim 55, where the specified proliferative disease is a malignant tumor.

57. The antibody according to claim 55, where the specified malignant tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsing�th aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

58. The antibody according to claim 55, where the specified antibody anywhereman with a cytotoxic agent.

59. The antibody according to claim 55, where the specified antibody anywhereman with a growth inhibitory agent.

60. The use of an antibody according to any one of claims.1, 16, 17, 19 and 20 to obtain drugs for the treatment of proliferative disease in the individual.

61. The use according to claim 60, where the specified proliferative disease is a malignant tumor.

62. The use according to claim 60, where the specified malignant tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

63. The use according to claim 60, where the specified antibody anywhereman with a cytotoxic agent.

64. The use according to claim 60, where the specified antibody anywhereman with a growth inhibitory agent.

65. Antibody p� any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for use in an effective amount for inhibiting cell growth, where the growth of these cells at least partially dependent on cell growth potentiating effect of CD79b.

66. The antibody or immunoconjugate according to claim 65, where the specified antibody anywhereman with a cytotoxic agent.

67. The antibody or immunoconjugate according to claim 65, where the specified antibody anywhereman with a growth inhibitory agent.

68. The use of an antibody according to any one of claims.16, 17, 19 and 20 or immunoconjugate according to claim 27 to obtain a medicine for inhibiting cell growth, where the growth of these cells at least partially dependent on cell growth potentiating effect of CD79b.

69. The use according to claim 68, where the specified antibody anywhereman with a cytotoxic agent.

70. The use according to claim 68, where the specified antibody anywhereman with a growth inhibitory agent.

71. The antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for use in an effective amount for therapeutic treatment of a tumor in a mammal, where the specified growth of the tumor is at least partially dependent on growth potentiating effect of CD79b.

72. The antibody or immunoconjugate according to claim 71, where the tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, recidivism�her indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

73. The antibody or immunoconjugate according to claim 71, where the specified antibody anywhereman with a cytotoxic agent.

74. The antibody or immunoconjugate according to claim 71, where the specified antibody anywhereman with a growth inhibitory agent.

75. The use of an antibody according to any one of claims.1, 16, 17, 19 and 20 or immunoconjugate according to claim 27 for obtaining a medicinal product for the treatment of a tumor in a mammal, where the specified growth of the tumor is at least partially dependent on growth potentiating effect of CD79b.

76. The use according to claim 75, where the tumor is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

77. The use according to claim 75, where the specified antibody anywhereman with a cytotoxic agent.

78. The use according to claim 75, where the specified antibody anywhereman with a growth inhibitory agent.

79. Immunoconjugate to claim 28 for use for inhibiting b-cell proliferation by affecting cell immunoconjugates in conditions under which the binding immunoconjugate with CD79b.

80. Immunoconjugate according to claim 79, where b-cell proliferation is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

81. Immunoconjugate according to claim 79, where the cell is xenotransplantation.

82. Immunoconjugate according to claim 79, where the exposure is carried out in vitro.

83. Immunoconjugate according to claim 79, where the exposure is carried out in vivo.

84. The use immunoconjugate according to claim 28 to obtain a medicine for inhibiting b-cell proliferation, where the introduction of a drug injected into the cells to influence cell immunoconjugates in the conditions under which the binding immunoconjugate with CD79b.

85. The use immunoconjugate according to claim 28 to obtain a medicine for inhibiting b-cell proliferation, where the product is introduced into the composition for introduction into a cell to influence cell immunoconjugates in the conditions under which the binding immunoconjugate with CD79b.

86. The use according to claims.84 and 85, where b-cell proliferation �ibrani from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), malcolmfeijten lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

87. The use according to claims.84 and 85, where the cell is xenotransplantation.

88. The use according to claims.84 and 85, where the exposure is carried out in vitro.

89. The use according to claims.84 and 85, where the exposure is carried out in vivo.

90. Method of determining the presence of CD79b in a sample suspected to contain CD79b, wherein said method includes the impact on the specified pattern of the antibody according to any one of claims.1, 16, 17, 19 and 20, and the definition of the specified binding of the antibody to CD79b in a specified sample, where the specified binding of the antibody to CD79b in a specified sample indicates the presence of said protein in a given sample.

91. A method according to claim 90, where the biological sample taken from a patient is assumed to be of b-cell-proliferative disease.

92. A method according to claim 91, where a cell-proliferative infringement is selected from lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), chalk�limfocitarnah lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

93. The antibody according to any one of claims.1, 16, 17, 19 and 20 and immunoconjugate according to claim 27 for use to associate a cell that expresses CD79b.

94. A method according to claim 93, where the specified antibody anywhereman with a cytotoxic agent.

95. A method according to claim 93, where the specified antibody anywhereman with a growth inhibitory agent.

96. A method of producing antibodies against CD79b, where the method comprises (a) cultivating a host cell selected from the group consisting of eukaryotic cell, and the cell SNO, in conditions suitable for expression of the polynucleotide encoding the antibody according to any one of claims.1, 16, 17, 19 and 20, and (b) isolation of antibody, where the eukaryotic cell is a human cell.

97. Composition for inhibiting b-cell proliferation, containing an effective amount of the antibody according to any one of claims.1, 16-20.

98. The composition of claim 97, where the composition comprises a carrier.

99. The polynucleotide encoding the antibody according to any one of claims.1, 16, 17, 19 and 20.

100. The expression vector containing the polynucleotide according to claim 99.

101. The selected cell host containing the vector according to claim 100 to obtain the antibody according to claims.1, 16, 17, 19 and 20.



 

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23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

2 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.

EFFECT: described is a method of the target product and culture medium preparation.

11 cl, 2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely, to obtaining inhibitors of adhesion and/or aggregation of platelets, and can be used in medicine. A polypeptide, used as a component of a pharmaceutical composition and in sets for screening of the inhibitors of platelet adhesion or aggregation, is obtained in a recombinant way with the application of a matrix of the salivary gland cDNA of Anopheles stephensi.

EFFECT: invention makes it possible to obtain the polypeptide, possessing inhibiting activity with respect to platelet aggregation and/or inhibiting activity with respect to platelet adhesion.

10 cl, 4 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering and biotechnology. Disclosed is a method of evaluating bioactivity of chemical compounds, where the first step includes transient transfection of cell line HEK 293 with plasmid vector pX-Y-neo (X is any eukaryote transcription factor, Y is a proteotypic peptide corresponding to said transcription factor), which contains a minimal human adenovirus type 5 promoter; a green fluorescent protein gene; a nucleotide sequence which codes the binding site of the transcription factor; a nucleotide sequence which codes the proteotypic peptide; a neomycin resistance gene; the second step includes determining the activity of the transcription factor via fluorescent analysis and chromatographic-mass spectrometer measurement of the content of the proteotypic peptide in the transfected cell culture in the presence of the test substance compared to a transfected intact cell culture.

EFFECT: invention provides fast and highly sensitive evaluation of bioactivity of chemical compounds.

2 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining insulin analogues, and can be used in medicine as a medication for reducing the glucose level in a patient's blood. An insulin analogue contains a polypeptide B-chain, including halogenated phenylalanine in B24 position, which provides an increased stability in comparison with non-halogenated insulin or insulin analogue. Halogenated phenylalanine represents ortho-monofluorophenylalanine, ortho-monobromophenylalanine or ortho-monochlorophenylalanine.

EFFECT: halogenation-conditioned insulin stabilisation makes it possible to simplify the treatment of patients with diabetes mellitus in developing countries, where there is no access to refrigerating equipment.

11 cl, 8 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and fundamental virology. Method includes obtaining chimeric virus by inserting gene of protein of icosahedral low-copy phloem-restricted virus (ILCPRV) envelope into effective viral vector based on tobamovirus RNA. After that plant is infected with obtained chimeric virus for it to multiply and accumulate in plant tissues. Finally, separation of chimeric virus from plant tissues is performed. Also described are plasmid for claimed method realisation, chimeric virus preparation, obtained by method described above, method of obtaining anti-serum to natural PLRV isolates and its application. Invention can be used in field of agriculture.

EFFECT: claimed is method for obtaining preparative quantities of viral particles, imitating virions of potato leaf roll virus (PLRV).

10 cl, 12 dwg

FIELD: biotechnologies.

SUBSTANCE: invention offers recombinant plasmid DNA coding a chimeric antibody against human tumour necrosis factor-alpha (TNF-alpha) based on pOptiVECTM-TOPO® plasmid. Invention refers to eukaryotic cell line as a producer of antibody to TNF-alpha, method of cell line obtainment by transfection of plasmid DNA according to the invention, and method of chimeric antibody obtainment for TNF-alpha by cultivation of cell line according to the invention.

EFFECT: increased synthesis level for antibodies against TNF-alpha by producer cells.

12 cl, 8 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, i.e. use of asparaginase and a method of foodstuff preparation on the basis of asparaginase. Asparaginase, which has an amino acid sequence at least 90% homologous to the amino acid sequence of <SEQ ID NO:2>, and which after an incubation period of 5 min. within the temperature range of 70°C to 100°C has a residual activity of 200 U/mg, may be used for preparing a foodstuff or a stimulant. The foodstuff preparation method involves the incubation of the foodstuff with the above mentioned asparaginase at the incubation temperature of at least 50°C. Where necessary, the foodstuffs shall be warmed up to a temperature by at least 10°C higher than the incubation temperature. Where necessary, asparaginase shall be separated from the foodstuffs or amidohydrolase shall be inactivated. If required, the separated asparaginase shall be reused.

EFFECT: invention enables the reduction of asparagine or acrylamide contents in the asparagine-containing foodstuffs.

15 cl, 5 dwg, 11 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and is a genetically modified strain of Streptomyces thermotolarences WSJ-IA, which produces isovaleryl spiramycin I. The present invention also discloses a method of producing said strain. The method includes steps of constructing a recombinant plasmid comprising a double gene ist-acyB2 and transforming the plasmid into the isovaleryl spiramycin I - producing strain WSJ-IA. The invention also discloses a method of producing isovaleryl spiramycin I by culturing the Streptomyces thermotolarences WSJ-IA strain in a culture medium.

EFFECT: present invention increases the output of the obtained isovaleryl spiramycin I.

3 cl, 3 dwg, 5 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. A disclosed polypeptide having the amino acid sequence which has a sequence identity of not less than 80% to the amino acid sequence shown in SEQ ID NO: 1 or 2, revealed in description, which polypeptide has a capable of expressing the polynucleotide activity. Also a polynucleotide encoding D-lactate dehydrogenase originated from DNA construct in which the polynucleotide and a promoter capable of expressing the polynucleotide are linked is introduced. Also described a transformant for production of lactic acid, or transformed yeast, in which the polynucleotide or the DNA construct is introduced. A method of producing D-lactic acid, which comprises the step of culturing the said transformant.

EFFECT: transformant capable of highly producing D-lactic acid compared to the D-lactic acid produced with host cell.

15 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.

23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. What is presented is a monoclonal anti-IFNAR1 antibodies with L234F, L235E and P331S Fc mutations of human IgG1 possessing a lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors as compared to a non-modified antibody. There are described the recovered nucleic acid providing expression of the above antibody containing a nucleotide sequence coding the antibody, and a pharmaceutical composition based on the above antibody.

EFFECT: using the invention provides the antibody possessing the lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors that provides reducing the undesired effector functions in treating chronic inflammation and autoimmune conditions.

9 cl, 34 dwg, 7 tbl, 36 ex

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