Antibodies and immunoconjugates and their application

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

 

This application is nepredvidennuiu application filed in accordance with 37 C.F.R. section 1.53(b), whose priority is claimed in accordance with 35 U.S.C. section 119(e) of provisional applications U.S. serial numbers 60/809328, filed may 30, 2006, 60/908941, filed on March 29, 2007, and 60/911829, filed April 13, 2007, the full content of which is thus given in the description by reference in full.

The SCOPE of the INVENTION

The present invention relates to antibodies to CD22 and their immunoconjugates. The invention also relates to methods of using antibodies to CD22 and their immunoconjugates.

BACKGROUND of the INVENTION

Lymphocytes are one of many types of cells produced in the bone marrow in the process of hemopoiesis. There are two populations of lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells). In this application lymphocytes of particular interest are B-cells.

B cells Mature in the bone marrow and leave the bone marrow, expressive on its surface antigennegative antibody. When a naive B-cell first encounters the antigen to which specific it-related membrane antibody, the cell begins to divide and its progeny differentiate into B-memory cells and effector cells, called plasma cells". B-memory cells of which have a longer lifespan and continue to Express associated with membrane antibody with the same specificity, as the original parent cell. Plasma cells do not produce associated with membrane antibody, but instead produce antibody in a form that can be secrete. Secreted antibodies are the main effector molecules of humoral immunity.

Associated with B-cell disorders include, as non-limiting examples, malignant lymphoma (non-Hodgkin's lymphoma, NHL), multiple myeloma and chronic lymphocytic leukemia (CLL, B-cell leukemia (CD5+ B-lymphocytes). Non-Hodgkin lymphoma (NHL), a heterogeneous group of malignant tumors, mainly arising from B-lymphocytes, represent approximately 4% of all newly diagnosed malignant tumors (Jemal, A. et al., CA-Cancer J. Clin., 52: 23-47, (2002)). Aggressive NHL includes approximately 30-40% of adult NHL (Harris, N.L. et al., Hematol. J. 1: 53-66 (2001)) and includes both diffuse B-cell lymphoma (DLBCL), lymphoma mantle cell (MCL), lymphoma peripheral T-cells and both anaplastic lymphoma. Advanced combined chemotherapy cures less than half of patients with aggressive NHL, and most patients ultimately die of the disease (Fisher, R.I. Semin. Oncol. 27(suppl. 12): 2-8 (2000)).

Associated with B-cell disorders include autoimmune diseases. Autoimmune diseases remain to inchesi important diseases in humans. As the name implies, autoimmune diseases is affected by its own immune system. Although particular types of autoimmune diseases pathological mechanisms are one of the main mechanisms include binding of certain antibodies (referred to herein as self-reactive antibodies or autoantibodies) with endogenous proteins of the organism. Doctors and scientists have identified more than 70 clinically certain autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, vasculitis, immunopositivity diabetes and lupus, such as systemic lupus erythematosus. Although many autoimmune diseases are rare - affecting less than 200,000 individuals in aggregate, these diseases affect millions of Americans, approximately five percent of the population, where the majority of the disease disproportionately affects women. The chronic nature of these diseases leads to huge social and financial burden.

Cytotoxic tools aimed at the surface antigens on B-cells, are an important focus of treatment is associated with B-cell malignancies. One such surface antigen of B-cells is a CD20. The first therapeutic antibody approved by the United States Food and Drug Administration for the treatment of recurrent Il is sustainable immature or follicular NHL, was rituximab (Rituxan; Genentech, Inc. (South San Francisco, CA) and IDEC Pharmaceutical Corp. (San Diego, CA), a chimeric (mouse/human) monoclonal antibody to CD20 (Leonard, J.P. et al., Clin. Cane. Res. 10: 5327-5334 (2004)).

Other B-cell antigens, such as CD19, CD22 and CD52, are targets of therapeutic potential for the treatment of lymphoma (Grillo-Lopez AJ, et al., Curr. Pharm. Biotechnol., 2: 301-11, (2001)). CD22 is a 135 kDa restrictively B-cells sialoglycoprotein expressed only on the surface of B-cells at late stages of differentiation (Dorken, B. et al., J. Immunol. 136: 4470-4479 (1986)). The predominant form of CD22 in humans is CD22-beta, which contains the extracellular domain of the seven domains of immunoglobulin superfamily (figure 1) (Wilson, G.L. et al., J. Exp. Med. 173: 137-146 (1991)). In another form, CD22-alpha, there are no domains of immunoglobulin superfamily, 3 and 4 (Stamenkovic, I. and Seed, B., Nature 345: 74-77 (1990)). It is shown that ligand binding to CD22 person associated with the domains of immunoglobulin superfamily 1 and 2 (also referred to as epitopes 1 and 2) (Engel, P. et al., J. Exp. Med. 181: 1581-1586, 1995).

When B-cell NHL expression of CD22 is in the range from 91% to 99% in aggressive and slow-growing populations, respectively (Cesano, A. et al., Blood 100: 350a (2002)). CD22 can function as a component of the activation complex of B-cells (Sato, S. et al., Semin. Immunol. 10: 287-296 (1998)) and adhesion molecules (Engel, Pl et al., J. Immunol. 150:719-4732 (1993)). B-cells of mice with absence of CD22 have a shorter life expectancy and increased apoptosis, indicating a key role of this antigen in the viability of B-cells (Otipoby, K.L. et al., Nature (Lond) 384: 634-637 (1996)). After binding to its natural ligand(s) or CD22 antibodies quickly internalized, providing a powerful co-stimulating signal in the primary B-cells and proapoptotic signals in neoplastic B-cells (Sato, S. et al., Immunity 5: 551-562 (1996)).

Antibodies to CD22 investigated as potential drugs for B-cell malignancies and other B-cell proliferative diseases. Such antibodies include CD22 RFB4 (Mansfield, E. et al., Blood 90: 2020-2026 (1997)), CMC-544 (DiJoseph, J.F., Blood 103: 1807-1814 (2004)) and LL2 (Pawlak-Byczkowska, E.J. et al., Cancer Res. 49: 4568-4577 (1989)). The antibody LL2 (previously called HPB-2) is a monoclonal antibody IgG2a mouse directed to the antigen CD22 (Pawlak-Byczkowska, E.J. et al., (1989), above). Immunohistochemical analysesin vitrodemonstrated the reactivity of the antibody LL2 regarding 50 out of 51 tested samples B-cell NHL, but not in relation to other malignant tumors or normal lymphoid tissues (Pawlak-Byczkowska (1989), above; Stein, R. et al., Cancer Immunol. Immunother. 37: 293-298 (1993)).

The use of conjugates of the antibody-drug for local delivery of cytotoxic or cytostatic means is, i.e. medicines for destruction or inhibition of tumor cells in the treatment of malignant tumors (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26: 151-172; U.S. patent 4975278), provides targeted delivery of a molecule drugs to tumors and accumulation inside them when systemic administration of these unconjugated drug can lead to unacceptable levels of toxicity to normal cells as well as tumor cells that need 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", in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.), p.475-506). Thus achieve maximum efficiency with minimal toxicity. How about fit when these strategies were reported and polyclonal antibodies, and monoclonal antibodies (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87). Medicines used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21: 183-87 (1986)). Toxins used in the conjugates of the antibody-toxin include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins, such as geldanamycin (Kerr et al., (1997) Bioconjugate Chem. 8(6): 781-784; Mandler et al., (2000) Journal of the Nat. Cancer Inst. 92(19): 1573-1581; Mander 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) and calicheamicin (Lode et al., (1998) Cancer Res. 58: 2928; Hinman et al., (1993) Cancer Res. 53: 3336-3342). Toxins can exert its cytotoxic and cytostatic effect through mechanisms including tubulin binding, DNA binding or inhibition of topoisomerase (Meyer, D.L. and Senter, P.D. "Recent Advances in Antibody Drug Conjugates for Cancer Therapy" in Annual Reports in Medicinal Chemistry, Vol.38 (2003) Chapter 23, 229-237). Some cytotoxic drugs during conjugation with large antibody or protein ligands of the receptors tend to become inactive or less active.

ZEVALIN® (zevalin®) (ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioactive isotope, consisting of monoclonal antibodies IgG1 Kappa mouse directed to CD20 antigen found on the surface of normal and malignant B-lymphocytes, and radioactive isotope111In or90Y related by timesaving linker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7): 766-77; Wiseman et al., (2002) Blood 99(12): 4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J. Clin. Oncol. 20(15): 3262-69). Although the zevalin has activity against B-cell non-Hodgkin lymphoma (NHL), introduction in most patients results in severe and prolonged cytopenias. In the year 2000 is for the treatment of acute myeloid leukemia by injection approved MYLOTARG™ (mylotarg™) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), conjugate antibody-drug consisting of CD33 antibodies person associated with calicheamicin (Drugs of the Future(2000) 25(7):686; U.S. patents№4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). For the treatment of malignant tumors expressing the antigen CanAg, such as a malignant tumor of the large intestine, pancreas, stomach and other developed cantuzumab mertansine (Immunogen, Inc.), conjugate antibody-drug consisting of huC242 antibody linked via a disulfide linker SPP to the molecule maytansinoids medicines DM1. In development for the potential treatment of prostate tumors is MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), conjugate antibody-drug consisting of monoclonal antibodies to specific membrane antigen prostate (PSMA)associated with the molecule maytansinoids medicines DM1. The same molecule maytansinoids medicines, DM1, through ridiculing linker, SMCC, bound with monoclonal antibody mouse, TA.1 (Chari et al., (1992) Cancer Research 52: 127-131). It was reported that this conjugate is 200 times less effective than the corresponding conjugate with a disulfide linker. In the document it is assumed that the linker SMCC was "darassalam".

From the marine mollusk,Dolabella auriculariaallocated to the short peptide compounds and detected they possess biological activity (Pettit et al., (1993) Tetrahedron 49: 9151; Nakamura et al., (1995) Tetrahedron Letters 36: 5059-5062; Sone et al., (1995) Journal Org. Chem. 60: 4474). Also the analogs of these compounds and some of them discovered that they have a biological activity (for review, see Pettit et al., (1998) Anti-Cancer Drug Design 13: 243-277). For example, auristatin E (US 5635483) is a synthetic analogue of the natural marine product dolastatin 10, means that inhibits tubulin polymerization by binding to the same site on tubulin, and anti-cancer drug vincristine (G.R. Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70: 1-79). Dolastatin 10, auristatin PE and auristatin E are linear peptides of four amino acids, three of which are unique to compounds of the class of dolastatin, and C-terminal amide.

Auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin, conjugatively (i) chimeric monoclonal antibodies cBR96 (specific to the antigen Lewis Y on carcinomas); (ii) cAC10 specific to CD30 on malignant neoplasms hematologic origin (Klussman, et al. (2004), Bioconjugate Chemistry 15(4): 765-773; Doronina et al. (2003) Nature Biotechnology 21(7): 778-784; "Monometylvaline Compounds Capable of Conjugation to Ligands"; Francisco et al. (2003) Blood 102(4): 1458-1465; US 2004/0018194); (iii) antibodies to CD20, such as Rituxan® (Rituxan®rituximab) (WO 04032828), for the treatment of expressing CD20 malignant tumours and immune disorders; (iv) antibodies to EphB2 2H9 and antibodies to IL-8 for the treatment of colorectal cancer (Mao, et al. (2004) Cancer Research 64(3): 781-788); (v) an antibody to E-selectin (Bhaskar et al. (2003) Cancer Res. 63: 6387-6394) and (vi) other antibodies to CD30 (WO 03/043583). Monomethylester (MMAE) also conjugatively with 2H9 antibody to EphB2R, which is a t/m tyrosinekinase receptor type 1 with high homology in mouse and man and which sverkhekspressiya in colorectal cancer cells (Mao et al. (2004) Cancer Res. 64: 781-788).

Monomethylaniline MMAF, variant of auristatin E (MMAE) with a phenylalanine at the C-end (U.S. patent No. 5767237; U.S. patent No. 6124431), is less potent than MMAE, but more potent during conjugation with monoclonal antibodies (Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, abstract number 623, presented March 28, 2004). Auristatin F phenylenediamine (AFP); MMAE with phenylalanine connected with mAb to CD70, 1F6, via C-Terminus 1F6 through phenylendiamine the spacer (Law et al., Proceedings of the American Association for Cancer Research, Volume 45, abstract number 625, presented March 28, 2004).

As a possible therapeutic compounds also investigated the conjugates of the antibody to CD22-toxin. For example, in previous publications as anticancer funds described containing a chain of ricin A immunotoxins, on ravennae to anti-CD22 (May, R.D. et al., Chemical Abstracts 106(21): 168656x pages 35-36 (1987); Ghetie, M.A. et al., Cancer Research 48: 2610-2617 (1988); and Amlot, P.L. et al., Blood 82(9):2624-2633 (1993)). If the toxin was a radioactive isotope, for epratuzumab, humanized version (grafted CDR) IgG1 LL2, shows the presence of therapeutic activity radioimmunoconjugates (Juweid, M.E. et al., Clin. Cancer Res. 5 (Suppl 10): 3292s-3303s (1999); Griffiths, G.L. et al., J. Nucl. Med. 44: 77-84 (2003); Linden, O. et al., Clin. Cancer Res. 5(suppl 10): 3287s-3291s (1999)).

In this area there is a need for additional drugs to treat a variety associated with B-cell malignancies, such as lymphomas, such as non-Hodgkin's lymphoma and other B-cell proliferative disorders. Particularly suitable for this purpose medicines include targeted to B cells against CD22 conjugates antibody-drug with significantly less toxicity, with an acceptable therapeutic efficacy. The present invention relates to these and other limitations and problems of the past.

Bringing in the application of any link is not an admission that the reference is prior art for this application. All cited in this document of references, including patents, patent applications and publications, in full included as references.

The INVENTION

Invented the e refers to antibodies to CD22 and how to use them.

In one aspect provided with the antibody binding to CD22, where the antibody contains at least one, two, three, four, five or six HVR selected from the

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO: 4;

(3) the HVR-H3 containing the amino acid sequence of SEQ ID NO: 6;

(4) the HVR-L1 containing the amino acid sequence of SEQ ID NO: 10;

(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO: 12; and

(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In another aspect, the antibody binding to CD22, contains (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 10, and (b) at least one, two, three, four or five HVR selected from the

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO: 4;

(3) the HVR-H3 containing the amino acid sequence of SEQ ID NO: 6;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO: 12; and

(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In another aspect, the antibody binding to CD22, contains (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 9, and (b) at least one, two, three, four or five HVR selected from the

(1) HVR-H1 containing the amino acid th is sequence SEQ ID NO: 2;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO: 4;

(3) the HVR-H3 containing the amino acid sequence of SEQ ID NO: 6;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO: 12; and

(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In another aspect, the antibody binding to CD22, contains (a) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6, and (b) at least one, two, three, four or five HVR selected from the

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO: 4;

(3) the HVR-L1 containing the amino acid sequence of SEQ ID NO: 9;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO: 12; and

(5) the HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In another aspect, the antibody binding to CD22, contains (a) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6, and (b) at least one, two, three, four or five HVR selected from the

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO: 4;

(3) the HVR-L1 containing the amino acid sequence of SEQ ID NO: 10;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO: 12; and

(5) the HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In one of the embodiments the antibody contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 10. In one of the embodiments, the antibody further comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 2, and HVR-H2 containing the amino acid sequence of SEQ ID NO: 4. In one of the embodiments, the antibody further comprises HVR-L2, containing the amino acid sequence of SEQ ID NO: 12, and HVR-L3, containing the amino acid sequence of SEQ ID NO: 14.

In certain embodiments of the exercise of any of the above antibodies further comprises at least one skeleton selected from a consensus framework subgroup III consensus VH and frame subgroup I VL.

In one aspect provided with the antibody binding to CD22, where the antibody contains a variable domain of the heavy chain with a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 16. In one of the embodiments, the antibody contains a variable domain of the heavy chain SEQ ID NO: 16.

In one aspect, the antibody further comprises a variable house the light chain sequence, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 17. In one of the embodiments, the antibody contains a variable domain light chain SEQ ID NO: 17.

In one aspect, the antibody further comprises a variable domain light chain sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 18. In one of the embodiments, the antibody contains a variable domain light chain SEQ ID NO: 18.

In one of the embodiments, the antibody contains a variable domain of the heavy chain with a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence SEQ ID NO: 16 and a variable domain light chain sequence, p is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17. In one of the embodiments, the antibody contains a variable domain of the heavy chain with a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence SEQ ID NO: 16 and a variable domain light chain sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the implementation of the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO: 16, and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 17. In one embodiment, the implementation of the variable domain of the heavy chain contains the amino acid sequence of alnost SEQ ID NO: 16, and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 18.

In certain embodiments of the implementation provided polynucleotide encoding any of the above antibodies. In one of the embodiments provided is a vector containing polynucleotide. In one of the embodiments provided is a host cell containing the vector. In one of the embodiments a host cell is eukaryotic. In one of the embodiments a host cell is a cell in the ovary of the Chinese hamster (CHO). In one of the embodiments provided is a method of obtaining antibodies to CD22, where the method comprises culturing the host cell under conditions suitable for expression of polynucleotide encoding the antibody, and isolating the antibody.

In one aspect provided with the antibody binding to CD22 expressed on the cell surface. In one of the embodiments the antibody binds to an epitope in the region of CD22 human or mouse containing domain 1 or domain 2, or domains 1 and 2. In one of the embodiments the cell is a cell of a mammal. In one of the embodiments the cell is a human cell. In one of the embodiments the cell is a malignant cell. In one of the variants of the s implementation of the cell is a B-cell. In one embodiment, the implementation of the malignant cell is a B-cell.

In certain embodiments of the exercise of any of the above antibodies is a monoclonal antibody. In one of the embodiments the antibody is an antibody fragment selected from Fab fragments, Fab'-SH, Fv, scFv, or (Fab')2. In one of the embodiments the antibody is humanized. In one of the embodiments the antibody is a human antibody.

In one aspect provides a method of detecting the presence of CD22 in a biological sample, where the method comprises bringing a biological sample into contact with any of the above antibodies under conditions that allow the binding of an antibody to CD22, and determining that there are a complex between the antibody and CD22. In one of the embodiments the biological sample contains B-cells. In one of the embodiments the biological sample obtained from a mammal with a B-cell infringement and/or B-cell proliferative disorder, including as non-limiting examples of lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lung lymph is, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell or mammal suspected of the violation.

In one aspect provides a method of diagnosing a cell proliferative disorders associated with increased expression of CD22, where the method includes bringing the test cells in contact with any of the above antibodies; determining the level of expression of CD22 by determining the binding of an antibody to CD22; and comparing the level of expression of CD22 test cell with the level of expression of CD22 control cell, where a higher level of expression of CD22 in the test cell compared to the control cell indicates the presence of cell proliferative disorders associated with increased expression of CD22. In one embodiment, the implementation of the test cell is a cell of a patient with suspicion on the presence of cell proliferative disorders, such as B-cell proliferative disorder. In one embodiment, the implementation of cellular proliferative violation is selected from B-cell disorders, including as non-limiting examples of lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slowly rastus the Yu NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell. In one of the embodiments the method comprises determining the level of expression of CD22 on the surface of the test cell and comparing the level of expression of CD22 on the surface of the test cell with the level of expression of CD22 on the surface of the control cells.

In one aspect provides a method of diagnosing a cell proliferative disorders associated with an increase in the number of cells, such as B-cells expressing CD22, where the method includes bringing the cells tested in the biological sample into contact with any of the above antibodies; determining the level of antibodies bound to the test cells in the sample by determining the binding of an antibody to CD22; and comparing the level of antibodies bound to cells in a control sample, where the level of bound antibody in the test and control samples normalized by the number expressing CD22 cells and where a higher level of bound antibody in the test sample by compared with the control sample indicates the presence of cell proliferative disorders associated with cells expressing CD22.

In one aspect provides a method defined who I soluble CD22 in blood or serum, where the method includes bringing the test sample of blood or serum of a mammal suspected presence of B-cell proliferative disorders in contact with the antibody to CD22 according to the invention and determining the increase of soluble CD22 in the test sample relative to the control sample of blood or serum of a normal mammal. In a specific embodiment, the method of determining suitable as the method for the diagnosis of B-cell proliferative disorders associated with increased soluble CD22 in the blood or serum of a mammal.

In one aspect, the antibodies of the invention include modified cysteine antibodies where one or more amino acids of the original antibody is replaced with the free amino acid cysteine as described in WO 2006/034488 (incorporated herein by reference in full). So you can modify, i.e. subjected to mutation, any form of antibodies to CD22. For example, you can modify the original Fab-fragment of the antibody so as to obtain the modified cysteine Fab, designated herein as "ThioFab". Similarly, you can modify the original monoclonal antibody to receipt ThioMab". It should be noted that due to the dimeric nature of IgG antibodies odnoshatrovaya mutation in toofab gives one obtained in d is the query result of constructing cysteine residue, while odnoshatrovaya mutation in Tioman gives two resulting design cysteine residue. Modified cysteine antibodies to CD22 according to the invention include monoclonal antibodies, humanized or chimeric monoclonal antibodies and antigenspecific antibody fragments, fused polypeptides and analogs that preferentially bind associated with cells CD22 polypeptide. Modified cysteine antibody may alternatively contain an antibody containing cysteine in the described in the present document position in the antibody or the Fab, the resulting design sequence and/or selecting antibodies, without requiring changes to the original antibody, such as through the design and selection of antibodies by phage display or by constructing a frame sequences and constant regions of the light chain and/or heavy chainsde novo. Modified cysteine antibody contains one or more free amino acids cysteine with the value of the reactivity of thiol groups in the range from 0.6 to 1.0; 0.7 to 1.0 or 0.8 to 1.0. The free amino acid cysteine is a cysteine residue, the resulting design in the original antibody and not part of a disulfide bridge. Modificirowan the e-cysteine antibodies suitable for attachment of cytotoxic and/or imaging compounds in the area of the built-cysteine, for example, through maleimide or halogenoacetyl. Nucleophilic reactivity of thiol functional group of the Cys residue in relation to maleimide group approximately 1000 times greater than that of any other functional groups of the amino acids in the protein, such as amino group of lysine residues or N-terminal amino group. Specific for thiol functional group in iodization and maleimide reagents may react with the amine groups, but need a higher pH (>9,0) and a longer reaction time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

In one embodiment, the implementation of the modified cysteine antibody to CD22 according to the invention contains the resulting design cysteine in any of the following provisions, where the position is a number by Kabat et al. in the 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 by the EU numbering in the heavy chain (including Fc-region) (see Kabat et al., (1991), above), where the constant region of light chain, indicated by underlining in figa, starts at position 108 (Kabat numbering), and the constant region of the heavy chain are indicated by underlining in figv and 17C, starts at position 118 (EU numbering). The position can also be specified in the form of its position in the sequential numbering of the amino acids Polner Smirnykh light chain or heavy chain, presented at figa-17C. One of the embodiments of the invention the antibody to CD22 contains the resulting design cysteine in LC-V205C (Kabat numbering: 205 Val; serial number 210 on figa modified so that in this position were Cys). The resulting design cysteine in the light chain on figa shown in bold double underline. One of the options for the implementation of the antibody to CD22 contains the resulting design cysteine in HC-A118C (EU number: Ala 118; serial number 121 on FIGU modified so that in this position were Cys). The resulting design cysteine in the heavy chain are shown in bold double underline on figv. One of the options for the implementation of the antibody to CD22 contains the resulting design cysteine Fc-S400C (EU number: Ser 400; serial number 403 on figs modified so that in this position were Cys). The resulting design cysteine in the Fc-region of the heavy chain shown in figs in bold double underline. In other embodiments, implementation of the resulting design cysteine heavy chains (including Fc-region) is located in any of these provisions: (EU numbering): 41, 88, 116, 118, 120, 171, 282, 375 or 400. Therefore clicks the zoom, changes of amino acids in these positions in the original antibody to CD22 according to the invention are: A41C, A88C, S116C, A118C, T120C, A171C, V282C, S375C or S400C. In other embodiments, implementation of the resulting design cysteine light chain is in any of these positions (Kabat numbering): 15, 43, 110, 144, 168, 205. Thus, changes of amino acids in these positions in the original antibody to CD22 according to the invention are: V15C, A43C, V110C, A144C, S168C or V205C.

Modified cysteine antibody to CD22 contains one or more free amino acids cysteine, where the modified cysteine antibody to CD22 binds to the CD22 polypeptide and received by a process comprising replacing one or more amino acid residues of the original antibody to CD22-cysteine, where the original antibody contains at least one HVR sequence selected from the

(a) sequence HVR-L1 RSSQSIVHSNGNTFLE (SEQ ID NO: 9), or sequences of HVR-L1 RSSQSIVHSVGNTFLE (SEQ ID NO: 10) (pigv);

(b) sequence HVR-L2 KVSNRFS SEQ ID NO: 12 (pigv);

(c) sequence HVR-L3 FQGSQFPYT (SEQ ID NO: 14) (pigv);

(d) sequence HVR-H1 GYEFSRSWMN (SEQ ID NO: 2) (figa);

(e) sequence HVR-H2 GRIYPGDGDTNYSGKFKG (SEQ ID NO: 4 (figa) and

(f) sequence HVR-H3 DGSSWDWYFDV (SEQ ID NO: 6) (figa).

In a specific aspect the invention relates to modified by cysteine antibody to CD22, which contains Adamu amino acid sequence, at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity of amino acid sequences with modified cysteine antibody with a full amino acid sequence as described herein or amino acid sequence modified by cysteine antibodies without the signal peptide, as described herein.

In yet another additional aspect of the invention relates to the selected modified by cysteine antibody to CD22, containing the amino acid sequence encoded by the nucleotide sequence for hybridization with a complementary DNA molecule that encodes (a) a modified cysteine antibody with a full amino acid sequence as described herein, (b) amino acid sequence modified by cysteine antibodies without the signal peptide, as described herein, (c) extracellular domain transmembrane protein modified with cysteine antibodies, signal peptides or without it, as described herein, (d) amino acid sequence encoded by any of sequences nekleenov the th acid, described herein, or (e) any other specifically defined fragment of the full amino acid sequence modified by cysteine antibodies, as described in this document.

In a specific aspect the invention relates to the selected modified by cysteine antibody to CD22 without N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as described herein. Methods for their preparation are also described in this document, where these methods include culturing the host cell containing the vector containing the appropriate encoding nucleic acid molecule, under conditions suitable for expression of modified cysteine antibodies, and the restoration of the modified cysteine antibodies from cell culture.

Another aspect of the invention relates to the selected modified by cysteine antibody to CD22 with a remote transmembrane domain or inaktivirovannye transmembrane domain. Methods for their preparation are also described in this document, where these methods include culturing the host cell containing the vector containing the appropriate encoding nucleic acid molecule under conditions approaching them for the expression of modified cysteine antibodies and recovery of modified cysteine antibodies from cell culture.

In other embodiments implementing the invention relates to the selected chimeric modified cysteine antibody to CD22 containing any of the described in this document modified with cysteine antibody, fused to a heterologous (non-CD22) polypeptide. Example of such chimeric molecules include any of the features described in this document modified with cysteine antibodies, fused with a heterologous polypeptide, such as limit epitope sequence or a Fc region of an immunoglobulin.

Modified cysteine antibody to CD22 can be a monoclonal antibody, antibody fragment, chimeric antibody, humanitariannet antibody, single-chain antibody or antibody, a competitive inhibition binding of the antibody to CD22 polypeptide with its respective antigenic epitope. Antibodies of the present invention may not necessarily be conjugated with inhibiting the growth of the agent or cytotoxic agent such as a toxin, including, for example, auristatin, an antibiotic, a radioactive isotope, a nuclease or other Antibodies of the present invention optionally can be obtained in CHO cells or bacterial cells, and they preferably inhibit the growth or proliferation and the and induce the death of cells, with which they are associated. For diagnostic purposes, the antibodies of the present invention can be mark detectable label, to attach to a solid substrate, or the like

In other embodiments, implementation of the present invention the invention relates to vectors containing DNA encoding any of the described in this document antibodies to CD22 and modified with cysteine antibodies to CD22. Also provided cell host containing any such vector. As an example, cell host may represent cells CHO cellsE. colior yeast cells. Additionally provided is a method of obtaining any of those described herein polypeptides, and it includes culturing host cells under conditions suitable for expression of the described polypeptide, and recovering the desired polypeptide from the cell culture.

Modified cysteine antibodies can be used in the treatment of malignant tumors, and they include antibodies specific for cell surface and transmembrane receptors and tumor-specific antigens (TAA). Such antibodies can be used in the form of free antibodies (unconjugated with drug or molecule-label) or in the form of conjugates of the antibody-drug (ADC). Modified cysteine antibodies according to the invention m is may be site-specifically and efficiently associated with reactive thiol reagent. Reacting with a thiol reagent can be a polyfunctional linker, tag, capture the fluorophore or the intermediate connection of a drug-linker. Modified cysteine antibody can be marked detectable label, mobilitat on solid-phase substrate and/or konjugierte with a molecule drugs. The reactivity of thiol groups can be shared for any antibody where it is possible to replace amino acids reactive amino acid cysteine in the ranges in the light chain selected from the ranges of amino acids: L-10 to L-20; L-38 to L-48; L-105 to L-115; L-139 to L-149; L-163 to L-173; and in ranges in the heavy chain selected from the ranges of amino acids: H-35 to H-45; H-83 to H-93; H-114 to H-127 H-170 to H-184, and in the Fc-region within the ranges selected from the range from H-268 to H-291; from H-319 to H-344; from H-370 to H-380 and from H-395 to H-405, where the numbering of the provisions of amino acids begins in position 1 on the numbering system of Kabat (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), and then continues as described in WO 2006034488. The reactivity of thiol groups can also be shared for certain domains of antibodies, such as the constant domain of the light chain (CL) and constant domains of the heavy chains, CH1CH2and CH3. Can prostituzione on cysteine, leading to the values of the reactivity of thiol groups of 0.6 and above in the constant domains of the α, δ, ε, γ and µ heavy chains source of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Such antibodies and their use is described in WO 2006/034488.

Modified cysteine antibodies according to the invention preferably retain antigennegative the ability of the variants of the original antibody wild type. Thus, the modified cysteine antibodies capable of binding, preferably specific to the antigens. Such antigens include, for example, tumor-specific antigens (TAA), proteins of the cell surface receptors and other molecules on the cell surface, transmembrane proteins, signaling proteins, factors that regulate cell viability, the factors regulating cell proliferation, molecules associated (e.g., known or suspected functionally contribute to) with the development or differentiation of tissues, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated (e.g., known or suspected functionally contributing) with angiogenesis. Tumor-specific antigen can be a factor of cluster of differentiation (i.e. protein CD, including the Aya as non-limiting examples of CD22). Modified cysteine antibodies to CD22 according to the invention retain the ability to bind the antigen of the variants of the original antibodies to CD22 wild type. Thus, the modified cysteine antibodies to CD22 according to the invention are capable of binding, preferably specific to the antigen CD22, including isoforms anti-CD22-beta and/or alpha person, including options, when such antigens expressed on the cell surface, including as non-limiting examples of B-cells.

The antibody according to the invention can be konjugierte with other reactive thiols means, in which the reactive group is, for example, maleimide, todatetime, pyridyldithio or other reactive thiols partner conjugation (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. Bioconjugate Techniques (1996) Academic Press, San Diego, p.40-55, 643-671). Partner can be a cytotoxic agent (e.g., a toxin, such as doxorubicin or pertussis toxin), a fluorophore, such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for imaging or radiotherapy metal, peptide or ones label or detektiruya label, or measuring the ground clearance of the vehicle, such as the hypoxia isomers of polyethylene glycol, the peptide that binds the third component, or another carbohydrate or a lipophilic agent.

In one aspect of the antibodies according to the invention can be konjugierte with any molecule-label, which can be covalently attached to the antibody via a reactive group of an activated group or a reactive thiol group of cysteine (Singh et al., (2002) Anal. Biochem. 304: 147-15; Harlow E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY; Lundblad R.L. (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, FL). Attached label may function to (i) ensure the program signal; (ii) interact with a second label to modify detected signal provided by the first or second label, e.g., to obtain FRET (resonance energy transfer fluorescence); (iii) stabilizing interactions or increase the affinity of binding to the antigen or ligand; (iv) impacts on mobility, e.g. electrophoretic mobility or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) providing an exciting group to modulate the affinity of ligand binding antibody/antigen or ion complexation.

Labeled modified cysteine antibodies can be used in diagnostic assays, such as the er, for detection of the expression of interest of antigen-specific cells, tissues, or serum. For diagnostic applications, the antibody, as a rule, mark detectable by the group. There are many labels that generally can be grouped into the following categories:

Radioactive isotopes (radionuclides), such as3H,11C,14C,18F,32P,35S64Cu68Ga86Y99Tc111In123I124I125I131I133Xe177Lu,211At or213Bi. Radiolabelled antibodies suitable for experiments aimed at the visualization of receptors. The antibody can be marked reagent ligands that bind, chelate or otherwise form a complex with a radioactive isotope of the metal, where the reagent is capable of reacting with thiol obtained by constructing cysteines in the 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 that can form a complex with a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). Radionuclides can be directed through the formation of complexes with conjugates of the antibody-drug according to the invention (Wu et al., (2005) Nature Biotechnology 23(9): 1137-1146).

Linker reagents such as DOTA-Maliki is (4-maleimidomethyl-DOTA), you can get a reaction aminobenzyl-DOTA with 4-maleimidomethyl acid (Fluka), activated isopropylcarbamate (Aldrich) by the method Axworthy et al., (2000) Proc. Natl. Acad. Sci. USA 97(4): 1802-1807). Reagents DOTA-maleimide react with the free amino acids of the cysteine-modified cysteine antibodies and form the antibody ligand that forms complexes with metals (Lewis et al., (1998) Bioconj. Chem. 9: 72-86). Chelating linker aiming reagents, such as DOTA-NHS (mono (N-hydroxysuccinimidyl ester) 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraoxane acid) are commercially available (Macrocyclics, Dallas, TX). Aimed at receptors visualization labeled with radionuclides antibodies may provide a marker of the activation of cascades of reactions through the detection and quantitative determination of the increasing accumulation of antibodies in tumor tissue (Albert et al., (1998) Bioorg. Med. Chem. Lett. 8: 1207-1210). Conjugated to radioactive metals after lysosomal degradation may remain inside the cells.

Metal-chelate complexes suitable as labels antibodies for imaging experiments, described in U.S. patent 5342606; U.S. patent 5428155; U.S. patent 5316757; U.S. patent 5480990; U.S. patent 5462725; U.S. patent 5428139; U.S. patent 5385893; U.S. patent 5739294; U.S. patent 5750660; U.S. patent 5834456; Hnatowich et al., (1983) J. Immunol. Methods 65: 147-157; Meares et al., (1984) Anal. Biochem. 142: 68-78; Mirzadeh et al., (190) Bioconjugate Chem. 1:59-65; Meares et al., (1990) J. Cancer 1990, Suppl. 10: 21-26; Izard et al., (1992) Bioconjugate Chem. 3: 346-350; Nikula et al., (1995) Nucl. Med. Biol. 22: 387-90; Camera et al., (1993) Nucl. Med. Biol. 20: 955-62; Kukis et al., (1998) J. Nucl. Med. 39: 2105-2110; Verel et al., (2003) J. Nucl. Med. 44: 1663-1670; Camera et al., (1994) J. Nucl. Med. 21: 640-646; Ruegg et al., (1990) Cancer Res. 50: 4221-4226; Verel et al., (2003) J. Nucl. Med. 44: 1663-1670; Lee et al., (2001) Cancer Res. 61: 4474-4482; Mitchell, et al., (2003) J. Nucl. Med. 44: 1105-1112; Kobayashi et al., (1999) Bioconjugate Chem. 10: 103-111; Miederer et al., (2004) J. Nucl. Med. 45: 129-137; DeNardo et al., (1998) Clinical Cancer Research 4: 2483-90; Blend et al., (2003) Cancer Biotherapy &Radiopharmaceuticals 18:355-363; Nikula et al., (1999) J. Nucl. Med. 40: 166-76; Kobayashi et al., (1998) J. Nucl. Med. 39: 829-36; Mardirossian et al., (1993) Nucl. Med. Biol. 20: 65-74; Roselli et al., (1999) Cancer Biotherapy &Radiopharmaceuticals, 14: 209-20.

(b) Fluorescent labels such as rare earth chelates (europium chelates), classes fluorescein, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; classes rhodamine, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; Texas red and their analogues. Fluorescent labels can be konjugierte with antibodies using methods described, for example, in Current Protocols in Immunology, above. Fluorescent dyes and reagents fluorescent labels include fluorescent dyes and reagents fluorescent labels, which are commercially available at Invitrogen/Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL).

(c) Available or described various labels, enzyme-substrate (U.S. patent 4275149). The enzyme generally catalyzes a chemical modification chromag the frame substrate, which can be measured in different ways. For example, the enzyme may catalyze a change in color of the substrate, which can be measured spectrophotometrically. An alternative enzyme may alter the fluorescence or chemiluminescence substrate. The techniques for measuring changes in fluorescence as described above. Chemiluminescent substrate during a chemical reaction becomes electronically excited, and may then emit light which can be measured (for example, using chemiluminometer), or to transfer energy to the fluorescent acceptor. Examples of enzymatic labels include luciferase (e.g., Svetlakov the luciferase and bacterial luciferase; U.S. patent 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, oxidase sugars (e.g. glucose oxidase, galactosidase and glucose-6-phosphatedehydrogenase), oxidase heterocycles (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides and other 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 combinations of enzyme-substrate include, for example,

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

(ii) alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and

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

Professionals in this field are numerous other combinations of enzyme-substrate. For an overview, see U.S. patent 4275149 and U.S. patent 4318980.

The label can indirectly to konjugierte with the side chain of amino acids, with an activated side chain of amino acids with modified cysteine antibody and the like, 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 selectively binds with streptavidin and thus, the label can be konjugierte with the antibody in such an indirect way. Alternatively, for the implementation of indirect conjugation of the label with the variant polypeptide variant polypeptide kongugiruut with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above, kongugiruut variant polypeptide against hapten (for example, antibody to digoxin). T is thus, you can make 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 applied in any known method of analysis, such as ELISA assays, competitive binding, direct and indirect sandwich assays and analyses of immunosurgery (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp.147-158, CRC Press, Inc.).

Detected label may be suitable for localization, visualization and quantification of event binding or recognition. Labeled antibodies according to the invention it is possible to determine the receptors on the cell surface. Another use detektirano labeled antibodies is a way of immunosafety-based granules, including algae pellets with a fluorescently labeled antibody and the detection of fluorescent signal upon binding of the ligand. Similar methods of detection are used, the effect of surface plasmon resonance (SPR) for measurement and detection of interactions of the antibody-antigen.

Detected labels such as fluorescent dyes, chemiluminescent dyes (Briggs et al., (1997) "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids", J. Chem. Soc., Perkin-Trans. 1: 1051-1058), provide the detected signal and, as a rule, applicable to labeled antibodies, preferably with the following properties: (i) labeled antibody Dol is but to give a very high signal with low background to a small number of antibodies can be had with high sensitivity to detect in cell-free and cell-based assays; and (ii) labeled antibody should be photostabilized so that the fluorescent signal could be observed, measured, and recorded without significant photobleaching. For applications, including the linking of cell surface labeled antibodies with the cell membrane or cell surfaces, in particular living cells, the label preferably (iii) have good solubility in water to achieve an effective concentration of conjugates and sensitivity of detection, and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes in the cells or not to cause premature cell death.

Direct quantification of the intensity of cellular fluorescence and counting events for fluorescent labeling, for example, binding of conjugates of peptide-dye from the cell surface, can be implemented in the system (FMAT® 8100 HTS System, Applied Biosystems, Foster City, Calif.), which automates the mixing and reading, non-radioactive assays with live cells or granules (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 includes analyses of the binding surface the receptor, analyses of immunosafety, solid-phase immunofluorescence assay (FLISA), cleavage by caspase (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosisin vivo", (1998) Proc. Natl. Acad. Sci. USA 95: 618-23; U.S. patent 6372907), 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 cytotoxic analysis. To identify positive or negative regulation of the molecule, which has the target on the cell surface, you can use the fluorimetric method of analysis in microvolumes (Swartzman, "A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology", (1999) Anal. Biochem. 271: 143-51).

Labeled antibodies according to the invention are suitable as biomarkers and probes for visualization of various ways and means of biomedical and molecular imaging, such as (i) MRI (magnetic resonance imaging); (ii) MicroCT (computed tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al., (2004) Bioconjugate Chem. 15: 41-49; (v) bioluminescence; (vi) fluorescence and (vii) ultrasound. Immunoscintigraphy is an imaging technique in which antibodies labeled with radioactive material administered to a patient, animal or person and get a picture of the site in the body where localized antibody (U.S. patent 658624). Imaging biomarkers can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic response to a therapeutic intervention. Biomarkers can be of several types: type 0 are natural historical markers of the disease and longitudinale correlated with known clinical indices, such as MRI examination of synovial inflammation in rheumatoid arthritis; markers of type I reveal the effect of the intervention in accordance with the mechanism of action, even if the mechanism may not be associated with clinical outcome; markers of type II function as a substitution endpoints, where the change of the biomarker or a signal from him suggests clinical benefit for "confirmation" of a given response, such as the measurement of bone erosion in rheumatoid arthritis by CT. Thus, imaging biomarkers can provide pharmacodynamic (PD) therapeutic information: (i) the expression of the target protein, (ii) about linking a therapeutic agent to a target protein, i.e. selectivity, and (iii) on the pharmacokinetic data clearance and half-life. The advantages of imaging biomarkersin vivoregarding laboratory biomarkers include non-invasive near the step, quantitative assessment throughout the body, re-dosing and evaluation, i.e. multiple time points and potentially portable impact from preclinical (animal) and clinical (human) results. For some applications biosalinity replace or minimize the number of experiments on animals in preclinical studies.

Methods tagging of peptides are 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, Fla.); De Leon-Rodriguez et al., (2004) Chem. Eur. J. 10: 1149-1155; Lewis et al., (2001) Bioconjugate Chem. 12: 320-324; Li et al., (2002) Bioconjugate Chem. 13: 110-115; Mier et al., (2005) Bioconjugate Chem. 16: 240-237.

Peptides and proteins labeled with two different molecules, the fluorescent reporter and the quencher in close enough, undergo resonance energy transfer fluorescence (FRET). Reporter group, as a rule, are fluorescent dyes that are excited by light radiation at a certain length of the e wave and transfer energy to the acceptor or quencher, the group, with the corresponding Stokes shift for emission at maximum brightness. Fluorescent dyes include molecules with a high degree of flavoring, such as fluorescein and rhodamine and their derivatives. Fluorescent reporter in the original peptide may be partially or largely suppressed by the quencher molecule. After cleavage of the peptide by peptidase or protease can be measured detective increase in fluorescence (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34).

Labeled antibodies according to the invention can also be used as an affinity agent. In this way labeled antibody immobilized on a solid phase, such as resin Sephadex or filter paper, well known in this field means. The immobilized antibody is in contact with the sample containing the purified antigen, and then the substrate is washed with a suitable solvent, which removes essentially all the material in the sample except the purified antigen that is bound to the immobilized variant polypeptide. Finally, the substrate is rinsed other suitable solvent, such as glycine buffer, pH 5.0, which releases the antigen from variant polypeptide.

Aiming reagents, generally have reactive functional group that can response the step (i) directly with the thiol of the cysteine-modified cysteine antibodies with the formation of labeled antibody, (ii) with the linker reagent with the formation of intermediate linker-label or (iii) to the linker antibody with the formation of labeled antibodies. Reactive functional group aiming reagents includes maleimide, halogenoacetyl, Succinimidyl ester of iodated (for example, NHS, N-hydroxysuccinimide), isothiocyanate, sulphonylchloride, 2,6-dichlorotriazinyl, pentafluoropropyl ester and phosphoramidite, although you can also use other functional groups.

Illustrative reactive functional group is a complex N-hydroxysuccinimidyl ester (NHS) Deputy carboxyl groups, detectable labels, such as Biotin or a fluorescent dye. Complex NHS ester label can be requested, allocate, clear, and/or describe, or you can get thein situand be subjected to the reaction with the nucleophilic group of an antibody. Typically, the carboxyl form of the label is activated by reaction with a specific combination of a carbodiimide reagent such as dicyclohexylcarbodiimide, diisopropylcarbodiimide or Orangevale reagent, e.g. TSTU (tetrafluoroborate O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea), HBTU (hexaphosphate O-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea) or HATU (hexaphosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea); activate the RA, such as 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide with getting complicated NHS ester label. In some cases, the label and the antibody can be linked through the activation and reaction of the label with the antibodyin situwith the formation of the conjugate label-antibody in a single phase.

Other activating and linking reagents include TBTU (hexaphosphate 2-(1H-benzotriazol-1-yl)-1-1,3,3-tetramethylene), TFFH (2-fluoro-hexaphosphate N,N',N",N"'-tetramethylurea), PyBOP (hexaphosphate benzotriazol-1-yl-oxy-Tris-pyrrolidinone, EEDQ (2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole and arylsulfonate, for example, triisopropylbenzenesulfonyl.

Connection albuminaemia peptide-Fab according to the invention:

In one aspect, the antibody according to the invention merged with aluminiuim protein. Binding to plasma proteins may represent an effective method for improving the pharmacokinetic properties of short-lived molecules. Albumin is the most represented protein in plasma. Albuminaemia peptides (ABP)that binds serum albumin, can alter the pharmacodynamics fused proteins with active domains, including changes to capture, penetration and diffusion in tissues. Editamaciejewska parameters can be changed through specific choice of an appropriate sequence binds serum albumin peptide (U.S. patent 20040001827). A number albuminaemia peptides identified by screening by phage display (Dennis et al., (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J. Biol. Chem. 277: 35035-35043; WO 01/45746). Compounds according to the invention include ABP sequences specified in (i) Dennis et al., (2002) J. Biol. Chem. 277: 35035-35043 in tables III and IV, page 35038; (ii) U.S. patent 20040001827 in [0076] SEQ ID nos: 9-22 and (iii) WO 01/45746 on pages 12-13, all of which are incorporated herein by reference in full. Albuminaemia (ABP)-Fab design by merging albuminaemia peptide from the C-end of the heavy chain Fab in the stoichiometric ratio 1:1 (1 ABP/1 Fab). It is shown that the Association of these ABP-Fab with albumin increased the half-life of antibodies in rabbits and mice more than 25 times. Thus, the above-described reactive Cys residues can be introduced in these ABP-Fab and used for site-specific conjugation with a cytotoxic drugs with subsequent studies on animalsin vivo.

Illustrative sequence albuminaemia peptides include as non-limiting examples of amino acid sequences listed in SEQ ID NO: 42-46:

CDKTHTGGGSQRLMEDICLPRWGCLWEDDFSEQ ID NO: 42
QRLMEDICLPRWGCLWEDDF SEQ ID NO: 43
QRLIEDICLPRWGCLWEDDFSEQ ID NO: 44
RLIEDICLPRWGCLWEDDSEQ ID NO: 45
DICLPRWGCLWSEQ ID NO: 46

Conjugates of the antibody-drug

In another aspect the invention relates to immunoconjugates or conjugates of the antibody-drug (ADC)containing antibody conjugated with a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e radioconjugates). In another aspect the invention additionally relates to methods of applying immunoconjugates. In one aspect of immunoconjugate contains any of the above antibodies to CD22, covalently attached to a cytotoxic agent or detectivemisa tool.

The use of conjugates of the antibody-drug for local delivery of cytotoxic or cytostatic funds, i.e. drugs for destruction or inhibition of tumor cells in the treatment of malignant tumors (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculecu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. patent 4975278), provides targeted delivery of a molecule drugs to tumors, and intracellular accumulation where systemic administration of these unconjugated drug, in addition to tumor cells that need to be removed, may result in unacceptable levels of toxicity to normal cells (Baldwin et al., (1986) Lancet pp.(Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.s), pp.475-506). Thus, it is desirable maximum efficacy with minimal toxicity. It was reported that these methods are suitable and polyclonal antibodies, and monoclonal antibodies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986), supra). Toxins used in the conjugates of the antibody-toxin include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins, such as geldanamycin (Mandler et al., (2000) Jour. 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) and calicheamicin (Lode et al., (1998) Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342). Toxins can manifest their citato the classical and cytotoxic effects through mechanisms, including tubulin binding, DNA binding or inhibition of topoisomerase. Some cytotoxic drugs, as a rule, are inactivated or become less active during conjugation with large antibodies or ligands of receptor proteins.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioactive isotope, consisting of monoclonal antibodies IgG1 Kappa mouse directed to CD20 antigen found on the surface of normal and malignant B-lymphocytes, and radioactive isotope111In or90Y associated timesaving linker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al., (2002) Blood 99(12): 4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J. Clin. Oncol. 20(15): 3262-69). Although the zevalin has activity against B-cell non-Hodgkin lymphoma (NHL), the introduction leads to a severe and prolonged cytopenias in most patients. In 2000, for the treatment of acute myeloid leukemia by injection approved MILOTARGTM(gemtuzumab ozogamicin, Wyeth Pharmaceuticals), conjugate antibody-drug composed of antibodies to huCD33 associated with calicheamicin (Drugs of the Future(2000) 25(7):686; U.S. patents№4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Phase II trials for the treatment of malignant tumors that Express CanAg, such as a malignant tumor of the Tolstoy of the intestine, pancreas, stomach and other passes cantuzumab mertansine (Immunogen, Inc.), conjugate antibody-drug composed of the huC242 antibody linked via the disulfide linker SPP to the molecule maytansinoids medicines DM1. In development for the potential treatment of prostate tumors is MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), conjugate antibody-drug composed of monoclonal antibodies to specific membrane antigen prostate (PSMA)associated with the molecule maytansinoids medicines DM1. Auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin, conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cACIO (specific to CD30 in hematological malignancies) (Doronina et al., (2003) Nature Biotechnology 21(7): 778-784) and are under therapeutic development.

This document describes a chemotherapeutic drug, suitable for immunoconjugates. Enzymatically active toxins and fragments thereof that can be used include the A chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin a chain, exotoxin A (fromPseudomonas aeruginosa), the A chain of ricin chain abrina, A chain of medecine, alpha sarcin, Bel and Aleurites fordii, diantimony proteins, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), inhibitor ofMomordica charantiaCurtin, krotin, the inhibitor ofSapaonaria officinalis, gelonin, Michelin, restrictocin, vanomycin, inomycin and trichothecenes. See, for example, WO 93/21232, published 28 October 1993. To obtain radioconjugates antibodies have a wide range of radionuclides. Examples include212Bi131I131In90Y and186Re. Conjugates of the antibody with a cytotoxic agent receive, by using a variety of bifunctional binding proteins funds, 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-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as the toluene-2,6-diisocyanate), and bis-compounds active fluorine (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., (1987) Science, 238:1098. Illustrative chelating agent for conjugation of the radionuclide to the antibody is labeled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) (WO94/11026).

Also in this document consider the conjugates of the antibody and one or more low molecular weight toxins such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecin and CC1065, and the derivatives of these toxins that have toxic activity.

Mitanin and maytansinoid

In some embodiments, the implementation immunoconjugate contains antibody (full-length or fragments) of the invention conjugated to one or more molecules of maytansinoids.

Maytansinoid are inhibitors of mitosis, which act by inhibiting the polymerization of tubulin. Maytansine was first isolated from the East African shrub,Maytenus serrata(U.S. patent No. 3896111). Subsequently discovered that certain microorganisms also produce maytansinoid, such as maytansine and esters maytansine C-3 (U.S. patent No. 4151042). Also described synthetic maytansines and its derivatives and analogues, for example, in U.S. patents№4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533.

Molecules maytansinoids medicines are attractive molecules in the conjugates of the antibody-drug because they are (i) relatively affordable to get through fermentation or hemicellulolytic, derivatization products of fermentation, (ii) amenable to derivatization of functional groups suitable for conjugation through nevalidni linkers to the antibody, (iii) stable in plasma, and (iv) effective against a number of lines of tumor cells.

Maytansine compounds suitable for use as molecules maytansinoid drugs, well known in this field, and they can be isolated from natural sources by known methods, to obtain by means of genetic engineering (see Yu et al., (2002) PNAS 99: 7968-7973), or maytansines and analogues maytansine known methods are synthetically.

Illustrative molecules maytansinoids medicines include molecules with modified aromatic ring, such as C-19-dechloro (U.S. patent 4256746) (obtained through the recovery of ansamitocins P2 hydride lithium aluminum); C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (U.S. patent No. 4361650 and 4307016) (obtained by demethylation usingStreptomycesorActinomycesor dechlorination using LAH); and C-20-dimethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. patent No. 4294757) (obtained by acylation using acylchlorides) and molecules with modifications in other provisions.

Illustrative molecules maytansinoids medicines also include molecules which such modifications as C-9-SH (U.S. patent 4424219) (obtained by reaction maytansine with H 2S or P2S5); C-14-alkoxymethyl(dimetoxy/CH2OR) (US patent 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. patent 4450254) (derived fromNocardia); C-15-hydroxy/acyloxy (U.S. patent 4364866) (obtained through the conversion maytansineStreptomyces); C-15-methoxy (U.S. patent No. 4313946 and 4315929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. patent No. 4362663 and 4322348) (obtained by demethylation maytansineStreptomyces) and 4,5-deoxy (U.S. patent 4371533) (obtained through the recovery maytansine trichloride titanium/LAH).

Illustrative embodiments of the molecules maytansinoids medicines include DM1; DM3 and DM4, with the following structures:

where the wavy line indicates the covalent joining of the sulfur atom of the medicinal product to the linker (L) conjugate antibody-drug. It was reported HERCEPTIN® (Herceptin®trastuzumab, an antibody to HER2), linked via SMCC with DM1 (WO 2005/037992, which fully incorporated herein by reference). Conjugate antibody-drug of the present invention can be obtained is described in her ways.

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

Illustrative conjugates antibody-drug where DM1 is connected through a linker BMPEO with Tilney group of antibodies, have the following structure and designation:

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

Immunoconjugate containing maytansinoid, methods for their preparation and their therapeutic use are described, for example, in U.S. patent No. 5208020; 5416064; 6441163 and European patent EP 0425235 B1, descriptions of which are incorporated herein by reference in full. In Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) described immunoconjugate containing maytansinoid designated DM1 associated with the monoclonal antibody C242 directed against colorectal cancer. It is revealed that the conjugate of vysokoletuchie for cultivated cells of large intestine cancer, and shows antitumor activity in the analysis of tumor growthin vivo. In Chad et al., Cancer Research 52: 127-131 (1992) described immunoconjugate in which maytansinoid via a disulfide linker anywhereman with the murine antibody A7 binding to an antigen cell lines cancer of the large intestine of man, or with another monoclonal antibody mouse TA.1 that binds to the oncogene HER-/neu. The cytotoxicity of the conjugate TA.1-maytansinoid testedin vitroin cell lines breast cancer human SK-BR-3 expressing 3×105surface antigens HER-2 on the cell. Drug conjugate has reached the degree of cytotoxicity, similar to the degree of cytotoxicity of free maytansinoid medicines, which can be increased by increasing the number of maytansinoid molecules per molecule of antibody. Conjugate A7-maytansinoid demonstrated low systemic cytotoxicity in mice.

Conjugates of the antibody to CD22-maytansinoid get through chemical binding of an antibody to a molecule maytansinoid without a significant reduction in the biological activity of the antibodies or molecules maytansinoid. See, for example, U.S. patent No. 5208020 (the description of which is thus included in the present description by reference in full). 3-4 molecules maytansinoid conjugated to one molecule of antibody, has demonstrated efficacy in enhancing cytotoxicity to target cells without negative impacts on the feature or the solubility of the antibody, although it can be expected that even one molecule of toxin/antibody increases cytotoxicity compared with the use of a single antibody. Maytansinoid well known in this field and we can Sintesi the work by known methods or to isolate from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 and other patent and non-patent publications referenced earlier in this document. Preferred maytansinoids are maytansines and analogues maytansine modified aromatic ring or other provisions of molecules maytansine, such as various esters maytansine.

There are many known in the field of the linker groups to obtain conjugates antibody-maytansinoid, including, for example, the linker group as described in U.S. patent No. 5208020, 6441163 or European patent 0425235 B1, Chari et al., Cancer Research 52: 127-131 (1992) and U.S. patent 2005/0169933 A1, descriptions of which are incorporated herein by reference in full. Conjugates of the antibody-maytansinoid containing linker component SMCC, can be obtained as described in patent application U.S. No. 11/141344, registered on may 31, 2005, "Antibody Drug Conjugates and Methods". Linker groups include disulfide groups, thioester groups, kislotolabilen group, photolabile group, peptidylarginine group or estradiollevelny group, as described in the above patents. Additional linker groups described and illustrated in this document.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bi is unctionally binding proteins funds 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-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-compounds active fluorine (such as 1,5-debtor-2,4-dinitrobenzene). Especially preferred connecting means include N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978)) and N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP) for the formation of disulfide bonds.

The linker can be attached to the molecule maytansinoid in different positions depending on the type of communication. For example, the ester bond can be formed by reaction with a hydroxyl group using traditional methods of binding. The reaction can be carried out at position C-3, in which the hydroxyl group at position C-14, modified with hydroxymethyl, C-15, modified hydroxyl group, and at position C-20, which is a hydroxyl group. In preferably the embodiment, the connection form on position C-3 maytansine or similar maytansine.

In one embodiment, the implementation of any antibody according to the invention (full-length or fragment) conjugated with one or more molecules of maytansinoids. In one of the embodiments immunoconjugate cytotoxic agent D is maytansinoid DM1. In one of the embodiments immunoconjugate the linker is a SMCC. In one embodiment, the implementation of the conjugate of the antibody-linker-drug is an antibody to CD22, as described herein, to which the SMCC linker covalently attached to a cytotoxic agent DM1.

Auristatin and dolastatin

In some embodiments, the implementation immunoconjugate contains the antibody of the invention conjugated to dolastatins or peptide analogues and derivatives of dolastatin, auristatin (U.S. patent No. 5635483; 5780588). It is shown that dolastatin and auristatin interfere with the activity of microtubules, the hydrolysis of GTP and division of nuclei and cells (Woyke et al., (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have antitumor (U.S. patent 5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965). Molecule drug dolastatin or auristatin can be linked to the antibody via the N-end (amino) or C-end (carboxyl) peptide group of medicines (O 02/088172).

Illustrative embodiments of auristatin include attached at the N end of a group of medicines monomethylaniline DE and DF, 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 cited as a reference in full.

Illustrative variant implementation of auristatin represents MMAE (where the wavy line indicates the covalent bond with the linker (L) conjugate antibody-drug).

Another illustrative variant implementation of auristatin is a MMAF, where the wavy line indicates the covalent bond with the linker (L) conjugate antibody-drug (U.S. patent 2005/0238649):

Additional illustrative embodiments of containing MMAE or MMAF and various linker components (described later in this document) have the following structures and abbreviations (where Ab indicates antibody, and p is a number from 1 to about 8):

Typically, the peptide groups of medicines can be obtained by forming a peptide bond between two or more amino acids and/or PE is tinyme fragments. Such peptide bonds can be obtained, for example, by the method of synthesis in the liquid phase (see E. Schröder and K. Lübke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press)that is well known in the field of peptide chemistry. Auristatin/dolastatin group of medicines can be obtained by the methods of U.S. patent 5635483; U.S. patent 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.

Calicheamicin

In other embodiments, implementation immunoconjugate contains the antibody of the invention conjugated to one or more molecules calicheamicin. Calicheamicin family of antibiotics are capable of forming double-stranded breaks in DNA in subpicomolar concentrations. To obtain conjugates calicheamicin family, see U.S. patents№5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all issued by American Cyanamid Company). Structural analogues calicheamicin that can be used include as non-limiting examples γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θ1I(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 ant the body, is QFA, representing antifolate. And calicheamicin, and QFA contain intracellular sites of action and have difficulty passing through the plasma membrane. Thus, the cell capture these funds due mediated antibody internalization significantly enhances their effect.

Other cytotoxic funds

Other antitumor agents that can be konjugierte with antibodies according to the invention include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of the tools, collectively known as the complex LL-E33288 described in U.S. patent 5053394, 5770710 and espiramicina (U.S. patent 5877296).

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

The present invention additionally relates to immunoconjugate formed by the antibody and the connection with nucleotidase activity (in the example, a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Ncasa).

For selective tumor destruction antibody may contain highly radioactive atom. To obtain radioconjugates antibodies there are many radioactive isotopes. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. When the conjugate is used for detection, it may contain 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 again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be entered in the conjugate by known methods. For example, the peptide can be biosynthetically or to synthesize by chemical amino acid synthesis using suitable amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as tc99mor I123That Re186That Re188and In111you can attach via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. For the introduction of iodine-mon to use the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57). In "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) described other ways.

Conjugates of the antibody and cytotoxic funds can be received with use of different binding proteins funds, 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-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine)derived bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-compounds active fluorine (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al. Science, 238:1098 (1987). Labeled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an illustrative chelating agent for conjugation of radionucleotide with the antibody. Cm. WO 94/11026. The linker can be a "biodegradable linker"facilitating release of the cytotoxic drug in the cell. For example, you can use colorability linker, pertinaciously linker, a photo lab the local linker, dimethyl linker or containing a disulfide linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. patent No. 5208020).

Compounds according to the invention include as non-limiting examples of the ADC obtained with cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (e.g., Pierce Biotechnology, Inc., Rockford, IL., U.S.A). Cm. pages 467-498, 2003-2004 Applications Handbook and Catalog.

Obtaining conjugates antibody-drug:

In the conjugates of the antibody-drug (ADC) according to the invention, the antibody (Ab) conjugated with one or more molecules of the drug (D), for example, molecules of the drug in amounts of from approximately 1 to approximately 20 per antibody, through a linker (L). The ADC of formula I can be obtained in several ways using reactions of organic chemistry, conditions, and reagents known to specialists in this area, including (1) the reaction of nucleophilic molecule antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a molecule drugs D; and (2) reaction of a nucleophilic group of the molecule drugs with bivalent the m of the linker reagent with the formation of D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. This document describes additional methods of obtaining the ADC.

Ab-(L-D)pThe formula I

The linker may consist of one or more linker components. Illustrative of the linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe), p-aminobenzeneboronic ("PAB"), N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC") and N-Succinimidyl(4-iodates)aminobenzoate ("Fairs are forthcoming-Siab"). For more linker components known in the field, and some are described in this document.

In some embodiments, the implementation of the linker may contain amino acid residues. Illustrative amino acid linker components include dipeptide, Tripeptide, tetrapeptide or 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 contained in the amino acid linker component, include the remains, found in nature, as well as minor amino acids and newstrategies in nature analogues Amin is acid, such as citrulline. Amino acid linker components 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 the protease plasmin.

Illustrative structure of the linker components is presented below (where the wavy line indicates the sites of covalent joining to other components of the ADC):

Additional illustrative linker components and reduction include (where the antibody (Ab) and the linker is represented, and p is a number from 1 to about 8):

The nucleophilic group of an antibody include as non-limiting examples of (i) the N-terminal amino group, (ii) amino side chain, for example, lysine, (iii) tirinya group of the side chain, for example cysteine, and (iv) hydroxyl or amino sugars, when the antibody is glycosylated. Amino group, tirinya and hydroxyl groups are nucleophilic and capable of reacting with the formation of covalent bonds with electrophilic groups on linker molecules and linker reagents including : (i) active esters such as NHS esters, esters, HOBt, halogenfree and loginengine; (ii) alkylhalogenide and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group. Certain antibodies contain recoverable megamachine disulfides, i.e. cysteine bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with reducing agent such as DTT (dithiothreitol). Thus, each cysteine bridge, theoretically, will form two reactive thiol of the nucleophile. Additional nucleophilic groups can be entered into the antibody by reaction of lysine with a 2-aminothiophenol (reagent Troth), which leads to the transformation of the amine in the thiol. Reactive tirinya group can be introduced into the antibody (or fragment) by introducing one, two, three, four or more cysteine residues (e.g., receiving mutant antibodies containing one or more non-natural amino acid cysteine residues).

Conjugates of the antibody-drug according to the invention can also be obtained by modifying antibodies with the introduction of electrophilic groups, which can react with nucleophilic substituents on the linker reagent or drug. Sugar glycosylated antibodies can oxidize, for example, using controllable periodic destruction oxidizing is agentov with the formation of aldehyde or ketone groups, which can react with amino groups of the linker reagents or molecules of the drug. Received group eminovic Schiff bases can form a stable connection or they can be recovered, for example, borhydride reagents with the formation of a stable amine linkages. In one embodiment, the implementation of the reaction of the carbohydrate portion of a glycosylated antibody with galactose oxidase or metaperiodate sodium can lead to the formation of protein carbonyl groups (aldehyde and ketone), which can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal residues of serine or threonine, can react with metaperiodate sodium, resulting in obtaining aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). Such aldehydes can react with a molecule drugs or nucleophile linker.

Similarly, the nucleophilic groups on the molecule drugs include as non-limiting examples of amino groups, tirinya, hydroxyl, hydrazide, Aksinya, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines groups capable of reaction with the formation of covalent bonds with electrophilic groups nalinkarn molecules and linker reagents including (i) active esters such as NHS esters, esters, HOBt, halogenfree and halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group.

In yet another aspect, the antibody contains one or more lysine residues that can be chemically modified by introduction of one or more sulfhydryl groups. Or antibody-based test portion of the molecule binds to the linker part of the molecule via a sulfur atom sulfhydryl groups. Reagents that can be used for modification of lysine include as non-limiting examples of N-Succinimidyl S-acetylthiourea (SATA) hydrochloride and 2-aminosilane (reagent Troth).

In another embodiment, the antibody may contain one or more carbohydrate groups that can be chemically modified so that they contain one or more sulfhydryl groups. Or antibody-based test portion of the molecule binds to the linker part of the molecule, such as an element Stretcher, through the sulfur atom of sulfhydryl groups, as described in this document.

In another embodiment, the antibody may contain one or more carbohydrate groups that can oxidize with obtaining aldehyde (-CHO) group (see, for example, Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). Relevant to the respective aldehyde can form a bond with the reactive center on the Stretcher element. Reactive centers on the Stretcher element, which can react with the carbonyl group on the antibody include as non-limiting examples of the hydrazine and hydroxylamine. Other protocols for modification of proteins by accession or Association of the elements of the medicines described in Coligan et al., Current Protocols in Protein Science, Vol.2, John Wiley & Sons (2002), incorporated herein by reference in full.

Methods of conjugation of linker molecules is a medicinal product with leading to cell proteins, such as antibodies, immunoglobulins or fragments thereof are, for example, in US5208020; US6441163; WO 2005037992; WO 2005081711 and WO 2006/034488 that, therefore, all included in the present description by reference in full.

Alternatively, you can get protein containing the antibody and cytotoxic agent, for example, by recombinant methods or peptide synthesis. A segment of DNA may contain the corresponding region encoding the two parts of the conjugate adjacent to each other or separated by a region that encodes a linker peptide which does not violate the desired properties of the conjugate.

In another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for utilization in the pre-targeting, where the conjugate of the antibody-receptor is administered to a patient with subsequent removal is receiving the unbound conjugate from the circulation using a cleanser, and then enter a "ligand" (e.g. avidin)which anywhereman with a cytotoxic agent (e.g., radionucleotide).

In one of the embodiments immunoconjugate cytotoxic agent, D, is auristatin formula DEor DF

DF

and where each of R2and R6represents methyl, each of R3and R4represents isopropyl, R7represents sec-butyl, each R8independently selected from CH3, O-CH3, OH, and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8-alkyl, or -(CH2)2-O-(CH2)2-O-(CH2)2-O-CH3; and R18represents-C(R8)2-C(R8)2-aryl; and

(d) p is in the range from approximately 1 to 8.

Below, embodiments of belong to any of the above immunoconjugates. In one of the embodiments immunoconjugate has activity against destruction of cellsin vitroorin vivo. In one of the embodiments the linker is attached to the antibody through Tilney group on the antibody. In one of the embodiments the linker is susceptible Rasse is of the protease. In one of the embodiments, the linker contains the dipeptide val-cit. In one of the embodiments, the linker contains a p-aminobenzyl group. In one embodiment, the implementation of p-aminoaniline group in the linker is located between the medicinal product and site of protease cleavage. In one embodiment, the implementation of p-aminoaniline group is a p-aminobenzeneboronic (PAB). In one of the embodiments, the linker contains 6-maleimidomethyl. In one embodiment, the implementation of the 6-maleimidomethyl in the linker is located between the antibody and the cleavage site of the protease. The above options exercise can be done one by one or in any combination with each other.

In one embodiment, the implementation of a drug selected from the MMAE and MMAF. In one of the embodiments immunoconjugate has the formula

where Ab represents any of the above antibodies to CD22, S represents a sulfur atom, and p is in the range from 2 to 5. In one of the embodiments immunoconjugate has the formula

where Ab represents any of the above antibodies to CD22, S represents a sulfur atom, and p is in the range from about 1 to about 6, from about 2 to AP is sustained fashion 5, from about 2 to about 6, from about 2 to about 4, from about 2 to about 3, from about 3 to about 4, from about 3 to about 5, from about 3 to about 6, or from about 4 to about 6.

Visualization techniques with labeled antibody:

In another embodiment of the invention modified with cysteine antibodies can be marked by tylnej groups of cysteine radionuclides, fluorescent dyes, runs bioluminescence molecules of the substrate, triggering the chemiluminescence of the substrate molecules, enzymes and other detectivesyme labels for experiments on visualization for diagnostic, pharmacodynamic and therapeutic applications. Usually labeled modified cysteine antibody, i.e. a "biomarker" or "probe", is administered by injection, perfusion or oral administration to a living organism, e.g. human, rodent or other small animal, perfusely organ or a tissue sample. Over time detects the distribution of the probe and are in the form of an image.

Industrial products:

In another embodiment of the invention provided an industrial product or a "kit"containing substances suitable for the treatment of the Oia described above diseases. Industrial product includes a container and a label or an insert in the packaging container or relating to him. Suitable containers include, for example, bottles, vials, syringes, blister packaging, etc. Containers can be obtained from a variety of materials, such as glass or plastic. The container contains a composition conjugate antibody-drug (ADC), which is effective for the treatment of pathological conditions, and may have a sterile inlet (for example, the container may be a package for intravenous solution or a bottle with a stopper that can be puncturing needle for subcutaneous injection). At least one active agent in the composition is an ADC. In the label or the liner in the packaging indicates that the composition is used to treat the selected state, such as a malignant tumor. Alternative or additional industrial product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-saline buffer, ringer's solution and dextrose. Additionally it may include other materials desirable from a commercial or user standpoint, including other buffers, diluents, filters, needles and syringes.

Farmaceuticas is their composition :

In one aspect is provided a pharmaceutical composition comprising any of the above immunoconjugates and a pharmaceutically acceptable carrier. In one aspect provides a method of treatment of B-cell proliferative disorders, where the method includes the administration to an individual a pharmaceutical composition. In one embodiment, the implementation of B-cell proliferative violation is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell. In one embodiment, the implementation of cellular proliferative violation associated with increased expression of CD22 on the surface of the cell.

In one aspect provides a method of inhibiting cell proliferation, where the method includes the effects on the cell by any of the above immunoconjugates under conditions that allow binding immunoconjugate with CD22. In one embodiment, the implementation of the B-cell is a tumor cell. In one of the embodiments the tumor cell is a B-cell of a mammal, with the presence or suspicion of the presence of B-cell proliferative disorders, selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell, the cell is a xenograft. In one embodiment, the implementation of the action takes placein vitro. In one embodiment, the implementation of the action takes placein vivo.

In one aspect provides a method of applying the antibody to CD22 according to the invention for analysis of serum soluble CD22 in mammals with leukemia or lymphoma for the diagnosis of B-cell leukemia or B-cell lymphoma, to monitor clinical progress or regression of the disease or to assess tumor mass or relapse. Such methods are described in U.S. patent 20050244828 (Kreitman, R.J. et al., the content is, therefore, incorporated by reference in full), which used a conjugate of antibody RFB4 to CD22 and PE38 toxin (fragment 38 exotoxin APseudomonas(see Kreitman, R.J. et al., NEJM 345: 241-247 (2001)).

BRIEF DESCRIPTION of FIGURES

Figa-1D: figa is a diagram CD22, showing seven immunoglobulin-like domains of the extracellular domain of the beta isoform. In the alpha from the Orme no domains 3 and 4. "TM" refers to the transmembrane domain. On FIGU depicts the amino acid sequence of the beta-form of CD22 (SEQ ID NO: 27). In the alpha form CD22 missing amino acids are shown in italics (encoding the domains 3 and 4 of the extracellular domain). The extracellular domain of the Mature form of the protein is underlined (SEQ ID NO: 28). Amino acids 1-21 represent a signal sequence, tsepliaeva from Mature forms. Figs represents the amino acid sequence of CD22-alpha (SEQ ID NO: 29). CD22 ECD-alpha is underlined (SEQ ID NO: 30). Figure 1D represents the amino acid sequence of CD22 cynomolgus monkey (cyno) (SEQ ID NO: 31). The first 19 amino acids CD22 cyno represent a signal sequence.

Figa-2B: figa represents the amino acid sequence of the variable region of the heavy chain of the antibody 10F4 to CD22 mouse according to the invention (m10F4), aligned with humanized version 1 antibody 10F4 (h10F4v1) and aligned with the sequence of the human subgroup III. HVR boxed (HVR-H1, HVR-H2, HVR-H3). Sequence covering HVR represent a frame sequence (from FR-H1 to FR-H4). Sequences are numbered according to the Kabat numbering. CDR by Kabat, Chothia and contact CDR indicated on the prisoners in the frame of the HVR. On FIGU shows the amino acid sequence of the variable region of the light chain of the antibody 10F4 the mouse CD22 according to the invention (m10F4), aligned with humanized version 1 antibody 10F4 (h10F4v1) and aligned with the sequence of human Kappa I. Version 2 and 3 gumanitarnogo antibody 10F4 (h10F4v2 and h10F4v3) Sekretareva Mature forms contain the same amino acid sequence. Antibodies h10F4v2 and h10F4v3 differ from h10F4v1 amino acid 28 HVR-L1 (N28V). HVR boxed. sequence FR-L1, FR-L2, FR-3 FR-L4 cover HVR (HVR-L1, HVR-L2, HVR-L3). Sequences are numbered according to the Kabat numbering. CDR by Kabat, Chothia and contact CDR indicated on the prisoners in the scope of the HVR.

On figa and 3B shows illustrative acceptor consensus framework sequence variable fragments of heavy chains of a person for use in implementing the present invention with sequence identifiers as listed below, where SEQ ID NO: FR are listed in the order FR-H1, FR-H2, FR-H3, FR-H4:

- consensus framework "A" VH subgroup I, the man without a CDR according to Kabat (SEQ ID NO: 26, 47, 48, 7);

- consensus frameworks "B", "C" and "D" VH subgroup I, the man without extended hypervariable regions (SEQ ID nos: 50, 51, 52, 7; SEQ ID NO: 50, 51, 52, 7, and SEQ ID NO: 50, 51, 53, 7);

- consensus framework "A" VH subgroup II man without CDR by Kabat (SEQ ID NO: 54, 55, 56, 7);

- consensus frameworks "B", "C" and "D" VH subgroup II man without extended hypervariable regions (SEQ ID nos: 57, 58, 56, 7 SEQ ID NO: 57, 58, 59, 7; and SEQ ID NO: 57, 58, 60, 7);

- to sensously frame "A" VH subgroup III person without CDR by Kabat (SEQ ID NO: 61, 62, 63, 7);

- consensus frameworks "B", "C" and "D" VH subgroup III person without extended hypervariable regions (SEQ ID nos: 64, 65, 63, 7; SEQ ID NO: 64, 65, 66, 7, and SEQ ID NO: 64, 65, 67, 7);

- frame "A" acceptor VH 1 person without CDR by Kabat (SEQ ID NO: 68, 62, 69, 7);

- frames "B" and "C" VH acceptor human without extended hypervariable regions (SEQ ID nos: 64, 65, 69, 7; and SEQ ID NO: 64, 65, 70, 7);

- frame "A" VH acceptor 2 people without CDR by Kabat (SEQ ID NO: 68, 62, 71, 7);

- frames "B", "C" and "D" VH acceptor 2 people without extended hypervariable regions (SEQ ID nos: 64, 65, 71, 7; SEQ ID NO: 64, 65, 72, 7, and SEQ ID NO: 64, 65, 73, 7).

On figa and 4B show illustrative acceptor consensus framework sequence of the variable fragments of the light chain (VL) of a person for use in implementing the present invention with sequence identifiers as follows:

- consensus framework (κv1-1) VL Kappa subgroup 1-1 man: SEQ ID NO: 74, 75, 76, 77;

- consensus framework (κv1) VL Kappa subgroup I of man: SEQ ID NO: 74, 78, 76, 77;

- consensus framework (κv2) VL Kappa subgroup II person: SEQ ID NO: 49, 79, 80, 77;

- consensus framework (κv3) VL Kappa subgroup III person: SEQ ID NO: 81, 82, 83, 77;

- consensus framework (κv4) VL Kappa subgroup IV person: SEQ ID NO: 84, 85, 86, 77.

Figa and 5B: figa given sequence alignment Fc-areas of natural sequences IgG brow the ESA humIgG1 (allotype not-A, SEQ ID NO: 38; and A allotype, where amino acid sequence SREEM in SEQ ID NO: 38 is replaced by SRDEL), humIgG2 (SEQ ID NO: 39), humIgG3 (SEQ ID NO: 40) and humIgG4 (SEQ ID NO: 41) with differences between the sequences are indicated by asterisks. Numbers above the sequences represent the numbering system of the EU. Also illustrative constant region Kappa. On FIGU presents full amino acid sequence (variable and constant region) of light and heavy chains gumanitarnogo antibodies to CD22 10F4v2, isotype IgG1. Underlined sections indicate constant domains.

On figa-6D shows the results of analyses on performance measurement ADC with CD22 in lympany cell lines. On figa shown that higher levels of CD22 on the cell surface correlates with a lower IC50anti-CD22-MCC-DM1 (high efficiency). On FIGU shown that increased the internalization of anti-CD22-MCC-DM1 correlates with a lower IC50anti-CD22-MCC-DM1. On figs shown that increased own the sensitivity of cells to the free drug correlates with a lower IC50anti-CD22-MCC-DM1. Fig.6D is a micrograph showing the internalization of fluorescently labeled antibodies to CD22 after binding to CD22 on the cell surface.

Figa-7B: figa is a graph showing the reduction op is Holi in vivoin the xenograft model which shows that administration of antibodies to CD22 mu10F4-smcc-DM1 and hu10F4v1-smcc-DM1 SCID mice transplanted with B-cell tumors of the person significantly reduces tumor volume. Load the medicinal product was approximately 4 and 4.6, see table 4. FIGU is a graph similar studies, but the load of the medicinal product was a bit smaller, around 2.9 and 3.0 (see table 5), and the effectiveness of mu10F4-smcc-DM1 and hu10F4v2-smcc-DM1 compared with control antibody and unconjugated mu10F4. Figs is a graph showing tumorin vivoin the xenograft model, where an anti-CD22-spp-DM1 was administered as shown in table 6.

Figa and 8B: figa is a graph for antibodies to CD22 5E8.1.8-smcc-DM1 and RFB4-smcc-DM1 entered in the xenografts of Ramos cells. FIGU is a graph for antibodies to CD22 5E8.1.8-smcc-DM1 and RFB4-smcc-DM1 entered in the xenografts BJAB-luc.

Fig.9 is a graph showing the relative impact on tumor volume depending on the time after the introduction of anti-CD22(RFB4)-smcc-DM1 at low, medium and high loads medicine.

Figure 10 is a graph showing the relative impact on tumor volume depending on the time after the introduction of anti-CD22(RFB4)-MC-vcPAB-MMAF or anti-CD22(RFB4)-MC-MMAF in Sinotrans entity Ramos.

11 is a graph that shows signs regulating the relative impact on tumor volume depending on the time after the introduction of anti-CD22(RFB4)-smcc-DM1 or MCvcPAB-MMAE.

Fig is a graph showing the relative impact on tumor volume depending on the time after injection of humanized variants of the anti-CD22 10F4 as immunoconjugates with MMAF or DM1, as described in table 12.

Figa-13C are graphs showing the relative impact on tumor volume depending on the time after the introduction of anti-CD22-smcc-DM1 or anti-CD22-MC-MMAF in various models xenografts B-cell lymphoma: SuDHL-4 (figa), DoHH2 (pigv) and Granta-519 (figs).

On Fig displays graphs domains CD22, remove the mapping of epitopes, as described in the examples. The domains are numbered 1-7. "TM" refers to the transmembrane domain.

On Fig given image modified with cysteine conjugates of the antibody to CD22-drug (ADC), where the molecule drugs attached to the resulting design cysteine group in the light chain (LC-ADC); the heavy chain (HC-ADC) and Fc region (Fc-ADC).

On Fig shows stages: (i) the restoration of the disulfide adducts of cysteine and miaocheng and intrachain disulfides in a modified cysteine antibody to CD22 (Tioman) in what stenopetalum TCEP (Tris hydrochloride-(2-carboxyethyl)phosphine); (ii) partial oxidation, i.e. re-oxidation to restore miaocheng and intrachain disulfides dhAA (dehydroascorbic acid); and (iii) conjugation re-oxidized antibodies with intermediate connection of a drug-linker to form a conjugate modified cysteine antibody to CD22-drug (ADC).

On figa-17C shows the amino acid sequence modified by cysteine antibodies to CD22 according to the invention, in which the light chain or heavy chain, or an Fc region modified with the introduction of cysteine in selected positions of amino acids. On figa shows the amino acid sequence of the light chain variant antibodies to CD22 10F4, in which valine at Kabat position 205 (position in the sequence - valine 210) is replaced by cysteine. On FIGU shows the amino acid sequence of the heavy chain variants of antibodies to CD22 10F4, in which the alanine in position on EU 118 (position in the sequence - alanine 121) is replaced by cysteine. On figs shows the amino acid sequence of the Fc region variant antibodies to CD22 10F4, in which the serine in position on EU 400 (position in the sequence - serine 403) replaced by cysteine. Each piece has a modified amino acid is shown in bold double underline. Underline single line means intercept ntie area. Variable regions are not underlined.

Figa-18E are diagrams FACS, demonstrating that binding of conjugates Tioman to CD22 and medicines (TDC) according to the invention, binding to CD22 expressed on the surface of BJAB cells-lucs, which is similar variants Tioman LC, HC and Fc Tioman, as well as conjugates with a variety of given drugs.

Fig is a graph which shows the change in mean tumor volume over time in the model on the xenograft treated with various TDC to CD22, distinguished by the position of the resulting design cysteine (LC, HC or Fc) and/or conjugation with drug (MMAF or MMAE). The models in the xenografts treated with TDC, antibody to CD22 10F4-LC-V210C-MCvcPAB-MMAE and antibody to CD22 10F4-HC-A121C-MCvcPAB-MMAE, shows a decrease in tumor volume during the study.

Figa is a graph which shows the change in mean tumor volume over time in xenograft lymphoma mantle cell Granta-519 human mice SCID CB17-treated heavy chain A118C TDC to CD22, conjugated with various molecules linker-drug and/or injected at various doses, as shown. Apparently, TDC antibody to CD22 10F4-HC(A118C)-MCvcPAB-MMAE is the most effective of the rotestirovana in this experiment means. FIGU is a graph which shows the change in mean tumor volume over time in xenograft follicular lymphoma DOHH2 mice SCID CB17 treated the same heavy chain A118C TDC to CD22, but in large doses. Apparently, TDC antibody to CD22 10F4-HC(A118C)-MCvcPAB-MMAE is the most effective of those tested in this experiment means. Figs is a graph showing the percentage change in mass in mice from the study with DOHH2 xenografts, demonstrating the absence of significant changes in weight during the first 14 days of the study.

Figa and 21B are bar charts showing changes in serum AST (aspartate aminotransferase) (figa) and serum neutrophils (pigv) on day 0 and 5, where he introduced the ADC containing fissionable and necessasary the linker.

Figa and 22B are graphs showing the reduction in the number of peripheral B-cells (CD20 cells+) in cynomolgus macaques, which were dosed out 10, 20 and 30 mg/kg of antibody to CD22 with MMAF (figa) and antibodies to CD22 with DM1 (pigv).

Figa and 23B are graphs showing the absence of significant changes in CD4+lymphocytes at 10, 20 and 30 mg/kg of antibody to CD22 with MMAF (figa) and antibodies to CD22 with DM1 (pigv).

On figa and 24B shows histological tissue samples almond-shaped glands Javanese Mac is to, where in the sample the almond-shaped gland of the animal, which was administered a dose of 10 mg/kg hu10F4v3-SMCC-DM1, decreases the number of B cells in germinal centers that are visible when the dosing control in the form of media (figa).

Figa is a chart showing the area of the follicles of the spleen, from which to take a sample of tissue for research, which shows that the ADC to CD22 in cynomolgus macaques removes B cells from stationary tissue. Cell division in its germinative center of the follicle of spleen cyno in dividing its germinative centers spleens cyno in animals that were injected dose of 10 mg/kg hu10F4v3-MC-MMAF, decreased (figv and 25C). Dividing naive B-cells in the same conditions was reduced (fig.25D and 25E).

DETAILED DESCRIPTION of embodiments of the INVENTION

Provided the selected antibodies that bind to CD22. Additionally provided immunoconjugate containing antibodies to CD22. Additionally provided are modified by cysteine antibodies to CD22 and their immunoconjugate. Antibodies and immunoconjugates according to the invention are suitable, for example, to diagnose or treat disorders associated with altered expression of, for example, increased expression of CD22. In certain embodiments of the implementation of the antibodies or immunoconjugates according to the invention is suitable for diagnosis or treatment of cell proliferative disorders is Oia, such as a tumor or a malignant tumor. In certain embodiments of the implementation of the antibodies or immunoconjugates according to the invention is suitable for detection CD22, for example, CD22 expressed on the cell surface.

Granted polynucleotide encoding antibodies to CD22. Given the vectors containing polynucleotide encoding antibodies to CD22, and provided cell host containing such vectors. Also provides compositions, including pharmaceutical preparations containing any one or more of polynucleotides, antibodies to CD22 or immunoconjugates according to the invention.

General procedures

The methods and procedures described or referred to in this document, as a rule, are widespread and are typically used by specialists in the field using traditional methodologies, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd, edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M.Ausubel, et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): Pcr 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I.Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J.Gait, ed., 1984): Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I.Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; ell and Tissue Culture: Laboratory Procedures (A.Doyle, J.B.Griffiths, and D.G.Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos, eds., 1987); PCR: The Polymerase Chain Reaction. (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E.Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.Janeway and P.Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zanetti and J.D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T.DeVita et al., eds., J.B.Lippincott Company, 1993).

DEFINITIONS AND ABBREVIATIONS

Definitions

"Isolated" antibody is an antibody that is identified and separated from the components of its natural environment and/or isolated from them. Contaminant components of its natural environment are substances that can interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other protein and non-protein solutions. In some embodiments, the implementation of the antibody purified (1) to more than 95% by weight of antibody as determined by the method of Lowry, and in some embodiments, the implementation of more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence in the application of the sequencing machine is rotating glass or (3) to homogeneity with SDS-PAGE in non or reducing conditions using the dye Kumasi blue or silver. The selected antibody includes the antibody in recombinant cellsin situas there is at least one component of the natural environment antibodies. However, as a rule, the selected antibody receive through at least one stage of cleaning.

"Isolated" nucleic acid molecule is a nucleic acid molecule that is separated from at least one other nucleic acid molecule with which it is normally associated, for example, in its natural environment. The selected nucleic acid molecule further includes a nucleic acid molecule contained in cells that normally Express the nucleic acid molecule, but the molecule of nucleic acid is outside the chromosome or chromosome, but in a position different from its natural position in the chromosome.

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

As used herein, the term "substantially similar" or "essentially the same"means sufficiently high similarity IU what do two numeric values (for example, one associated with an antibody according to the invention and the other associated with a reference/compare antibody) so that the person skilled in the art may consider the difference between two values of low or no biological and/or statistical significance within the context of specified values measured biological characteristics (e.g., Kd values). The difference between these two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20% and/or less than about 10% depending on the reference/comparison value.

As used herein, the phrase "substantially reduced" or "substantially different" means a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparison molecule) so that the specialist in this area can be considered as the difference between the two values is statistically significant within the context of specified values measured biological characteristics (e.g., Kd values). The difference between these two values is, for example, greater than about 10%, greater than about 20%, more than AP is sustained fashion 30%, greater than about 40% and/or greater than about 50%, depending on the values for reference/comparison of the molecule.

As used herein, the term "vector"is intended to refer to the nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to the circular double-stranded DNA, which can be ligitamate additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be legirovanyh in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are administered (e.g., bacterial vectors with bacterial plot start replication and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host and, thus, be replicated together with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors herein referred to as "recombinant expressing vectors" or just "expressing vectors. As a rule, Express the dominant vectors, suitable methods of recombinant DNA, are often in the form of plasmids. In the present description "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

"Polynucleotide" or "nucleic acid", used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be embedded in a polymer of DNA or RNA polymerase or via synthetic reactions. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If present, the modified nucleotide can be performed before or after Assembly of the polymer. The nucleotide sequence may be interrupted dinucleotide components. Polynucleotide may contain the modification(s)performed after synthesis, such as the conjugation of the label. Other types of modifications include, for example, "kierowanie, the replacement of one or more naturally occurring nucleotides a similar, mezhnukleotidnyh modifications, such as modifications with uncharged linkages (e.g., methylphosphonate, fosfotriefirnym, phosphoamide, carbamates, etc) and with charged linkages (e.g., phosphorothioate, phosphorodithioate etc), modification containing side groups, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine and so on), modification intercalators (e.g., acridine, psoralen, etc.), modification, containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc), modification, containing alkylating tools, versions with modified linkages (e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotide(s). In addition, you can override any of the hydrophilic group, usually present in sugars, for example, phosphonate groups, phosphate groups, they may be protected by the standard protective groups or activated with additional links to additional nucleotides, and can be conjugated to a solid or semi-solid substrate. 5'- and 3'-terminal OH can be phosphorylated or substituted amines or organic kapinowski groups with the number of carbon atoms from 1 to 20. Other hydroxyl can also be derivatization standard protective groups. Polynucleotide can also contain a form, similar to the sugars ribose and deoxyribose, which, as a rule, is swesty in this area, including, for example, 2'-O-methyl, 2'-O-allyl-, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, α-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and basic nucleoside analogues, such as methylribose. One or more fosfolipidnyh relations can be replaced by alternative linking groups. These alternative linking groups include as non-limiting examples of 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(formatall"), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all communication polynucleotide must be identical. The preceding description applies to all polynucleotides mentioned in this document, including RNA and DNA.

As used herein, "oligonucleotide"typically refers to short, usually single-stranded, as a rule, synthetic polynucleotides, the length of which is typically, but not necessarily, be less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" the e are mutually exclusive. The above description for polynucleotides equally and fully applicable to oligonucleotides.

"Percent (%) amino acid sequence identity" with respect to the reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence of the candidate that are identical with amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing, if necessary, passes, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of determining the identity of the sequences. Alignment to determine percent identity of amino acid sequences can be of various well-known specialists in this field by, 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 sequence alignment, including any algorithms needed to achieve maximal alignment between the compared full-sized sequences. However, for the purposes of the present document value % identity am nekisnotnice sequences are obtained from the use of computer programs compare sequences ALIGN-2. A computer program comparing the sequences ALIGN-2 created at Genentech, Inc. and the source code is submitted with documentation to the user 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 in public Genentech, Inc., South San Francisco, California or may be compiled from the source code. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All parameters of sequence comparison are set by the program ALIGN-2 and do not change.

In those cases, when comparing amino acid sequences used ALIGN-2, % amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (which, alternatively, can be formulated as the fact that the given amino acid sequence A is or contains a certain % amino acid sequence identity with the amino acid sequence B) is calculated as follows:

100 multiplied by the ratio X/Y,

where X represents the number of amino acid residues, calculated by the program sequence alignment ALIGN-2 in that program's alignment of A and B as identical matches, and where Y represents the total number of amino acid on Titkov in B. It is clear that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all values % identity of amino acid sequences used herein are obtained as described in the immediately preceding paragraph using the computer program ALIGN-2.

"Surface marker on B-cells" or "surface antigen of B-cells" herein is an antigen expressed on the surface of B-cells, which can serve as a target for the antagonist, who is associated with him, including as non-limiting examples of antibodies to surface antigen of B-cells, or soluble form of the surface antigen of B-cells capable of countering the binding of the ligand with naturally occurring B-cell antigen. Illustrative surface markers of B-cells include surface markers 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 surface markers of B-cells include RP105, FcRH2, B-cell CR2, CR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, at btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287. Surface marker on B-cells of particular interest, preferably expressed on B-cells compared to other B-cell tissues of a mammal and can be expressed in the precursors of B cells and Mature B-cells.

As used herein, the term "CD22" refers to any natural CD22, which can be any vertebrate, including mammals such as primates (e.g. humans, cynomolgus macaques (cyno)) and rodents (e.g. mice and rats), unless otherwise specified. The term encompasses "full-size", reprezentirovanii CD22, as well as any form of CD22, which is the result of processing in the cell. The term also encompasses naturally occurring variants of CD22, for example, splicing variants, allelic variants and isoforms. The main isoform CD22 (CD22) contains 847 amino acids and seven immunoglobulin-like regions in the extracellular domain (see Wilson, G.L. et al., J. Exp. Med. 173: 137-146 (1991)). Minor isoform, CD22-alpha, contains 647 amino acids and in its extracellular domain lacking the immunoglobulin-like domains 3 and 4 (see Stamenkovic, I. and Seed, B., Nature 345: 74-77 (1990)and Wilson et al., (1991), above). Amino acid sequence CD22 shown in figv, where the underlined region represents the extracellular D. the men (ECD), and the area displayed in italics indicates amino acids that are missing in the sequence of the extracellular domain of CD22 alpha. On figs presents the amino acid sequence CD22 in which ECD is underlined. Amino acid sequence from amino acid 1 to amino acid 21 is a signal sequence, tsepliaeva from the Mature form of the protein. In one embodiment, the implementation of CD22 expressed on the cell surface, as on the surface of normal B-cells or neoplastic B-cells. On fig.1D presents the amino acid sequence CD22 cynomolgus macaque.

"Antibody" (Ab) and "immunoglobulin" (Ig) are glycoproteins with similar structural characteristics. While antibodies demonstrate specificity of binding to a specific antigen, immunoglobulin and include antibodies and other antibody molecules that are, as a rule, there is no antigenic specificity. The polypeptides of the latter type are formed, for example, at low levels the lymphatic system and elevated levels of myeloma.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent ant the body, multivalent antibodies are polyspecific antibodies (for example, bespecifically antibodies, provided that they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody may be chimeric, human, humanized and/or affinity Mature.

The term "antibody to CD22" or "antibody binding to CD22" refers to an antibody that is capable of binding to CD22 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent with targeted delivery to CD22. Preferably, the degree of binding of antibodies to CD22 to an unrelated protein, non-CD22, is less than approximately 10% of the binding of an antibody to CD22 as measured, for example, by radioimmunological assay (RIA). In certain embodiments of the implementation of the antibody, binding to CD22, has a dissociation constant (Kd) ≤1 ám ≤100 nm ≤10 nm, ≤1 nm or ≤0.1 nm. In certain embodiments of the implementation of the antibody to CD22 binds to the epitope of CD22, which is conservative in CD22 in different species.

"Variable region" or "variable domain" antibody refers to aminocentesis the domains of the heavy or light chain antibodies. Variable domain of the heavy chain can be described as "VH". Variabel the hydrated domain light chain can be described as "VL". These domains usually are the most variable parts of the antibodies and contain antigennegative areas.

The term "variable" refers to the fact that certain parts of the variable domains differ in sequence among antibodies and are used in the binding and specific recognition of each particular antibody for its particular antigen. However, the variability is distributed throughout the variable domains of antibodies are not evenly. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVR) in the variable domains of the light chains and heavy chains. More highly conserved part of the variable domains are called the frame regions (FR). The variable domains of the original heavy and light chains contain four areas FR, basically taking a configuration of a beta-layer, connected by three CDRs, which form loops connecting, and in some cases forming part of the structure of the beta-layer. CDRs in each chain are held together in close proximity regions FR and together with the CDRs from the other chain are involved in the formation antigennegative site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody santhanam, but they have different effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species on the basis of amino acid sequences of their constant domains can be categorized into one of two distinct types, called Kappa (κ) and lambda (λ).

Depending on the amino acid sequence of the constant domain of their heavy chains of antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2. The constant domains of the heavy chains, corresponding to different classes of immunoglobulins are called α, δ, ε, γ and µ, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, for example, Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). The antibody may be part of a larger fused molecule, formed by covalent or non-covalent coupling of antibodies with one or more other proteins or peptides.

The terms "full-size antibody", "intact antibody" and "whole antibody" is used in this paper is e used interchangeably to refer to antibodies in its essentially intact form, and not in the form of fragments of antibodies, as defined below. The terms, in particular, relate to the antibody heavy chains that contain an Fc region.

"Antibody fragments" contain only a portion of an intact antibody, where it retains at least one, and most or all of the functions normally associated with this part when it is in an intact antibody. In one of the embodiments the antibody fragment contains antigennegative the site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, the antibody fragment, for example, a fragment containing the Fc region, retains at least one biological functions normally associated with the Fc-region, when it is in an intact antibody, such as FcRn binding, modulation of time half-life of antibodies, ADCC function and binding of complement. In one embodiment, the implementation of a fragment of the antibody is a monovalent antibody has a half-life ofin vivoessentially similar for the intact antibody. For example, such an antibody fragment may contain antigennegative shoulder associated with the Fc sequence capable of ensuring the stability of the fragmentin vivo.

Cleavage of antibodies with papain leads to the formation of two Ident is cnyh antigenspecific fragments, called "Fab"fragments, each with one antigennegative plot, and a residual "Fc"fragment, whose name reflects its ability to easy crystallization. Treatment with pepsin results in the formation of fragment (Fab')2containing two antigenspecific plot and is still capable of cross-linking antigen.

"Fv" is the minimum antibody fragment that contains a complete antigennegative plot. In one embodiment, the implementation of the double-stranded molecule Fv consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain in a tight non-covalent Association. In the molecule of single-chain Fv (scFv) single variable domain of the heavy chain and one variable domain light chain can be covalently linked by a flexible peptide linker such that the light and heavy chain can associate in a "dimeric" structure analogous to the structure of double-stranded molecules Fv. In this configuration, the three CDRs of each variable domain interact with the formation antigennegative area on the surface of the dimer VH-VL. Together, the six CDR provide the antibody binding specificity of the antigen. However, even a single variable domain (or half of an Fv, containing only three CDRs specific for an antigen) has the ability dissolved, in order to recognize and bind antigen, although with lower affinity than the entire binding site.

The Fab fragment contains the variable domains of the heavy and light chains, and it also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fragments, Fab' differ from Fab fragments by the addition of a few residues at the C-end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH in this document represents the designation for Fab'in which the cysteine residue(s) of the constant domains bear a free Tilney group. Fragments (Fab')2antibody source was given as pairs of Fab fragments', which contain between them a hinge cysteine. Also known other chemical connectors fragments of antibodies.

Antibody fragments "single-chain Fv" or "scFv" contain domains of antibody VH and VL, where these domains are in the same polypeptide chain. Typically, the scFv polypeptide between VH and VL domains further comprises a polypeptide linker, which enables the scFv to form the desired binding of the antigen structure. For an overview, see scFv Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

The term "diately" refers to small fragments of antibodies with two antihistamine areas where these fragments contain the variable domain of the heavy chain (VH)associated with alabilirim domain light chain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and get two antigenspecific plot. Diately can be bivalent or bespecifically. Diately more fully described, for example, in EP 404097; WO 93/1161; Hudson et al., (2003) Nat. Med. 9: 129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). In Hudson et al., (2003) Nat. Med. 9: 129-134 also described Triatel and tetrathele.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group essentially homogeneous antibodies, i.e., the individual antibodies comprising the group are identical except for possible mutations, for example, naturally occurring mutations that may be present in minor amounts. Thus, the definition of "monoclonal" indicates the characteristic of antibodies as not a mixture of different antibodies. In certain embodiments of the exercise of such monoclonal antibody generally includes the antibody containing a polypeptide sequence that binds to the target where the binding target polypeptide sequence obtained by the method, which includes the selection of linking one target polypeptide sequence of m is Oresta polypeptide sequences. For example, the selection process can be a selection of unique clone of many clones, such as a pool of hybridoma clones, phage clones or clones of recombinant DNA. It should be understood that the selected binding the target sequence can be further modified, for example, to increase the affinity against the target for the humanization of binding the target sequence, to increase production in cell culture, to reduce its immunogenicityin vivoto obtain polyspecific antibodies, etc. and that the antibody containing the modified binding the target sequence is also a monoclonal antibody according to this invention. Unlike drugs polyclonal antibodies, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody of the preparation of monoclonal antibodies directed towards a single determinant on the antigen. In addition to their specificity, the preparations of monoclonal antibodies have the advantage that they are usually not contaminated by other immunoglobulins.

The definition of "monoclonal" indicates the characteristic of the antibody as being obtained essentially of a homogeneous group of antibodies, and it should not be construed as requiring receipt of antibody which is specific way. For example, monoclonal antibodies for use in the present invention can be obtained in a variety of ways, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), the methods of recombinant DNA (for example, see U.S. patent No. 4816567), phage display technology resulting in (for example, see Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004) and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), and methods for producing human antibodies or similar to human antibodies in animals that have parts or all of the loci or genes of human immunoglobulins, coding sequences of human immunoglobulins (for example, see WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. patent№5545807; 5545806; 5569825; 5625126; 5633425; 5661016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to correspond with the respective sequences of antibodies obtained from a particular species or belonging to a specific 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 as 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)).

"Humanized" forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-owned human immunoglobulin. In one of the embodiments humanitariannet antibody is a human immunoglobulin (recipient antibody)in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody)such as mouse, rat, rabbit or non-human Primate with the desired specificity, affinity and/or capacity. In some cases, the corresponding non-human residues replace the remnants of the framework region (FR) of a human immunoglobulin. Furthermore, humanized antibodies may contain residues that do not exist is in the recipient antibody or in the donor antibody. These modifications can be performed to further enhance the effectiveness of the antibody. Basically humanitariannet antibody contains essentially all of at least one, and typically two, variable domain, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops not belonging to the human immunoglobulin and all or substantially all of the FR represent the FR sequence of a human immunoglobulin. Humanitariannet antibody optionally also contains at least part of a constant region of immunoglobulin (Fc), typically part of a constant region of human immunoglobulin. For more detail, 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). Also see the following review articles and cited links: 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).

"Human antibody" is an antibody that contains the amino acid sequence corresponding to the amino acid sequence of the antibody produced by a human, and/or received by any of the methods for producing human antibodies, as described herein. This definition of human antibodies specifically excludes humanitariannet antibody containing not belonging to the Euro is ovako antigennegative remains.

When used herein the term "hypervariable region", "HVR" or "HV" refers to regions of the variable domain of antibodies, which are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies greatest variety of the six hypervariable regions demonstrate H3 and L3, and guess what, specifically, H3 plays a unique role in making the antibodies of high specificity. Xu et al., (2000) Immunity 13: 37-45; Johnson and Wu (2003) in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, NJ). Indeed, naturally occurring antibodies camel, consisting only of heavy chains, functional and stable in the absence of light chain. Hamers-Casterman et al., (1993) Nature 363: 446-448; Sheriff et al., (1996) Nature Struct. Biol. 3: 733-736.

This document is used in the treatment of a number of descriptions of the hypervariable regions. Define complementarity region (CDR) at Kabat 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)). Instead, Chothia indicates the position of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). Hypervariable region AbM represent a compromise between Kabat CDRs by and strukturnymi loops by Chothia and their use in modeling software antibodies Oxford Molecular's AbM. "Contact" hypervariable region based on a comprehensive analysis of available crystal structures. Below are the remnants of each of these hypervariable region.

LoopKabatAbMChothiaContact
L1L24-L34L24-L34L26-L32L30-L36
L2L50-L56L50-L56L50-L52L46-L55
L3L89-L97L89-L97L91-L96L89-L96
H1H31-H35BH26-H35BH26-H32H30-H35B
(Numbering according to Kabat)
H1H31-H35H26-H35H26-H32H30-H35
(Chothia numbering)
H2 H50-H65H50-H58H53-H55H47-H58
H3H95-H102H95-H102H96-H101H93-H101

Hypervariable region may contain "extended hypervariable region"as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. In each of these definitions remains variable domains are numbered according to Kabat et al., above. Hypervariable region HVR-H1 and HVR-H2 of antibody 10F4 to CD22 according to the invention are H26-H35 and H49-H65 using the numbering according to Kabat.

"Framework" or "FR" residues are those residues of the variable domains, which differ from the residues of the hypervariable regions as defined in this document.

The term "residue numbering variable domain as in Kabat" or "the numbering of the positions of the amino acids as in Kabat" and its variants refers to the numbering system used for the variable domains of the heavy chain or variable light chain domains in the view of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual linear amino acid series is here may contain fewer amino acids or more amino acids, corresponding shortening of, or insertion into FR or HVR variable domain. For example, the variable domain of the heavy chain can include the insertion of one amino acid (residue 52a according to Kabat) after residue 52 H2 and inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 FR heavy chain. The residue numbering according to Kabat this antibody can be determined by alignment by regions of homology to the sequence of the antibody with the "standard" numbered according to Kabat sequence.

"The free amino acid cysteine" refers to amino acid residue cysteine, which is inserted bioengineering way to the original antibody, is Tilney functional group (-SH) and not paired as or is not part of the otherwise intramolecular or intermolecular disulfide bridge.

The term "value thiol reactivity" is a quantitative characteristic of reactivity of free amino acids cysteine. The value of thiol reactivity represents the percentage of free amino acids cysteine in a modified cysteine antibody, which reacts with reactive thiol reagent and converted into a maximum value of 1. For example, the free amino acid cysteine in a modified cysteine antibody, which with 100% output is m reacts with reactive thiol reagent, such as biotinylating reagent, to form a labeled Biotin antibody has a value of thiol reactivity of 1.0. Another amino acid cysteine, is inserted in the same or another of the original antibody, which is 80% yield reacts with reactive thiol reagent, has a value of thiol reactivity of 0.8. Another amino acid cysteine, is inserted in the same or another source antibody that does not react with the reactive thiol reagent, has a value of thiol reactivity 0. Determine the value of thiol reactivity of specific cysteine can be performed by means of ELISA analysis, mass spectroscopy, liquid chromatography, autoradiography or other quantitative analytical tests. Reacts with thiol reagents, allows the capture of modified cysteine antibodies and comparison and quantitative analysis of the reactivity of cysteine include Biotin-PEO-maleimide ((+)-biotinyl-3-maleimidomethyl-3,6-dioxaoctyl, Oda et al., (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.) Biotin-BMCC, PEO-iodization, iodates-1C-Biotin and Biotin-HPDP (Pierce Biotechnology, Inc.) and Na-(3-multimediaplayer)biocytin (MPB, Molecular Probes, Eugene, OR). Other commercial sources for biotinylation, bifunctional and polyfunctional linker reagents VK is ucaut Molecular Probes, Eugene, OR and Sigma, St. Louis, MO.

"The original antibody" is an antibody containing the amino acid sequence on the basis of which one or more amino acid residues substituted by one or more cysteine residues. The original antibody may contain natural sequence or the sequence of the wild type. The original antibody may contain preexisting modification of the amino acid sequence (such as additions, deletions and/or substitutions) relative to other natural, wild-type or modified forms of antibodies. The original antibody may be directed to the interest of the target antigen, for example, to biologically important polypeptide. Also consider antibodies directed to polipeptides antigens (such as associated with tumors glycolipid antigens; see U.S. patent 5091178).

This document uses the following abbreviations, and they have these definitions: BME is a beta mercaptoethanol, Boc represents N-(tert-butoxycarbonyl), cit is citrulline (2-amino-5-breedopedia acid), dap is dalapon, DCC is a 1,3-dicyclohexylcarbodiimide, DCM represents dichloromethane, DEA is diethylamin, DEAD is diethylazodicarboxylate is at, DEPC is diethylphosphoramidite, DIAD is diisopropylsalicylic, DIEA represents N,N-diisopropylethylamine, dil is daisosasen, DMA is a dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is a simple dimethyl ether of ethylene glycol (or 1,2-dimethoxyethane), DMF represents N,N-dimethylformamide, DMSO is a sulfoxide, doe is draftin, dov is a N,N-dimethylamine, DTNB is a 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is a hydrochloride of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EEDQ is a 2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is an acid ethyl ester, Fmoc represents N-(9-fluorenylmethoxycarbonyl), gly represents glycine, HATU is hexaphosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea, HOBt represents 1-hydroxybenzotriazole, HPLC is a high-performance liquid chromatography, ile represents isoleucine, lys is a lysine, MeCN (CH3CN) is acetonitrile, MeOH performance is made by a methanol, Mtr is a 4-insidefamily (or 4-methoxytrityl), nor is a (1S,2R)-(+)-norephedrine, PAB represents the p-aminobenzoylamino, PBS is a phosphate-saline buffer (pH 7), PEG represents a polyethylene glycol, Ph represents phenyl, Pnp is a p-nitrophenyl, MC represents a 6-maleimidomethyl, phe represents L-phenylalanine, PyBrop is hexaphosphate bromo-Tris-pyrrolidinone, SEC represents exclusion chromatography, Su represents succinimide, TFA represents triperoxonane acid, TLC is a thin-layer chromatography, UV is a UV and val is a valine.

"Affinity matured" antibody is an antibody with one or more changes in one or more HVR, which leads to improvement in the affinity of the antibody against the antigen compared to the original antibody, in which no such change(s). In one embodiment, the implementation of the affine Mature antibody possesses nanomolar or even picomolar affiniscape for the target antigen. Affine Mature antibodies get known in this field means. In Marks et al., Bio/Technology 10: 779-783 (1992) describes affinity maturation by permutation domains VH and VL. Random mutagenesis HVR and/or ka is the red residues described in 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 an "antagonist" antibody is an antibody that inhibits or reduces biological activity associated antigen them. Specific blocking antibodies or antagonist antibodies largely or completely inhibit the biological activity of the antigen.

As used herein, "agonist antibody"is an antibody that mimics at least one type of functional activity of interest polypeptide.

"Effector functions" antibody refers to those kinds of biological activity which is inherent in the Fc region (Fc region with the natural sequence or a Fc region of the variant amino acid sequence) antibodies, and vary depending on the isotype of the antibody. Examples of effector functions of antibodies include C1q binding and is caused by complement cytotoxicity; binding Fc receptor; mediated antibody-dependent cell cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g. B-cell receptor and activation of B-cells.

"Fc receptor" or "FcR" refers to a receptor which binds to Fc region of antibodies. In some embodiments, the implementation of FcR is a natural FcR person. In some embodiments, the implementation of FcR represents the FcR, which binds IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and forms of alternative splicing of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), with similar amino acid sequences, which mainly differ in their cytoplasmic domains. Activating receptor FcγRIIA contains in its cytoplasmic domain immunoreceptor activating motif-based tyrosine (ITAM). Inhibiting receptor FcγRIIB contains in its cytoplasmic domain immunoreceptor inhibitory motif-based tyrosine (ITIM) (see Daëron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). In this document the term "FcR" covers other FcR, including FcR, which will be identified in the future.

The term "Fc receptor" or "FcR" 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)) and the regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997, Hiton 2004). Binding to human FcRnin vivoand the half-life in serum of human FcRn polypeptide with high affinity binding can be assessed, for example, in transgenic mice or transfected cell lines human expressing human FcRn, or primates, which have introduced variants of the Fc polypeptides.

In WO 00/42072 (Presta) described variants of the antibodies with enhanced or reduced binding to FcR. The contents of this patent publication are incorporated herein by reference in full. See also Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

"Effector cells" are cells, expressing one or more FcR and carry out effector functions. In certain embodiments of the exercise of the cells Express at least FcγRIII and perform effector function(s) ADCC. Examples of human leukocytes mediating ADCC include mononuclear cells of peripheral blood (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. Effector cells can be isolated from a natural source, for example from the blood.

"Due to antibody-mediated cell cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound Fc-receptors (FcR), located on certain cytotoxic cells (e.g., eating is the only killer (NK) cells, neutrophils and macrophages), allowing these cytotoxic effector cells specifically bind to a carrier antigen-cell target, and then kill the target cell with cytotoxins. Primary cells mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess ADCC activity of interest molecules can be analyzed ADCCin vitrosuch as analysis, described in U.S. patent No. 5500362 or 5821337, or U.S. patent No. 6737056 issued by Presta. Suitable for such analyses effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer (NK) cells. Alternative or additionally, ADCC activity of interest molecules can be estimatedin vivofor example, in animal models, such as the model described in Clynes et al., PNAS (USA) 95: 652-656 (1998).

"Due to complement cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical pathway of complement is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass)that are associated with them recognizable antigen. To assess activation of complement can be analysed CC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202: 163 (1996).

Variants of polypeptides with altered amino acid sequences of the Fc-region is enlarged or reduced ability to bind C1q described in U.S. patent No. 6194551B1 and WO 99/51642. The content of these patent publications are incorporated herein by reference in full. Also see Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

The term "containing the Fc region polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin, which contains the Fc-region. C-terminal lysine (residue 447 according to the numbering system of the EU) Fc-region can be removed, for example, when cleaning the polypeptide or by recombinant construct encoding a polypeptide nucleic acid. Thus, a composition comprising a polypeptide with Fc-region according to this invention may include polypeptides with K447, all of the remote K447, or a mixture of polypeptides with the K447 residue and without it.

"Acceptor framework of human rights" for the purposes of the present document is a frame containing the amino acid sequence of frame VL or VH obtained from the skeleton of the human immunoglobulin or the consensus framework of human rights. The acceptor framework of human rights "derived from" a skeleton of the human immunoglobulin or consensus skeleton man can contain the same their amino acid is an explicit sequence or it may contain pre-existing changes in amino acid sequence. In some embodiments, the implementation of a number of preexisting substitutions of amino acids is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Where VH are preexisting replacement amino acids, preferably these replacements occur only in three, two or one of the provisions 71H, 73H and 78H; for example, amino acid residues in these positions can represent 71A, 73T and/or 78A. In one of the embodiments of the acceptor framework VL person is identical to the sequence of frame VL immunoglobulin or consensus sequence of a skeleton of a man.

"The consensus skeleton man" represents a frame, which represents the most frequently occurring amino acid residues at the choice of frame sequences of the VL or VH of a human immunoglobulin. As a rule, the choice of the sequences of the VL or VH immunoglobulin conduct of subgroups of sequences of the variable domains. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the implementation of the VL subgroup is a subgroup Kappa I as in Kabat et al., above. In one embodiment, the implementation of the VH subgroup is predstavljaet a subgroup III as in Kabat et al., above.

"The consensus framework VH subgroup III contains a consensus sequence derived from the amino acid sequences of subgroup III variable domains of the heavy chains in Kabat et al., above. In one embodiment, the implementation of the amino acid sequence of the consensus framework VH subgroup III contains at least part of each or all of the following sequence:

EVQLVESGGGLVQPGGSLRLSCAAS (FR-H1, SEQ ID NO: 1)-HVR-H1-

WVRQAPGKGLEWV (FR-H2, SEQ ID NO: 3)-HVR-H2-

RFTISADTSKNTAYLQMNSLRAEDTAVYYC (FR-H3, SEQ ID NO: 5)-HVR-H3-

WGQGTLVTVSS (FR-H4, SEQ ID NO: 7).

"The consensus framework VL subgroup I contains a consensus sequence derived from the amino acid sequence in sub-group I variable domains of the light chains Kappa in Kabat et al., above. In one embodiment, the implementation of the amino acid sequence of the consensus framework VL subgroup I contains at least part of each or all of the following sequence:

DIQMTQSPSSLSASVGDRVTITC (FR-L1, SEQ ID NO: 8)-HVR-L1-

WYQQKPGKAPKLLIY (FR-L2, SEQ ID NO: 11)-HVR-L2-

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (FR-L3, SEQ ID NO: 13)-HVR-L3-FGQGTKVEIK (FR-L4, SEQ ID NO: 15).

"Secretory signal sequence" or "signal sequence" refers to a nucleic acid sequence that encodes a short signal peptide, which can be used to direct the newly synthesized interest of proteins is through the cell membrane, typically, the inner membrane or the inner or outer membrane of prokaryotes. Therefore, the protein of interest, such as the polypeptide of light or heavy chains of immunoglobulin secreted in periplasm prokaryotic host cells or the medium for cultivation. The signal peptide encoded secretory signal sequence can be endogenous to the host cells, or may be exogenous, including signal peptides found expressed with the polypeptide in nature. The secretory signal sequence, typically located on aminocore expressed polypeptide and, as a rule, the enzymatic removed between the biosynthesis and secretion of the polypeptide from the cytoplasm. Thus, the signal peptide, as a rule, is not present in the product of the Mature protein.

Typically, the affinity of binding" refers to the power of the total non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g., antigen). As used herein, unless otherwise indicated, "the affinity of binding" refers to the natural affinity of binding, which reflects the interaction of 1:1 between the partners of a pair of binding (e.g., antibody and antigen). The affinity of a molecule X for its partner Y, as p is Avila, you can imagine the dissociation constant (Kd). The affinity can be measured well-known in this field of ways, including the methods described herein. Low-affinity antibodies, usually slowly bind antigen and tend to be easy dissociation, whereas high-affinity antibodies, typically bind antigen faster and tend to stay connected for longer. In this area there are many ways to measure the affinity of binding, any of which can be used for the purposes of the present invention. Specific illustrative embodiments of described below.

In one embodiment, the implementation of the "Kd" or "Kd value" according to this invention is measured by analyzing the binding of radioactive antigens (RIA)performed with the Fab version of interest antibody and its antigen as described in the following analysis. The binding affinity of FABS for antigen in solution is measured by balancing Fab with a minimal concentration of labeled (125I) antigen in the presence of tetraoxa dilutions of unlabeled antigen, then spending the capture associated antigen to the antibody coated to Fab tablet (Chen, et al., (1999) J. Mol. Biol. 293: 865-881). To create conditions for the analysis of tablets for micrometrology (Dynex) cover during the night of 5 µg/ml exciting antic the La to Fab (Cappel Labs) in 50 mm 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 readsorbing tablet (Nunc No. 269620), 100 PM or 26 mm [125I]-antigen are mixed with serial dilutions of interest Fab (for example, in accordance with the assessment of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57: 4593-4599). Then interest Fab incubated over night; however, incubation can be continued over a longer period (for example, approximately 65 hours) to ensure that equilibrium is achieved. Then the mixture is transferred to an exciting tablet for incubation at room temperature (for example, within one hour). Then the solution is removed and the plate washed eight times with 0.1% Tween-20 in PBS. After drying of the tablets add 150 ál/well of scintillator (MicroScint-20; Packard) and on tablets spend counting on a gamma counter Topcount (Packard) for ten minutes. The concentration of each Fab, giving less than or equal to 20% of the maximum binding is chosen for use in the analysis of competitive binding.

In another variant implementation, the Kd or Kd value is measured by use of analysis of surface plasmon resonance using BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using CM5 chips with immobilized antigen at~10 response units (RU). In summary, karboksimetilirovaniya dextranase biosensor chips (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) associated protein antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 µm). After injection of the antigen is injected 1 M ethanolamine to block unreacted groups. For kinetic measurements is injected twofold serial dilution of Fab (0.78 nm to 500 nm) in PBS with 0.05% of Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl/min Rate of Association (kon) and the rate of dissociation (koff) are calculated using a simple model linking the Langmuir one-to-one (BIAcore Evaluation Software version 3.2) by simultaneous build sensogram Association and dissociation. The equilibrium dissociation constant (Kd) calculated as the ratio koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol. 293: 865-881. If the rate of education in the analysis of surface plasmon resonance exceed 106M-1with-1then the rate of formation can be determined using the method of quenching the fluorescence, which measures the increase or decrease of the radiation intensity of fluorescence (excitation = 295 nm; irradiation is s = 340 nm, bandwidth of 16 nm) at 25°C 20 nm of the antibody to the antigen (in the form of Fab) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as equipped with a stopped-flow spectrophotometer (Aviv Instruments) or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with the use of the cuvette with stirring.

"The rate of formation", "rate of Association" or "kon" according to this invention can also be defined as described above with the use of system BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ).

"Violation" is any pathological condition or disease that can favorably impact when applying a treatment substance/molecule or method according to the invention. It includes chronic or acute disorders, including pathological conditions which predispose the mammal to the disorder. Non-limiting examples of disorders to treatment by the present document include malignant conditions, such as B-cell proliferative disorders and/or B-cell tumors, such as lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, volontariato the hydrated leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell.

The terms "cell proliferative violation" and "proliferative violation" refers to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the implementation of cellular proliferative violation is a malignant tumor.

As used herein, a "tumor" refers to any growth and proliferation of neoplastic cells, malignant or benign, and all pre-malignant and malignant cells and tissues. The terms "cancer", "cancerous", "cell proliferative violation", "proliferative violation" and "tumor" are not mutually exclusive, as specified in this document.

The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation, or describe it. Examples of malignant tumors include as non-limiting examples, a malignant B-cell proliferative disorders, where B-cell proliferative violation is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell. On the other malignant condition, for example, carcinoma, lymphoma (e.g., jackinsky and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such malignant tumors include squamous cell malignant tumor, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, malignant tumor of the abdominal cavity, liver-cell malignant tumor, gastrointestinal tumor, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, 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, hepatic carcinoma, leukemia and other lymphoproliferative disorders and various types of head and neck cancer.

"B-cell malignancy" in this document includes nahodkinskuju lymphoma (NHL), including well-differentiated/f is likalaruku NHL, small cell (SL) NHL, intermediate degree of differentiation/follicular NHL, intermediate degree of differentiation of diffuse NHL, high differentiation immunoblastic NHL, well-differentiated lymphocytic NHL, well-differentiated NHL from small non-digestible cell NHL with a massive defeat, lymphoma, mantle cell, AIDS-related lymphoma and macroglobulinemia waldenstrom, nahodkinskuju lymphoma (NHL), Hodgkin's disease with predominance of lymphocytes (LPHD), small cell lymphoma (SLL), chronic lymphocytic leukemia (CLL), slow-growing NHL, including relapsing slow-growing NHL and refractory to rituximab slow-growing NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; lymphoma mantle cell and other hematological malignancies. These malignancies can be treated with antibodies directed to surface markers of B-cells, such as CD22. Such diseases addressed in this document subject to treatment with antibodies directed to surface B-cell marker, such as CD22, and the introduction includes the introduction of unconjugated ("free") antibody or an antibody conjugated with a cytotoxic agent, as the op is Sano in this document. Such diseases addressed in this document, subject to the combined treatment, including antibody to CD22 or conjugate antibody to CD22-drug according to the invention in combination with another antibody or conjugate antibody-drug, another cytotoxic agent, therapeutic radioactivity or other treatment carried out simultaneously or sequentially. For illustrative method of treatment according to the invention the antibody to CD22 according to the invention is administered in combination with antibody to CD20, immunoglobulin, or CD20 binding fragment, together or sequentially. Antibody to CD20 can be a free antibody or conjugate antibody-drug. In the embodiment, the combined treatment antibody to CD22 is an antibody of the present invention, and antibody to CD20 is a Rituxan® (Rituxan®rituximab).

As used herein, the term "non-Hodgkin's lymphoma or NHL" refers to a malignant tumor of the lymphatic system, other than jackinsky lymphomas. Hodginsii lymphoma, usually can be distinguished from non-Hodgkin's lymphomas by the presence of cells, reed-Sternberg at jackinsky lymphoma and the absence of these cells in non-Hodgkin lymphomas. Examples of non-Hodgkin lymphoma covered what Ermin, as used herein, include any nahodkinskuju lymphoma, which is a specialist in this field (e.g., an oncologist or pathologist) identifies as nahodkinskuju in accordance with well-known in this area classification schemes such as the scheme of the Revised European-American Limphoma (REAL), as described in the Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). In particular, see the listings on Fig, 11.58 and 11.59. More specific examples include as non-limiting examples of relapsing or refractory NHL, edge high-NHL, NHL stage III/IV, resistant to chemotherapy NHL, lymphoblastic leukemia and/or lymphoma of the predecessors of B-cells, small cell lymphoma, B-cell chronic lymphocytic leukemia, and/or prolymphocytic leukemia, and/or small cell lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, lymphoma of B-cell marginal zone lymphoma splenic marginal zone, newslove lymphoma marginal zone MALT, nodular lymphoma of the marginal zone, hairy cell leukemia, plasmacytoma/myeloma plasma cells, immature/follicular lymphoma, intermediate degree of differentiation/follicular NHL, lymphoma mantle cell lymphoma centers of follicles (follicular), ex is offered by the degree of differentiation of diffuse NHL, diffuse both B-cell lymphoma, aggressive NHL (including aggressive edge NHL and aggressive relapsing NHL), NHL, relapsing after autologous transplantation of stem cells or refractory to her primary mediastinal both B-cell lymphoma, primary exudative lymphoma, poorly differentiated immunoblastic NHL, well-differentiated lymphocytic NHL, well-differentiated NHL from small non-digestible cell NHL with a massive defeat, lymphoma Berkata, leukemia of the predecessors (peripheral) large granular lymphocytes, mushroom avium and/or syndrome Cesari, cutaneous lymphoma, anaplastic both lymphoma, angiocentric lymphoma.

"Autoimmune disease" herein is a disease or disorder arising from and directed against its own tissues or organs of an individual, or certain symptoms or manifestations of, or arising from his status. When many of these autoimmune and inflammatory disorders may exist a number of clinical and laboratory markers, including as non-limiting examples, hypergammaglobulinemia, high levels of autoantibodies, sediment complexes antigen-antibody in the tissues, the improvement after corticosteroid or immunodeficiency is acupressure treatment and aggregates of lymphoid cells in the affected tissues. Without limitation to any theory regarding mediated B-cell autoimmune disease, suppose that B-cells are pathogenic effect in autoimmune diseases through many mechanical ways, including production of autoantibodies, the formation of immune complexes and activation of dendritic and T-cells, the synthesis of cytokines that direct the release of chemokines and providing focus for ectopic politogenesis. Each of these pathways may participate in the pathogenesis of autoimmune disease in varying degrees.

"Autoimmune disease" can be an organ-specific disease (i.e. immune response directed against specific organ systems such as the endocrine system, haematopoiesis, skin, the cardiopulmonary system, the gastrointestinal tract and liver system, kidneys, thyroid, ears, neuromuscular system, the Central nervous system and so on) or a systemic disease that can affect multiple organ systems (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, and so on). Preferred such diseases include autoimmune rheumatologic disorders (such as rheumatoid arthritis, Sjogren syndrome, scleroderma, lupus such as SLE and lupus nefr is t, polymyositis/dermatomyositis, cryoglobulinemia syndrome antiphospholipid antibodies and psoriatic arthritis), autoimmune gastrointestinal 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 coeliac disease), vasculitis (such as ANCA-negative 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, expected outcome, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as glomerulonephritis syndrome? and illness Berger), autoimmune dermatologic disorders (such as psoriasis, urticaria, rash, common bladderwort, bullous pemphigoid and cutaneous lupus erythematous), hematologic disorders (such as thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune the diseases of the ear (for example, such as disease of the inner ear and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison disease and autoimmune thyroid disease (e.g., graves ' disease and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren syndrome, graves ' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Specific examples of other autoimmune diseases, as defined herein, which in some cases include autoimmune diseases listed above, include as non-limiting examples, arthritis (acute and chronic rheumatoid arthritis, including juvenile rheumatoid arthritis and stages such as rheumatoid synovitis, 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, of still's disease, arthritis of the spine, osteoarthritis, chronic progredient course arthritis, de is almyroudis arthritis, primary chronic arthritis, reactive arthritis, menopausal arthritis, arthritis due to the reduction of estrogen and ankylosing spondylitis/rheumatoid spondylitis), autoimmune lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis such as spotted psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, Allergy, including allergic diseases such as hay fever and syndrome Jobe, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, rash, dermatitis herpetiformis, coins dermatitis, seborrheic dermatitis, non-specific dermatitis, primary contact dermatitis from annoying substances and atopic dermatitis, x-linked syndrome of Hyper IgM, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS), such as sinapticeski MS, primary progressive MS (CPD is) and remitirse-relapsing MS (RRRS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis, atoxicity sclerosis, optionalemail (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmuneautoimmune gastrointestinal disease, gastrointestinal inflammation, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenosis colitis, polypous colitis, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), inflammation of the intestine, gangrenous pyoderma, nodular erythema, primary sclerosing cholangitis, respiratory distress syndrome, including respiratory distress syndrome, adult or acute respiratory failure (ARDS), meningitis, inflammation of all or part of the choroid of the eye, iritis, chorioidea, autoimmune hematological violation, graft versus host, angioedema, such as hereditary angioedema, damage to the cranial nerves as in meningitis, herpes pregnant, pemphigoid pregnant, inflammatory itching, autoimmune early cessation of ovulation, sudden hearing loss due to pathological autoimmune condition, IgE mediated diseases such as anaphylaxis and allergic and atopic rhinitis, e is cephalic, such as Rasmussen encephalitis and limbic encephalitis and/or encephalitis of the brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, agranulocytosis uveitis, valanchery uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with nephrotic syndrome and without it, such as chronic or acute glomerulonephritis such as primary GN, immunopositivity GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrane - or membranous proliferative GN (MPGN), including type I and type II, and rapidly progressive GN (RPGN), proliferative nephritis, autoimmune policlasista endocrine failure, balanitis, including limited plasmacytosis balanitis, balanoposthitis, centrifugal erythema annulare, ashy dermatosis, erythema multiforme, annular granuloma, brilliant zoster, sclerotic atrophic lichen, simple chronic zoster, thorn versicolor, tinea flat, scaly ichthyosis, epidermolizei hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, food allergies, allergies to medications, allergies to insects, rare allergic disorders such as mastocytosis, allergic reaction, eczema including Allergy is a mini or atopic eczema, statsnow eczema, disgestrotical eczema and bubble palmoplantar eczema, asthma such as bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory response, immune reactions against foreign antigens such as blood group A-B-On the fetus during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, deficiency of leukocyte adhesion, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematous, lupus alopecia, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus disseminated erythematous, juvenile (type I) diabetes mellitus, including child IDDM, diabetes mellitus in adults (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic violation of large arteries, immune responses associated with acute or delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, Wegener, including lymphomatoid Wegener, agranulocytosis, vasculitis (including vasculitis, large with the courts, such as polymyalgia rheumatica and giant cell (Takayasu's) arteritis diagnostics, vasculitis medium-sized vessels, such as Kawasaki syndrome and polyarteritis polyarteritis/periarteritis nodosa, immunovaccine, CNS vasculitis, cutaneous vasculitis, due to hypersensitivity vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis and systemic necrotizing vasculitis, ANCA-negative vasculitis, and ANCA-associated vasculitis, such as the syndrome Cerca-Strauss (CSS), Wegener's granulomatosis and microscopic polyangiitis), temporal arteritis diagnostics, aplastic anemia, autoimmune aplastic anemia, positive Coombs test anemia, anemia diamond-Blackfan, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison disease, true red cell anemia or aplasia (PRCA), a deficiency of factor VIII, hemophilia A, autoimmune neutropenia(s), cytopenia, such as pancytopenia, leukopenia, diseases involving diabetes leukocytes, inflammatory disorders of the CNS, Alzheimer's disease, Parkinson's disease, syndrome of multiple organ damage, such as secondary syndromes septicemia, trauma or hemorrhage, mediated by complexes of antigen-antibody disease, disease of antibodies to basal membrane cloud the points syndrome antibodies to phospholipids, motoneuron, allergic neuritis, a disease/syndrome behceta, syndrome Castleman syndrome?, Raynaud's syndrome, Sjogren syndrome, and syndrome of Stevens-Johnson, pemphigoid or pemphigus, such as bullous pemphigoid, scar (mucous membranes) pemphigoid, skin pemphigoid, common bladderwort, paraneoplastic pemphigus, layered pemphigus, pemphigus pemphigoid mucosa and pemphigus erythematous, acquired bullous bullosa, eye inflammation, preferably allergic ocular inflammation, such as allergic conjunctivitis, bullous disease, linear IgA, autoimmune induced inflammation of the conjunctiva, autoimmune polyendocrinopathy, illness or Reiter syndrome, thermal injury due to an autoimmune condition, preeclampsia, disorder caused by immune complexes, such as jade immune complexes of antibody-mediated nephritis, neirolepticalkie disorders, polyneuropathies, chronic neuropathy such IgM polyneuropathies or IgM mediated neuropathy, thrombocytopenia (as developed in patients with myocardial infarction), including thrombotic thrombocytopenic purple (TTP), post-transfusion purple (PTP), heparin-induced thrombocytopenia, and autoimmune or immune posredovanju thrombocytopenia, including, for example, idiopathic thrombocytopenic purple (ITP)including chronic or acute ITP, scleritis such as idiopathic keratoscleritis, 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, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune disease of the thyroid gland, idiopathic hypothyroidism, Grave's disease, Grave's disease of the eyes (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 or the syndrome of Lambert-Eaton syndrome frozen human or frozen individual, encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as associated with thymoma myasthenia gravis, spinal cerebellar degeneration, neuromyotonia, or needs to be opsoclonus myoclonus syndrome (OMS), and sensory it is Opatija, multi focal motor neuropathy, Sheehan 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), obliterating brodholt (no transplant) vs NSIP, Guillain-Barre syndrome, a disease Berger (IgA nephropathy), idiopathic IgA nephropathy, dermatitis, linear IgA, acute febrile neutrophilic dermatosis, pedagogiczny pustular dermatosis, transient contritione dermatitis, cirrhosis, such as primary biliary cirrhosis and pneumonocytes, autoimmune enteropathy syndrome, gluten enteropathy or the celiac disease, refractory sprue, idiopathic sprue, cryoglobulinemia, such as mixed cryoglobulinemia, amyotrophic lateral sclerosis (ALS; disease low Gehrig's disease (als), 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 the syndrome Kogan/recipricocity interstitial keratitis, bell's palsy, a disease/sweet syndrome, rosacea autoimmune, pain associated with shingles, amyloidosis, netlocation the th lymphocytosis, primary lymphocytosis, which includes B-cell lymphocytosis (e.g., benign monoclonal gammopathy and the monoclonal gammapathy undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, kalapati, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, paroxysmal paralysis and kalapati CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune Hepatology violation, fibromyalgia, multiple endocrine failure, syndrome Schmidt, adrenalin, atrophy of the stomach, presenilny dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinizing the polyneuropathy, Dressler syndrome, focal alopecia, total alopecia, CREST syndrome (calcinosis, the phenomenon of Reynard, esophageal dyskinesia, sclerodactyly and telangiectasia), male and female autoimmune infertility, e.g., due to antibodies to sperm, mixed lesions of the connective tissue, Chagas disease, rheumatic fever, habitual miscarriage, easy farmer, polymorphic erythema, postcardiotomy syndrome, Cushing's syndrome, a mild poultry farmers, llurgicheskij granulomatous vasculitis, soft limfotsity vasculitis syndrome Alport, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, cyanosis, schistosomiasis, ascariasis, aspergillosis, syndrome Santera syndrome Kaplan, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, fibrous mediastinal, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent towering erythema, erythroblasts fruit, eosinophilic faceit, syndrome, Shulman syndrome, still's, flares, cycle such as chronic cycle, heterochronic cycle, iridocyclitis (acute or chronic) or cycle of Phew, purpura Shenleyn's disease, infection with the human immunodeficiency virus (HIV), SCID, acquired immune deficiency syndrome (AIDS), Echovirus infection, sepsis (system inflammatory response syndrome (SIRS)), endotoxicosis, pancreatitis, Tiresias, parvovirus infection, infection with the rubella virus, postvaccine syndromes, congenital rubella infection, infection with Epstein-Barr infectious parotitis syndrome Evan, autoimmune gonads, chorea of Sydenham, poststreptococcal jade, obliteratus is obliterans, thyrotoxicosis, the " dryness " of the spinal cord, chorionic, the giant cell rheumatica, chronic hypersensitive pneumonitis, conjunctivitis, such as spring Qatar, keratoconjunctivitis sicca and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, nephropathy with minimal changes, soft family damage and damage due to ischemia-reperfusion injury, reperfusion of transplanted organ, an autoimmune reaction to the retina, inflammation of the joints, bronchitis, chronic pulmonary obstruction/obstruction of the respiratory tract, silicosis, atty, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular insufficiency, such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, spermiogenesis, autoimmune hemolysis, disease Beck, the cryoglobulinemia, Dupuytren's contracture, phacoanaphylaxis endoftheline, allergic enteritis, leprosy nodular erythema, idiopathic facial palsy, chronic fatigue syndrome, rheumatic fever, a disease of Hamman-rich, sensorineural hearing loss, the paroxysmal hemoglobinuria, hypogonadism, regional REIT, radiation, infectious mononucleosis, transverse myelitis, primary the idiopathic myxedema, nephrosis, sympathetic ophthalmic, neonatal ophthalmic, Opticheskie, granulomatous orchitis, pancreatitis, acute multiple sciatica, gangrenous pyoderma, thyroiditis Quervain acquired atrophy of the spleen, the non-malignant thymoma, lymphovascular limit, vitiligo, toxic shock syndrome, food poisoning, conditions involving infiltration of T-cells, deficiency of leukocyte adhesion, immune response associated with acute or delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diabetes, the syndrome of multiple organ failure, disease, mediated by complexes of antigen-antibody, a disease caused by antibodies to basal membrane of the glomeruli, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed lesions of the connective tissue, nephrotic syndrome, insult, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, idiopathic adult hypoparathyroidism (AOIH), cardiomyopathy such as common cardiomyopathy acquired bullous bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or negniy sinusitis, acute or chronic sinusitis, ethmoid the th, the frontal, maxillary sinuses or sphenoidal sinusitis, allergic sinusitis associated with eosinophils violation, such as eosinophilia, pulmonary infiltrates eosinophilia syndrome eosinophilia-myalgia syndrome Leffler, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma or granulomas containing eosinophils, anaphylaxis, spondyloarthropathies, seronegative spondyloarthropathies, polyendocrine autoimmune disease, sclerosing cholangitis, sclerosis, episcleritis, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia childhood syndrome Wiskott-Aldrich syndrome ataxia-telangiectasia, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, such as chronic arthroleptis, lymphadenitis, the answer is a decrease in blood pressure, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, breach of allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, Vespa is sustained fashion dermatitis, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other inflammatory disorders of the Central nervous system, ocular and orbital inflammatory disorders, associated with transfusion of granulocytes syndromes induced by cytokines toxicity, narcolepsy, acute severe inflammation, chronic untreatable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvula and endometriosis. Such diseases considered in this document is subject to treatment by administration of antibodies that bind to surface marker for B-cells, such as CD22, and treatment includes the introduction of unconjugated ("free") antibody or an antibody conjugated with a cytotoxic agent, as described herein. Such diseases are also considered herein as being subject to treatment by combined treatment, including antibody to CD22 or conjugate antibodies to CD22-drug according to the invention in combination with another antibody or conjugate antibody-drug, another cytotoxic agent, radiation or other drug administered simultaneously or consequently is.

As used herein, "treatment" (and variations such as "cure" or "treat"refers to clinical intervention aimed at changing the natural course of the disease in an individual, or changes in cells exposed to the treatment, and it can be done or for prophylaxis or during the clinical pathological process. Desirable effects of treatment include preventing occurrence or recurrence of the disease, relieve symptoms, reduce any direct or indirect pathological consequences of the disease, prevention of metastasis, decrease the speed of disease progression, relief or temporary weakening of the state when disease and remission or improved prognosis. In some embodiments, the implementation of the antibodies according to the invention are used to delay the development of diseases or disorders or to slow the progression of disease or impairment.

"Individual" is a vertebrate. In certain embodiments of the implementation of the vertebrate is a mammal. Mammals include as non-limiting examples of farm animals (such as cows), sports animals, animals (such as cows, dogs and horses), primates, mice and rats. In certain embodiments of the implementation of the ml is kopituse is a person.

"Effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

"Therapeutically effective amount" of a substance/molecule of the invention can vary depending on factors such as stage of disease, age, sex and weight of the individual, and the ability of a substance/molecule to cause the desired response in the individual. Therapeutically effective amount includes an amount by which any toxic or detrimental effects of the substance/molecule were outweighed by therapeutically positive effects. "Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily as a prophylactic dose is used in subjects prior to or at the early stage of the disease, the prophylactically effective amount will be less than therapeutically effective amount.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes the death or destruction of cells. The term is intended to include radioactive the s isotopes (for example, At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalatory, enzymes and fragments thereof such as nucleases, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, toxins, inhibiting growth funds molecule drugs and the various antitumor or anticancer means described below. The following are other cytotoxic funds. Antitumor agent causes destruction of tumor cells.

Toxin is any substance that can have a devastating effect on the growth or proliferation of the cell.

"Chemotherapeutic agent" is a chemical compound suitable for the treatment of malignant tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide CYTOXAN® (cytoxan®); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, t is the cue as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL® (Marinol)); beta-lapachone; lapachol; colchicine; Betulinol acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN® (Cosmegen®)), CPT-11 (irinotecan, CAMPTOCAR® (camptosar®)), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllin acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy mustard; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin nimustine and ranimustine; antibiotics such as andinavia antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (194)); dynemicin, including dynemicin a; spiramycin; and neocarzinostatin chromophore and related chromoprotein andinavia antibiotic chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morpholino doxorubicin, cyanomethane doxorubicin, 2-pyrroline doxorubicin and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate 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; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane, testolactone; antigranulocyte tools such as aminoglutethimide, mitotane, trilostane; compensate the s folic acid, such as prolinnova acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; 2-acylhydrazides; procarbazine; polysaccharide complex PSK® (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 (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); thiotepa; taxoid, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ without cremophor designed with albumin nanoparticle drug paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novant is n; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and lacavalier.

Also in this definition included protivokomarinye funds, which act to regulate, reduce, block, or inhibit the action of hormones that can stimulate the growth of malignant tumors and are often administered in the form of system or General treatment. They can imagine themselves hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene EVISTA®, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene FARESTON®; protivoprolezhnevyie means; negative regulators of estrogen receptors (ERD); means operating with the suppression or shutting down the ovaries, for example, agonists of growth hormone-releasing factor, luteinizing hormone (LHRH), is such as acetate leuprolide LUPRON® and ELIGARD®, goserelin acetate, acetate of buserelin and triptorelin; other anti-androgens such as flutamide, nilutamide and bikalutamid; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutetimid, acetate megestrol MEGASE®, exemestane AROMASIN®, formestane, fadrozole, vorozole RIVISOR®, letrozole, FEMARA® and anastrozole ARIMIDEX®. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate DIDROCAL®, NE-58095, zoledronicaa acid/zoledronate ZOMETA®, alendronate FOSAMAX®, AREDIA pamidronate®, tiludronate SKELID® or risedronate ACTONEL®; and troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, particularly those which inhibit expression of genes in the transmission path of a signal involved in berantai cell proliferation, such as PKC-alpha, Raf, H-Ras and the receptor for epidermal growth factor (EGF-R); vaccines such as THERATOPE vaccine® and gene therapy vaccines, for example, the vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX® rmRH; ditosylate of lapatinib (low molecular weight inhibitor of two tyrosinekinase ErbB-2 and EGFR, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of asanoha above.

"Growth inhibitory agent" when used herein refers to the compound or compositions that inhibit the growth of cells (such as cell expressing CD22)in vitroorin vivo. Thus, the growth inhibitory agent can be a tool that significantly reduces the percentage of cells (such as cells expressing CD22) in S-phase. Examples of inhibiting the growth of funds include funds that are blocking the passage of the cell cycle (in a place other than S phase), such as a means of inducing arrest in G1 and arrest in phase M. Classical blockers M-phases include periwinkle (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. The same tools that carry out the arrest in G1, also give a side effect of arrest in S-phase, for example, alkylating DNA tools, 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", Murakami et al., (WB Saunders: Philadelphia, 1995), especially on page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived and the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the Assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

The term "intracellular metabolite" refers to a compound resulting from the metabolic process or metabolic reactions within the cells of the conjugate antibody-drug (ADC). Metabolic process or metabolic reaction can be an enzymatic process, such as proteolytic cleavage of the peptide linker ADC or hydrolysis of functional groups, such as hydrazone, ester or amide. Intracellular metabolites include as non-limiting examples of antibodies and free drug that undergoes intracellular cleavage after joining, diffusion, capture, or transport into the cell.

The terms "derived intracellular and intracellular cleavage" refers to a metabolic process or reaction inside a cell with a conjugate of the antibody-drug (ADC), through which breaks the covalent connection, i.e. the linker between the molecule drug (D) and antibody (Ab), which leads to the separation of free lcars the public funds from the antibody inside the cell. Thus, the split-off group of the ADC are intracellular metabolites.

The term "bioavailability" refers to the system availability (i.e. levels in blood/plasma) given the number of drugs introduced to the patient. Bioavailability is an absolute term that refers to the dimension of time (speed) and total number (quantity) of a medicinal product which reaches the General flow of the entered dosage forms.

The term "cytotoxic activity" refers to unichtozhayuschego cells, cytotoxic or growth inhibitory effect of conjugate antibody-drug or intracellular metabolite conjugate antibody-drug. Cytotoxic activity can be expressed as the value of the IC50, which represents the concentration (molar or mass) per unit volume at which survives half cells.

"Alkyl" represents a C1-C18the hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are 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-Butert-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)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

As used herein, the term "C1-C8-alkyl" refers to saturated or unsaturated hydrocarbons with straight or branched chain with the number of carbon atoms from 1 to 8. Representative "C1-C8-alkyl" group includes the t as non-limiting examples methyl, -ethyl, -n-propyl, n-butyl, -n-pentyl, n-hexyl, n-heptyl, -n-octyl, ' n ' nonyl and-n-decyl; while branched C1-C8-alkali include as non-limiting examples,- isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C1-C8-alkali include as non-limiting examples,- vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylene, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -PROPYNYL, -1-butynyl, -2-butynyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-etylhexyl, 2,2-dimethylpentyl, 2,4-dimethylpentyl, 2.5-diethylhexyl, 3,5-dimethylhexane, 2,4-dimethylpentyl, 2-methylheptan, 3-methylheptan, n-heptyl, isoheptyl, n-octyl and isooctyl. C1-C8is an alkyl group can be unsubstituted or substituted by one or more groups that include as non-limiting examples of-C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -SO3R', -S(O)2R', -S(O)R', -OH, -g the lågen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Alkenyl" represents a C2-C18the hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms, at least one site of unsaturation, i.e. a double bond sp2carbon-carbon. Examples include as non-limiting examples of the ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7) and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

"Quinil" represents a C2-C18the hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms, at least one site of unsaturation, i.e. a triple bond sp carbon-carbon. Examples include as non-limiting examples of acetylene (-C=CH) and propargyl (-CH2C=CH).

"Alkylene" refers to a saturated, branched or linear or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers formed by removing two hydrogen atoms from the same or two different carbon atoms in the source alkane. Typical alkylene radicals include as non-limiting examples of the methylene (-CH2-), 1,2-ethyl (-CH2/sub> CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2and so on

"C1-C10-alkylene" is a straight saturated hydrocarbon group of the formula -(CH2)1-10-. Examples of C1-C10-alkylene include methylene, ethylene, propylene, butylene, pentile, hexylen, reptile, octiles, Nonlin and decalin.

"Albaniles" refers to an unsaturated branched or unbranched hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers formed by removing two hydrogen atoms from the same or two different carbon atoms in the original alkene. Typical alkenylamine radicals include as non-limiting examples of 1,2-ethylene (-CH=CH-).

"Akinyan" refers to an unsaturated, branched or unbranched hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers formed by removing two hydrogen atoms from the same or two different carbon atoms in the source alkyne. Typical akinleye radicals include as non-limiting examples of acetylene (-C≡C-), propargyl (-CH2C≡C-) and 4-pentenyl (-CH2CH2CH2C≡C-).

"Aryl" refers to carbocyclic aromatic groups is. Examples of aryl groups include as non-limiting examples of phenyl, naphthyl and anthracene. Carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or substituted by one or more groups that include as non-limiting examples of-C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

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

in which the phenyl group can be unsubstituted or substituted by up to 4 groups, including as non-limiting example,- C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Arylalkyl" refers to an acyclic alkyl, the radical, in which one of the hydrogen atoms associated with carbon atom, typically with a limit and the and sp 3-carbon atom, substituted aryl radical. Typical arylalkyl groups include as non-limiting examples are 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, alkenylphenol or alkenylphenol group, arylalkyl group consists of 1-6 carbon atoms, and the aryl group has from 5 to 14 carbon atoms.

"Heteroaromatic" refers to an acyclic alkyl, the radical, in which one of the hydrogen atoms associated with carbon atom, typically with a limit or sp3-carbon atom, substituted heteroaryl radical. Typical heteroallyl groups include as non-limiting examples of 2-benzimidazolylthio, 2-purolater etc. Heteroallyl group contains from 6 to 20 carbon atoms, for example, an alkyl group, including albanello, alkenylphenol or alkenylphenol group, heteroallyl group consists of 1-6 carbon atoms, and the heteroaryl group has from 5 to 14 carbon atoms and from 1-3 heteroatoms selected from N, O, P and S. the Heteroaryl group heteroallyl group may be a monocycle with the number of ring members from 3 to 7 (from 2 to 6 carbon atoms) or bicycl with what kolichestvo ring members from 7 to 10 (4 to 9 carbon atoms and 1 to 3 heteroatoms, selected from N, O, P and S), for example, bicyclo[4,5], [5,5], [5,6] or [6,6].

"Substituted alkyl", "substituted aryl" and "substituted arylalkyl" mean alkyl, aryl and arylalkyl, respectively, in which one or more hydrogen atoms are each independently replaced with the Deputy. Typical substituents include as non-limiting examples of-X, -R, -O-, -OR, -SR, -S-, -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, NC(=O)R, -C(=O)R, -C(=O)NR2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3-, -PO3H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2where each X independently represents a halogen: F, Cl, Br or I; and each R independently represents-H, C2-C18-alkyl, C6-C20-aryl, C3-C14-heterocycle, protecting group or molecule procarcinogen funds. Allenova, Alcanena and akinlana group, as described above, can also be substituted in this way.

"Heteroaryl" and "heterocycle" refers to a cyclic system in which one or more ring atoms are heteroatoms, for example nitrogen, oxygen and sulfur. Heterocyclic radical contains from 1 to 20 atoms of carbon and from 1 to 3 heteroatoms, selected from N, O, P and S. the Heterocycle may be a monocycle with the number of ring members from 3 to 7 (from 2 to 6 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S) or Bicycle with the number of ring members from 7 to 10 (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example, bicyclo[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.

Examples of heterocycles include, as examples, not limitations, pyridyl, dihydropyridin, tetrahydropyranyl (piperidyl), thiazolyl, tetrahydrothiophene, oxidized grey tetrahydrothiophene, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianeptine, indolyl, indolinyl, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydropyranyl, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6H-1,2,5-tedisamil, 2H,6H-1,5,2-detainer, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xant the Nile, femoxetine, 2H-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, honokalani, hintline, cinnoline, pteridine, 4aH-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, furutani, phenoxazines, isopropanol, bromanil, imidazolidinyl, imidazolyl, pyrazolidine, pyrazoline, piperazinil, indolinyl, isoindolyl, hinokitiol, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazole, Kendall, benzoxazolyl and satanail.

As an example, and not limitations on carbon heterocycles are linked in position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5 or 6 pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5 or 6 pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4 or 5 oxazole, imidazole or thiazole, position 3, 4 or 5 isoxazol, pyrazole or isothiazole, position 2 or 3 of aziridine, position 2, 3 or 4 azetidine, position 2, 3, 4, 5, 6, 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 of isoquinoline. Even more typically associated carbon heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-feast of dainel, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

As an example, and not limitation, related nitrogen heterocycles are connected to position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of the research and position 9 carbazole or β-carboline. Even more typically related nitrogen heterocycles include 1-ezyrider, 1-azetidin, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

"C3-C8-heterocycle" refers to an aromatic or non-aromatic C3-C8carbocycle, in which from one to four cyclic carbon atoms are independently replaced by a heteroatom from the group consisting of O, S and N. Representative examples of C3-C8-heterocycle include as non-limiting examples benzofuranyl, benzothiophene, indolyl, benzimidazolyl, coumarinyl, ethenolysis, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, chinoline, pyrimidinyl, pyridinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolin, isoxazolyl and tetrazolyl. C3-C8-heterocycle may be unsubstituted or substituted groups in the number to seven, including as non-limiting examples, -C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"C3-C8-heterocycle" refers to C3-C8-heterocyclic group, as defined above, where one of the hydrogen atoms of the heterocyclic group substituted communication. C3-C8-heterocycle may be unsubstituted or substituted groups up to six, including as non-limiting examples, -C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Carbocycle" means a saturated or unsaturated cyclic system with the number of carbon atoms from 3 to 7 in the form of a monocycle or with the number of carbon atoms from 7 to 12 in the form of Bicycle. Monocyclic carbocycle contain from 3 to 6 cyclic atoms, even more typically 5 or 6 cyclic atoms. Bicyclic carbocycle contain from 7 to 12 cyclic atoms, for example, arranged in the form of bicyclo[4,5], [5,5], [5,6] or [6,6], ili or 10 cyclic atoms, located in the form of bicyclo [5,6] or [6,6]. Examples of monocyclic carbon cyclic systems include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, l-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl and cyclooctyl.

"C3-C8-carbocycle" is a 3-, 4-, 5-, 6-, 7 - or 8-membered saturated or unsaturated non-aromatic carbocyclic ring system. Representative C3-C8-carbocycle include as non-limiting examples cyclopropyl-cyclobutyl-cyclopentyl, cyclopentadienyl, cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadiene, -1,3,5-cycloheptatriene, -cyclooctyl and-cyclooctadiene. Group C3-C8-carbocycle may be unsubstituted or substituted by one or more groups that include as non-limiting examples of-C1-C8-alkyl, -O-(C1-C8-alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"C3-C8-carbocycle" refers to the group of C3-C8-carbocycle defined above, where one of the atoms of odor the Yes group carbocycle substituted connection.

"Linker" refers to a chemical group that contains covalent bond or a chain of atoms that covalently attach the antibody to the molecule drugs. In various embodiments, the implementation of linkers include a divalent radical such as alkerdeel, areldil, heteroaryl, groups such as -(CR2)nO(CR2)n-repeating unit alkyloxy (for example, polietilene, PEG, polymethylenes), alkylamino (for example, polyethylenimine, Jeffamine™); and esters and amides of dibasic acids, including succinate, succinamide, diglycolate, malonate and caproamide.

The term "chiral" refers to molecules which have the property of incompatibility with its reflection, while the term "achiral" refers to molecules that are compatible with its reflection.

The term "stereoisomers" refers to compounds that have the same chemical composition, but differ in the arrangement of atoms or groups in space.

"Diastereoisomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactively. See the si diastereomers can be divided analytical methods with high resolution, such as electrophoresis and chromatography.

"Enantiomers" refers to stereoisomers of the compounds, which are analagies mirror images of each other.

Stereochemical definitions and conventions used in this document, for the most part consistent with S.P.Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound to denote the absolute configuration of the molecule near its chiral center(s) use the prefixes D and L or R and S. the Prefixes d and l or (+) and (-) are used to designate the sign of rotation of plane-polarized light by the compound, where (-) or l means that the connection is levogyrate. The connection to the prefix (+) or d is Pervouralsk. For a given chemical structure of these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer is also possible to define as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. The mixture of enantiomers of 50:50 is designated as a racemic mixture or a racemate, which can form the I, when a chemical reaction or process is not stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refers to an equimolar mixture of two enantiomeric molecules devoid of optical activity.

"Leaving group" refers to a functional group that can substitute another functional group. In this area some well-known leaving group, and examples include as non-limiting examples of the halides (e.g. chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluensulfonyl (tosyl), trifloromethyl (triplet) and triftormetilfullerenov.

Reduction

LINKER COMPONENTS:

MC = 6-maleimidomethyl

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

The citrulline = 2-amino-5-breedopedia acid

PAB = p-aminobenzeneboronic (example "auto" linker component)

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

MC(PEG)6-OH = maleimidomethyl-polyethylene glycol (can be attached to zisteinom antibodies)

SPP = N-Succinimidyl-4-(2-pyridylthio)pentanoate

SPDP = N-Succinimidyl-3-(2-pyridyldithio)propionate

SMCC = Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

IT = they who nationan

CYTOTOXIC DRUGS:

MMAE = monomethylaniline E (MW 718)

MMAF = option auristatin E (MMAE) with a phenylalanine at the C-end medicines (MW 731,5)

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

MMAF-TEG = MMAF with tetraethylene glycol, in ester bonds with phenylalanine

MMAF-NtBu = N-tert-butyl, attached in the form of amide to the C-end of MMAF

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

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

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

Additional abbreviations are as follows: AE is auristatin E, Boc represents N-(tert-butoxycarbonyl), cit is citrulline, dap is dalapon, DCC is a 1,3-dicyclohexylcarbodiimide, DCM represents dichloromethane, DEA is diethylamin, DEAD is diethylazodicarboxylate, DEPC is diethylphosphoramidite, DIAD is diisopropylsalicylic, DIEA represents N,N-diisopropylethylamine, dil is daisosasen, DMA is a dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is a dimethyl ether of ethylene glycol (or 1,2-dimethoxy the Tang), DMF represents N,N-dimethylformamide, DMSO is a sulfoxide, doe is draftin, dov is a N,N-dimethylamine, DTNB is a 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is a hydrochloride of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EEDQ is a 2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is an acid ethyl ester, Fmoc represents N-(9-fluorenylmethoxycarbonyl), gly represents glycine, HATU is hexaphosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea, HOBt represents 1-hydroxybenzotriazole, HPLC is a high-performance liquid chromatography, ile represents isoleucine, lys is a lysine, MeCN (CH3CN) is acetonitrile, MeOH is a methanol, Mtr is a 4-insidefamily (or 4-methoxytrityl), nor is a (1S,2R)-(+)-norephedrine, PBS is a phosphate-saline buffer (pH 7,4), PEG represents a polyethylene glycol, Ph represents phenyl, Pnp is a p-nitrophenyl, MC represents a 6-maleimidomethyl, phe represents L-phenylal the NIN, PyBrop is hexaphosphate bromo-Tris-pyrrolidinone, SEC represents exclusion chromatography, Su represents succinimide, TFA represents triperoxonane acid, TLC is a thin-layer chromatography, UV is a UV and val is a valine.

COMPOSITIONS AND METHODS for THEIR preparation

The invention relates to antibodies that bind CD22. Also immunoconjugates containing anti-CD22 antibodies. Antibodies and immunoconjugates according to the invention can be used, for example, for the diagnosis or treatment of disorders associated with altered expression of, for example, increased expression of CD22. In specific embodiments, the implementation of antibodies or immunoconjugates of the present invention can be used to diagnose or treat disorders associated with impaired cell proliferation, for example, malignant tumors.

Anti-CD22 antibodies

In one aspect the invention relates to antibodies that bind to CD22. In some embodiments, the implementation of the provided antibodies that are associated with the adult form of CD22 human and cynomolgus monkey (cyno). In one such embodiment, the Mature form of human CD22 has amino acid sequence SEQ ID NO: 27. The main Mature isoform of the person with whom contains the extracellular domain, containing seven Ig-like domains, and has the amino acid sequence of SEQ ID NO: 28. In another embodiment, the minor isoform of human CD22 with missing extracellular domains 3 and 4 has the amino acid sequence of SEQ ID NO: 29. Amino acid sequence of the extracellular domain of the minor isoform represents SEQ ID NO: 30. CD22 cyno has the amino acid sequence of SEQ ID NO: 31. In some embodiments, the implementation of the antibody to CD22 binds to a Mature form of CD22 expressed on the cell surface. In some embodiments, the implementation of the antibody to bind with Mature form of CD22 expressed on the cell surface and inhibits cell growth. In some embodiments, the implementation of the antibody to CD22 binds to a Mature form of CD22 expressed on the cell surface and inhibits cell proliferation. In certain embodiments of the implementation of the antibody to CD22 binds to a Mature form of CD22 expressed on the cell surface and induces death of cells. In some embodiments, the implementation of the antibody to CD22 binds to a Mature form of CD22 expressed on the surface of malignant cells. In some embodiments, the implementation of the antibody to CD22 binds to a Mature form of CD22, which sverkhekspressiya on the surface of malignant cells sravnenie the normal cells of the same tissue origin. In some embodiments, the implementation of the antibody to CD22 is conjugated to a cytotoxin or a detectable label and binds to CD22 on the cell surface. In some embodiments, the implementation of the conjugate antibody-toxin inhibits cell growth. In some embodiments, the implementation of the conjugate antibody is detected by the label implies that the cell expressing on its surface CD22, becomes detectablein vitroorin vivo.

In one aspect the antibody to CD22 is a monoclonal antibody. In one aspect the antibody to CD22 is a fragment of the antibody, e.g., Fab fragment, Fab'-SH, Fv, scFv, or (Fab')2. In one aspect the antibody to CD22 is a chimeric, humanitariannet antibody or a human antibody. In one aspect of any of the antibodies to CD22, described herein, is purified.

In this document is provided for illustrative monoclonal antibodies obtained from ragovoy library. Antigen used for screening libraries were the polypeptide with the sequence of amino acid sequences SEQ ID NO: 28 or SEQ ID NO: 30, the respective extracellular domains (ECD) CD22 beta and alpha. Antibodies obtained by screening libraries that are affine Mature.

In one aspect provided by the mod is olonline antibodies which compete with 10F4.4.1 mouse humanitarianism 10F4v1 and v3, and 5E8.1.8 mouse for binding to CD22. Also provides monoclonal antibodies that bind the same epitope as 10F4.4.1 mouse, humanized 10F4v1 and v3, and 5E8.1.8 mouse.

In one aspect of the invention provided polynucleotide encoding antibodies to CD22. In certain embodiments of the implementation of the given vectors containing polynucleotide encoding antibodies to CD22. In certain embodiments of the implementation of the given cell host containing such vectors. In another aspect of the invention is provided a composition containing antibodies to CD22 or polynucleotide encoding antibodies to CD22. In certain embodiments of the implementation of the composition according to the invention is a pharmaceutical drug for the treatment of cell proliferative disorders, such as listed in this document violations.

Introduction and preparation of antibodies

In one embodiment, the implementation of the antibody to CD22 or conjugate antibody to CD22-drug (including as non-limiting examples, the conjugate Tioman to CD22-drug according to the invention) according to the invention is administered in combination with an antagonist of the surface antigen of B-cells. Introduction "in combination with" one or more additional therapeutic means on which includes simultaneous (concurrent) and consecutive introduction of any order. In one embodiment, the implementation of the introduction is sequential or alternating. In another embodiment, the introduction is simultaneous, parallel, or combined in one and the same drug. In one of the embodiments, the antagonist of the surface antigen of B-cells is an antibody or antigen binding fragment. In one of the embodiments, the antagonist of the surface of B-cells is a conjugate of the antibody-drug.

The drugs on this document as necessary for the particular indication to be treated, can contain more than one active compound, preferably an active connection to a complementary activity that does not affect each other adversely. For example, in addition to the antibody to CD22, conjugate the antibody to CD22-drug or CD22 binding to the Oligopeptide may be desirable to include one additional drug antibody, for example, the second antibody to CD22, which binds a different epitope on the CD22 polypeptide, or a second antibody that binds to another surface antigen B-cell, or an antibody to some other target such as a growth factor, which acts on the specific growth of a malignant tumor. Alternative or additionally, the composition can be the t further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, protivogelmintnoe tool and/or cardioprotector. Such molecules, respectively, are combined in amounts that are effective for the designated purposes.

Currently, depending on the stage of a malignant tumor, the treatment of malignant tumors includes one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy and chemotherapy. Therapy with antibodies to CD22, conjugate the antibody to CD22-drug or oligopeptides to CD22 may be especially desirable in elderly patients who are poor tolerate the toxicity and side effects of chemotherapy and metastatic disease, when radiation therapy has limited usefulness. Aimed at the tumor antibodies to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 according to the invention is suitable to facilitate expressing CD22 malignant tumors at initial diagnosis or at relapse. For therapeutic applications the antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 can be used separately or in combined treatment, such as hormones, antiangiogenic drugs or radiation is uchennymi compounds or in conjunction with surgery, cryotherapy and/or radiotherapy. Treatment with antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 can be combined with other forms of conventional therapy, consistently with traditional therapy, before or after it. In this method according to the invention for the treatment or relief of a malignant tumor patient with a malignant tumor, you can enter the antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 in conjunction with the treatment of one or more previously known chemotherapeutics. Antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 is administered with a therapeutically effective dose of a chemotherapeutic drug. In another embodiment, the antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22 is administered in conjunction with chemotherapy to enhance the activity and efficacy of chemotherapeutic agents. In The Physicians' Desk Reference (PDR) describes the dosage of these funds, which are used to treat a variety of malignant tumors. Dosing regimens and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective, depend on the specific malignant tumor to be treated, the degree of illness is evania and other factors, well known to practitioners in this field, and the doctor can easily be determined.

In one specific embodiment, the patient is given a conjugate containing an antibody to CD22, conjugate the antibody to CD22-drug or Oligopeptide to CD22, conjugated with a cytotoxic agent. Preferably immunoconjugate associated with protein CD22, internalized cell, resulting resulting in increased therapeutic efficacy immunoconjugate destruction of malignant cells with which it is associated. In one embodiment, the implementation of the cytotoxic agent strikes or blocks of nucleic acid into a malignant cell. Examples of cytotoxic agents are described above and include auristatin, maytansinoid, calicheamicin, ribonuclease and endonuclease DNA or their biologically active derivatives.

Antibodies to CD22, the conjugates of the antibody to CD22-drug or oligopeptides to CD22, or their conjugates with a toxin is administered to the human patient by known methods, such as intravenous administration, e.g., in the form of a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneally, intracerebrally, subcutaneous, intravascular, intrasynovial, intrathecal, oral, local or way of inhalation is mi. Preferred are intravenous or subcutaneous administration of the antibody, conjugate the antibody to CD22-drug or oligopeptides to CD22.

With the introduction of antibodies to CD22, conjugate the antibody to CD22-drug or oligopeptides to CD22 can be combined with other treatment regimens. The combined introduction includes co-administration with use of certain drugs or one pharmaceutical drug and consistent introduction in any order, preferably where there is a time period while both (or all) active funds at the same time exhibit their biological activity. Preferably the combined treatment leads to a synergistic therapeutic effect.

Also, it may be desirable to combine the introduction of antibodies to CD22 or antibody conjugates of the antibody to CD22-drug or oligopeptides to CD22 with antibodies directed to another tumor antigen or surface antigen of B-cells associated with a particular malignancy.

In yet another embodiment, therapeutic methods of treatment of the present invention include the combined introduction of the anti-CD22 antibody (or antibodies), conjugate(s), anti-CD22 antibodies and drugs or oligopeptides(s) together with one or more chimiotherapie the practical means or means, any abscopal growth, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include phosphate estramustine, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyurethane (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics, as well as the combination of means such as, but not limited to, CHOP or FOLFOX. Protocols for obtaining and dispensing for such chemotherapeutic agents can be used in accordance with the manufacturer's instructions or as determined empirically by practitioners. Protocols for obtaining and dispensing for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

The antibody is administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intra-lungs, intranasal and/or intranidus introduction. Parenteral administration include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Also provided by intrathecal injection (relative to the intrathecal delivery of antibodies to CD20 see, for example, patent application U.S. 2002/0009444, Grillo-Lopez, A). Preferably, the dosing is carried out intravenously or subcutaneously.

The second drug in the contain with the initial impact and/or later the effects of therapeutic antibodies or immunoadhesins, where the combined introduction includes co-administration, using separate drugs or a single pharmaceutical preparation, and consistent introduction in any order, preferably where there is a time period while both (or all) active funds at the same time exhibit their biological activity.

Although therapeutic antibody to CD22, conjugate the antibody to CD22-drug immunoadhesin or other biological agent for the treatment of autoimmune diseases can be entered as the only means, as a rule, therapeutic antibody or immunoadhesin combine with one or more secondary drugs. For example, for RA and other autoimmune diseases, antibody, immunoadhesin or other biological medicinal product is preferably combined with any one or more of IMMUNOSUPRESSIVE funds, chemotherapeutic agents, BAFF antagonists, antagonists or antibodies to integrins and/or cytokines listed in the definitions above; any one or more disease modifying Antirheumatic drugs (DMARDs), such as hydroxychloroquine, sulfasalazin, methotrexate, Leflunomide, azathioprine, D-penicillamine, gold (oral), gold (intramuscular), mi is Oticon, cyclosporine; Immunoadsorbent staphylococcal protein A; intravenous immunoglobulin (IVIG); non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoid (e.g., by injection into the blood vessels); a corticosteroid (e.g., methylprednisolone and/or prednisone); folate; antagonist for the antibody to the tumor necrosis factor (TNF), for example, etanercept/ENBREL™, infliximab/REMICADE™, D2E7 (Knoll) or CDP-870 (Celltech); an antagonist of IL-1R (e.g., Kineret); antagonist 1L-10 (for example, inadequacy); modulator blood clotting (for example, WinRho); an antagonist of IL-6/antibody to TNF (CBP-1011); CD40 antagonist (for example, IDEC 131); antagonist Ig-Fc-receptor (MDX33); an immunomodulator (e.g., thalidomide or ImmuDyn); antibody to CD5 (for example, H5g1.1); inhibitor of macrophages (e.g., MDX, 33); co-stimulating blocker (for example, BMS 188667 or taurima); inhibitor of complement (e.g., h5G1.1, 3E10 or antibody to accelerating the decay factor (DAF)); antagonist IL-2 (zxSMART); inhibitor of EGFR (see definition above); a tyrosine kinase inhibitor (see definition above); antiangiogenic agent (for example, antibody to VEGF, such as bevacizumab); antibodies to CD22, such as LL2 or epratuzumab (LYMFOCIDE®; Immunomedics), including epratuzumab Y-90 (Juweid et al., Cancer Res 55(23 Suppl):5899s-5907s (1995)), antibody to CD22 Abiogen (Abiogen, Italy), CMC 544 (Wyeth/Celltech), combaticon (UT Soutwestern), BL22 (NIH) and LympoScan Tc99 (Immunomedics); anti-Christ. elom to EpCAM, such as 17-1A (PANOREX®); antibody ∀v∃3 (e.g., VITAXIN®; Medimmune); antibody to CD37, such as TRU016 (Trubion); antibody to IL-21 (Zymogenetics/Novo Nordisk); antibody to B-cells (Impheron); focused on B-cell MAb (Immunogen/Aventis); 1D09C3 (Morphosys/GPC); LymphoRad 131 (HGS); antibody to Lym-1 Y-90 (USC); LIF 226 (Enhanced Lifesci.); antibody to BAFF (for example, WO 03/33658); antibody to BAFF receptor (for example, WO 02/24909); BR3 antibody; antibody Blys, such as belimumab; LYMPHOSCD22-B™; ancalimon to Lym-1 (USC/Peregrine); ISF 154 (UCSD/Roche/Tragen); gomulicki (Idec 152; Biogen Idec); antibody to the receptor of IL-6, such as talisuna (ACTEMRA™; Chugai/Roche); antibody to IL-15, such as HuMax-Il-15 (Genmab/Amgen); antibody to the receptor for chemokines, such as antibody to CCR2 (e.g., MLN1202; Millieneum); antibody to complement, such as antibody to C5 (for example, eculizumab, 5G1.1; Alexion); oral preparation of human immunoglobulin (e.g., IgPO; Protein Therapeutics); antibody to IL-12, such as ABT-874 (CAT/Abbott); Teplitskaya (BMS-224818); B-cell vaccine; DN-BAFF (Xencor); CRx-119 (CombinatoRx); BAFF antagonist Amgen; pentostatin (Pfizer); IC-485 (ICOS); antagonist chemokines, such as T-487 (tularik) or reticulosa (AVR-118); SCO-323 (SCIOS); antagonist integrins 683699, Tanabe, NGD-2001-1 (Neurogen); SCIO-469 (SCIOS); BIRB-796 (Boehringer Ingelheim); VX702, VX850 (Vertex); an antagonist of leukotriene B-4 (such as ameloot, BIIL-284; BI); modulator of microtubules (Paxceed; Angiotech); inhibitor of proteases (MBS561392; BMS); AGIX-4207 (Atherogenics); ISIS-104838 (ISIS/Elan); MFG-IRAP (Univ. Pitt.); trap IL-1 (RGN-303; Regeneron/Novartis); ODA is Lukina (Wyeth); the everolimus (certican; Novartis); AMEVIVE (Biogen Idec); ORG-39141 (Organon); FK-506 (Fujisawa); and an antagonist of IL-2 (tacrolimus; Fujisawa).

Detailed description of illustrative antibodies to CD22 is as follows:

1. Specific embodiments of the antibodies to CD22

In one aspect the invention relates to an antibody that contains at least one, two, three, four, five or six HVR selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 4; (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO: 6; (d) HVR-L1 containing the amino acid sequence of any one of SEQ ID NO: 9, 10, 19, 20, 21, 22, 23; ((e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 12 and (f) HVR-L3 containing the amino acid sequence selected from SEQ ID NO: 14.

In one aspect the invention relates to the antibody to CD22, containing at least one, at least two, or all three of HVR sequence VH selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 4; (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO: 6. In one aspect the invention relates to the antibody to CD22, containing HVR-H1 containing the amino acid series is here SEQ ID NO: 2. In one aspect the invention relates to the antibody to CD22, containing HVR-H2 containing the amino acid sequence of SEQ ID NO: 4. In one aspect the invention relates to the antibody to CD22, containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO: 6.

In one aspect the invention relates to the antibody to CD22, containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO: 6, the HVR-H1 containing the amino acid sequence selected from SEQ ID NO: 2.

In one aspect the invention relates to the antibody to CD22, containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO: 6, and HVR-H2 containing the amino acid sequence selected from SEQ ID NO: 4.

In one aspect the invention relates to the antibody to CD22, containing HVR-H1 containing the amino acid sequence of SEQ ID NO: 2, and HVR-H2 containing the amino acid sequence of SEQ ID NO: 4.

In one aspect the invention relates to the antibody to CD22, containing HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; HVR-H2 containing the amino acid sequence of SEQ ID NO: 4; and HVR-H3 containing the amino acid sequence of SEQ ID NO: 6.

In one aspect the invention relates to the antibody to CD22, containing at least one, at least two or all three members is Telenesti HVR LL, selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 12; and (c) HVR-L3 containing the amino acid sequence selected from SEQ ID NO: 14. In one aspect the invention relates to the antibody to CD22, containing HVR-L1 containing the amino acid sequence selected from SEQ ID NO: 9. In one aspect the invention relates to the antibody to CD22, containing HVR-L1 containing the amino acid sequence selected from SEQ ID NO: 10. In one aspect the invention relates to the antibody to CD22, containing HVR-L1 containing the amino acid sequence selected from SEQ ID nos: 19-23. In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N28 replaced by V (amino acid replacement N28V, giving the sequence of SEQ ID NO: 10). In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N28 replaced by A (amino acid replacement N28A, giving SEQ ID NO: 19). In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N28 replaced by Q (amino acid replacement N28Q giving SEQ ID NO: 20). In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N28 substituted by S (amino acid replacement N28S, giving SEQ ID NO: 21). In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N8 replaced by D-amino acid replacement N28D, giving SEQ ID NO: 22). In one aspect of the HVR-L1 contains the amino acid sequence of SEQ ID NO: 9, where N28 replaced by I (amino acid replacement N28I giving SEQ ID NO: 23). In one aspect the invention relates to the antibody to CD22, containing HVR-L1 containing the amino acid sequence of any one of SEQ ID NO: 9, 10, 19, 20, 21, 22, 23. In one aspect of the HVR-L1 represent any one of SEQ ID NO: 9, 10, 19, 20, 21, 22 or 23 and the amino acid in position N30 (asparagine at position 30) replaced by A (amino acid replacement N30A). In one aspect of the HVR-L1 represent any one of SEQ ID NO: 9, 10, 19, 20, 21, 22 or 23 and the amino acid in position N30 (asparagine at position 30) replaced by Q (amino acid replacement N30Q).

In one aspect the invention relates to the antibody to CD22, containing (a) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6, and (b) HVR-L3 containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the implementation of the antibody to CD22 further comprises (a) HVR-H1 containing SEQ ID NO: 2, and HVR-H2 containing SEQ ID NO: 4.

In one aspect the invention relates to the antibody to CD22, containing (a) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6, and (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the implementation of the antibody to CD22 further comprises (a) HVR-H1 containing SEQ ID NO: 2, and HVR-H2, sotiriadou is SEQ ID NO: 4.

In one aspect the invention relates to the antibody to CD22, containing (a) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6, and (b) HVR-L1 containing the amino acid sequence selected from SEQ ID NO: 9, 10, 19, 20, 21, 22 and 23. In some embodiments, the implementation of the antibody to CD22 further comprises (a) HVR-H1 containing SEQ ID NO: 2, and HVR-H2 containing SEQ ID NO: 4. In some embodiments, the implementation of the amino acid sequence of SEQ ID NO: 9, 10, 19, 20, 21, 22 or contains 23 amino acid replacement N30A or N30Q. In some embodiments, the implementation of the antibody to CD22 further comprises HVR-L2, containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the implementation of the antibody to CD22 further comprises HVR-L3 containing the amino acid sequence of SEQ ID NO: 14.

In one aspect the invention relates to the antibody to CD22, containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 4; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 containing the amino acid sequence selected from SEQ ID NO: 9, 10, 19, 20, 21, 22, 23; ((e) HVR-L2 containing amino acid sequence of SEQ ID NO: 12; and HVR-L3, containing the amino acid sequence of SEQ ID NO: 14. In some embodiments implementing the invention additionally relates to aminokislotnoi sequence SEQ ID NO: 9, 10, 19, 20, 21, 22 or 23, is selected as the HVR-L1, a modified amino acid substitution N30A or N30Q.

In one aspect the invention relates to the antibody to CD22, containing the variable domain of the heavy chain, containing SEQ ID NO: 16 (see figa, h10F4v1). In one aspect the invention relates to the antibody to CD22, containing the variable domain of the light chain containing SEQ ID NO: 17 (see figv, h10F4v1). In one aspect the invention relates to the antibody to CD22, containing the variable domain of the light chain containing SEQ ID NO: 18 (see figv, h10F4v3).

In one aspect the invention relates to the antibody to CD22, containing a heavy chain containing SEQ ID NO: 34 (see figa, m10F4). In one aspect the invention relates to the antibody to CD22 containing light chain containing SEQ ID NO: 35 (see figv, m10F4).

In one aspect the invention relates to the antibody to CD22, containing 1, 2, 3, 4, 5 or 6 of the HVR sequences of the antibodies 10F4.4.1 obtained through hybridoma deposited in the ATCC and having inventory number PTA-7621.

In one aspect the invention relates to the antibody to CD22, containing 1, 2, 3, 4, 5 or 6 of the HVR sequences of the antibodies 5E8.1.8 obtained through hybridoma deposited in the ATCC and having inventory number PTA-7620.

Antibody to CD22 can contain any frame sequence of the variable domain, provided that the antibody retains sposobnosti to bind CD22. For example, in some embodiments, the implementation of antibodies to CD22 according to the invention contain a consensus framework sequence of the heavy chain subgroup III person. In one embodiment, the implementation of these antibody consensus framework sequence of the heavy chain contains the replacement(s) at positions 71, 73 and/or 78.

In one of the embodiments of these antibodies, position 71 is a, A, position 73 is a T and/or position 78 is an A. In one embodiment, the implementation of these antibodies contain the frame sequence of the variable domain of the heavy chain huMAb4D5-8, for example, SEQ ID NO: 1, 3, 5, 7 (FR-H1, FR-H2, FR-H3, FR-H4, respectively). huMAb4D5-8 commercially known as GERCEPTIN® antibody to HER2, Genentech, Inc., South San Francisco, CA, USA; also mentioned in U.S. patent No. 6407213 and 5821337 and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93. In one such embodiment, these antibodies also include consensual frame sequence light chain human ☐I. In one such embodiment, these antibodies contain the frame sequence of the variable domain of the light chain of huMAb4D5-8, for example, SEQ ID NO: 8, 1, 13, 15 (FR-L1, FR-L2, FR-L3, FR-L4, respectively).

In one embodiment, the implementation of the antibody to CD22 contains a variable domain of a heavy chain containing a frame sequence and a hypervariable region, where resna sequence contains the sequence FR-H1-FR-H4 SEQ ID NO: 1, 3, 5 and 7, respectively, and HVR H1 contains the amino acid sequence of SEQ ID NO: 2; HVR-H2 contains the amino acid sequence of SEQ ID NO: 4, and HVR-H3 contains the amino acid sequence selected from SEQ ID NO: 6. In one embodiment, the implementation of the antibody to CD22 contains the variable domain of the light chain containing the frame sequence and hypervariable region where the frame sequence contains the sequence FR-L1-FR-L4 SEQ ID NO: 8, 11, 13 and 15, respectively; HVR-L1 contains an amino acid sequence selected from SEQ ID NO: 9, 10, 19, 20, 21, 22 and 23, where any one of SEQ ID nos: 9-10 or 19-23 may contain amino acid replacement N30A or N30Q; HVR-L2 contains the amino acid sequence of SEQ ID NO: 12, and HVR-L3 contains the amino acid sequence selected from SEQ ID NO: 14. In one of the embodiments the antibody variable domain of the heavy chain contains SEQ ID NO: 16, and the variable domain of the light chain contains SEQ ID NO: 17 or 18.

In some embodiments implementing the invention relates to the antibody to CD22, containing the variable domain of the heavy chain containing the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the implementation of the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but the antibody containing the amino acid sequence retains the ability to bind to CD22. In some embodiments, implementation of the sequence SEQ ID NO: 16 substituted, inserted or deleted only from 1 to 10 amino acids. In some embodiments, replace, insert and delete are conducted in areas outside the HVR (i.e., in FR). In some embodiments, the implementation of the antibody to CD22 contains a variable domain of a heavy chain containing the amino acid sequence selected from SEQ ID NO: 16.

In some embodiments implementing the invention relates to the antibody to CD22, containing the variable domain of the heavy chain, as shown below.

1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala SerGly Tyr Glu Phe Ser Arg Ser Trp Met AsnTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe Lys GlyLys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala ArgAsp Gly Ser Ser Trp Asp Trp Tyr Phe Asp TyrTrp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 113 (SEQ ID NO: 16) (HVR residues are underlined).

In some embodiments, the sequence HVR and FR heavy chains contain the following:

HVR-H1 (Gly Tyr Glu Phe Ser Arg Ser Trp Met Asn, SEQ ID NO: 2)

HVR-H2 (Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe Lys Gly, SEQ ID NO: 4)

HVR-H3 (Asp Gly Ser Ser Trp Asp Try Tyr PheAsp Tyr, SEQ ID NO: 6)

FR-H1 (Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser, SEQ ID NO: 1)

FR-H2 (Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val, SEQ ID NO: 3)

FR-H3 (Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg, SEQ ID NO: 5)

FR-H4 (Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser, SEQ ID NO: 7)

In some embodiments implementing the invention relates to the antibody to CD22, containing the variable domain of the light chain, as shown below.

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysArg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Phe Leu GluTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile TyrLys Val Ser Asn Arg Phe SerGly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr CysPhe Gln Gly Ser Gln Phe Pro Tyr ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO: 17) (HVR residues are underlined, and the position N28 printed in bold)

or

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysArg Ser Ser Gln Ser Ile Val His Ser Val Gly Asn Thr Phe Leu GluTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile TyrLys Val Ser Asn Arg Phe SerGly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr CysPhe Gln Gly Ser Gln Phe Pro Tyr ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO: 18) (HVR residues are underlined, and the position N28 printed in bold).

In some embodiments, the sequence light chain HVR contain the following:

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Phe Leu Glu, SEQ ID NO: 9)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Val Gly Asn Thr Phe Leu Glu, SEQ ID NO: 10)

HVR-L1 (Arg Ser Se Gln Ser Ile Val His Ser Ala Gly Asn Thr Phe Leu Glu, SEQ ID NO: 19)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Gln Gly Asn Thr Phe Leu Glu, SEQ ID NO: 20)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Asn Thr Phe Leu Glu, SEQ ID NO: 21)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Asp Gly Asn Thr Phe Leu Glu, SEQ ID NO: 22)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly Asn Thr Phe Leu Glu, SEQ ID NO: 23)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly Ala Thr Phe Leu Glu, SEQ ID NO: 32)

HVR-L1 (Arg Ser Ser Gln Ser Ile Val His Ser Ile Gly Gln Thr Phe Leu Glu, SEQ ID NO: 33)

HVR-L2 (Lys Val Ser Asn Arg Phe Ser, SEQ ID NO: 12)

HVR-L3 (Phe Gln Gly Ser Gln Phe Pro Tyr Thr, SEQ ID NO: 14).

In some embodiments, the sequence (FR light chain contains the following:

FR-L1 (Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys, SEQ ID NO: 8);

FR-L2 (Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr, SEQ ID NO: 11);

FR-L3 (Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys, SEQ ID NO: 13)

FR-L4 (Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg, SEQ ID NO: 15).

In one aspect the invention relates to the antibody to CD22, containing the variable domain of the light chain containing the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence selected from SEQ ID NO: 17 or 18. In some embodiments, the implementation of the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions, additions or deletions relative to the reference sequence, but the antibody containing the amino acid after outermost, retains the ability to bind to CD22. In some embodiments, implementation of a sequence selected from SEQ ID NO: 17 or 18, substituted, inserted or deleted only from 1 to 10 amino acids. In some embodiments, replace, insert and delete are conducted in areas outside the HVR (i.e., in FR). In some embodiments, the implementation of the antibody to CD22 contains the variable domain of the light chain containing the amino acid sequence selected from SEQ ID NO: 17 or 18.

In one aspect the invention relates to the antibody to CD22, containing (a) a variable domain of a heavy chain containing the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence selected from SEQ ID NO: 16; and (b) the variable domain of the light chain containing the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence selected from SEQ ID NO: 17 or 18. In some embodiments, the implementation of the amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions, additions or deletions relative to the reference sequence, but the antibody containing the amino acid sequence retains the ability to bind to CD22. In some embodiments, is sushestvennee in the reference sequence substituted, inserted or removed only from 1 to 10 amino acids. In some embodiments, replace, insert and delete are conducted in areas outside the HVR (i.e., in FR). In some embodiments, the implementation of the antibody to CD22 contains a variable domain of a heavy chain containing the amino acid sequence of SEQ ID NO: 16, and the variable domain of the light chain containing the amino acid sequence selected from SEQ ID NO: 18.

In one aspect the invention relates to the antibody to CD22, containing (a) one, two or three VH HVR selected from VH HVR presented on figa, and/or (b) one, two or three VL HVR selected from HVR LL, presented at figv. In one aspect the invention relates to the antibody to CD22, containing the variable domain of the heavy chain selected from the variable domains of the heavy chain, presented on figa, and the variable domain of the light chain selected from the variable domains of the light chain, presented on figv.

In one aspect the antibody to CD22 according to the invention contains 1, 2, 3, 4, 5 or 6 of the hypervariable regions of the antibodies 5E8.1.8 obtained by hybridoma deposited in the ATCC and having inventory number PTA-7620.

2. Antibody fragments

The present invention relates to fragments of antibodies. Antibody fragments can be obtained by conventional methods, such as enzymatic cleavage, or recomb Nannini ways. Under certain circumstances there are advantages to using fragments of antibodies, but not of whole antibodies. The smaller size of the fragments provides quick clearance and may lead to improved access to solid tumors. For an overview of some fragments of antibodies, see Hudson et al., (2003) Nat. Med. 9:129-134.

To obtain fragments of antibodies developed different ways. Traditionally, these fragments get through proteolytic cleavage of the original antibody (for example, see 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 produced directly by recombinant host cells. All antibody fragments Fab, Fv and ScFv can be Express inE. coliand to secrete fromE. colithus, providing a light receiving large amounts of these fragments. Antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternatively, fragments, Fab'-SH can be directly recovered from theE. coliand chemically bind with the formation of fragments (Fab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach fragments (Fab')2you can directly select from the culture of the recombinant host cells. In U.S. patent No. 5869046 described fragment Fab and (Fab')2with increased half-life ofin vivo containing residues of the epitope binding receptor salvation. Practitioners demonstrate other ways of obtaining fragments of antibodies. In certain embodiments of the implementation of the antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185; U.S. patent No. 5571894 and 5587458. Only Fv and scFv are molecules with intact antihistamine areas devoid of constant regions; thus, they may be suitable for reduced nonspecific binding when usingin vivo. You can construct fused scFv proteins in obtaining merger effector protein with amino - or C-end of the scFv. Cm. Antibody Engineering, ed. Borrebaeck, above. The antibody fragment may also be a "linear antibody, for example as described in U.S. patent No. 5641870. Such linear antibodies can be monospecificity or especificada.

3. Humanized antibodies

This invention relates to a humanized antibody. In this area there are various ways to humanitarian antibodies not owned by the person. For example, humanitariannet antibody may contain one or more amino acid residues introduced into it from a source that does not belong to man. These non-human amino acid residues are commonly referred to as "import" residues, which typically is of erut from the "import" variable domain. Essentially, humanitarian can be carried out by way of Winter with colleagues (Jones et al., (1986) Nature 321:522-525; Riechmann et al., (1988) Nature 332:323-327; Verhoeyen et al., (1988) Science 239:1534-1536), by substituting the sequences of the hypervariable region corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where essentially less than one intact human variable domain of the human substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies, as a rule, are human antibodies in which some hypervariable residues region and possibly some FR residues substituted by residues from analogous sites of antibodies rodents.

The choice of variable domains of human light and heavy chains, for use in obtaining humanized antibodies may be important to reduce antigenicity. In accordance with the so-called method of "best approximation" of the sequence of the variable domain of the antibody rodent sceneroot relatively complete library of known sequences of the variable domains of a human. Then the sequence of the person that is closest to the sequence of the rodent, taking as a framework for gumanitarnogo antibody (Sims et al., (1993) J. Imunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196:901). In another method uses a particular framework, derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same frame can be used for some other humanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., (1993) J. Immunol, 151:2623).

As a rule, it is also important that antibodies humanitarianly with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal in accordance with one method of humanized antibodies produced by way of analysis of the source sequences and various imaginary humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and well known to specialists in this field. Available computer programs which illustrate and display probable three-dimensional conformational structures of selected possible sequences of immunoglobulins. The study of these images enables you to analyze the possible role of the residues in the functioning of the possible sequences of immunoglobulin, i.e. the analysis of residues that influence the ability of the immunoglobulin candidate to bind its antigen. So the m way you can select and combine the remains of FR from the recipient and import sequences so as to achieve the desirable characteristics of the antibodies, such as increased affinity for the antigen(s)target. Typically, the remains of the hypervariable region are directly and most substantially involved in the effect on binding to the antigen.

4. Human antibodies

Human antibodies to CD22 according to the invention can be designed by combining the sequence(s) of the variable domain of an Fv clone selected from derived from human libraries, phage display, with the known sequences of the constant domains of a human, as described above. Alternatively, the hybridoma method, it is possible to obtain monoclonal human antibodies to CD22 according to the invention. Cell line human myeloma and heteromalla cell line mouse-human to obtain monoclonal human antibodies are described, e.g., Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

Currently you can obtain transgenic animals (e.g. mice)that after immunization able to production of the full range of human antibodies in the absence of endogenous production of immunoglobulins. For example, described that the homozygous deletion of the gene connection region is (J Hheavy chain antibodies in chimeric and mutated in the germline of mice results in complete inhibition of endogenous production of antibodies. The transfer of genes of the immunoglobulin germline man this mutant in the germline of mice results in stimulation of antigen for production of antibodies person. For example, see, Jakobovits et al., Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

Also for obtaining human antibodies from antibodies, non-human, e.g., rodent, where the human antibody has similar appendectomy and specificnosti relative to the source, not belonging to the human antibodies, it is possible to use a permutation of genes. In accordance with this method, also referred to as a "fingerprint epitope", the variable region of the heavy or light chain fragment does not belong to a human antibody obtained by the method of phage display as described in this document, replace the repertoire of genes V-domain of a person, receiving a group of chimeric scFv or Fab with does not belong to the human chain/chain person. Selection with antigen leads to the secretion of chimeric scFv or Fab with does not belong to the human chain/chain person, where the repertoire circuits person recovers antigennegative plot, destroyed by deleting the appropriate line not belonging to h is one circuit in the primary clone from the phage display technique, i.e. the epitope causes (imprints) the choice of the partner chain man. When the process is repeated for the remaining substitution does not belong to the human chain, get a human antibody (see PCT WO 93/06213, published April 1, 1993). Unlike traditional humanitarian non-human antibodies by inoculation CDR, this method provides a fully human antibody that does not contain residues of FR or CDR that comes from man.

5. Bespecifically antibodies

Bespecifically antibodies are monoclonal antibodies that have the binding specificity of at least two different antigens. In certain embodiments of the implementation bespecifically antibodies are human antibodies or humanized antibodies. In certain embodiments implement one of specificdate binding is a binding specificity for CD22 and the other specificity of binding is a binding specificity for any other antigen. In certain embodiments of the implementation bespecifically antibodies may bind to two different epitopes CD22. Bespecifically antibodies can also be used to localize cytotoxic agents to cells expressing CD22. These antibodies are shoulder, bind CD22, and shoulder, the light is yuusei cytotoxic agent, for example, such as saporin, anti-interferon-α, Vinca alkaloid, a chain of ricin A, methotrexate or radioactive isotope hapten. Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically antibody (Fab')2).

Methods of obtaining bespecifically antibodies known in the field. Traditionally, the recombinant getting bespecifically antibodies based on simultaneous expression of two pairs of heavy chain-light chain immunoglobulin, where the two heavy chains have different specificnosti (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random sorting of light and heavy chains of immunoglobulins these hybridoma (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which, as a rule, carried out at the stages of affinity chromatography, is quite difficult, and the product yield is low. Such methods are described in WO 93/08829, published 13 may 1993, and in Traunecker et al., EMBO J., 10:3655 (1991).

In accordance with another approach, the variable domains of the antibodies with the desired specificnosti binding (antigennegative sites of antibody-antigen) merge with a constant domain sequence of an antibody. Merger, for example, Prov is changed with a constant domain of the heavy chain of the immunoglobulin, containing at least part of the areas of the hinge, CH2 and CH3. In certain embodiments of the implementation, at least one m is the first constant region of the heavy chain (CH1)containing the site necessary for binding to the light chain. DNA encoding the fusion heavy chain immunoglobulin and, if desired, the light chain of the immunoglobulin, is inserted in a separate expressing vectors and together transferout in a suitable organism, the host. This provides greater flexibility in adjusting the respective proportions of the three polypeptide fragments in the variants of implementation, when the unequal treatment of the three polypeptide chains used in the construction provide the optimum output. However, when the expression of at least two polypeptide chains in equal relations leads to high outputs or when the relationship does not have special significance, coding sequences for two or all three polypeptide chains can be inserted into one expressing vector.

In one embodiment, the implementation of this approach bespecifically antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one arm, and with a pair of hybrid heavy chain-light chain immunoglobulin (providing a second binding specificity) in the other shoulder. Revealed is, that this asymmetric structure facilitates the separation of the desired especifismo connections from unwanted combinations of chains of immunoglobulin, as the presence of light chain immunoglobulin in only one half of bespecifically molecules provides an easy way of separation. This approach is described in WO 94/04690. Learn more about getting bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210(1986).

In accordance with another approach, you can construct a contact surface between a pair of antibody molecules to maximize the percentage of heterodimers, which is recovered from a culture of recombinant cells. The contact surface includes at least part of the domain CH3 constant domain of the antibody. In this way one or more small side chains of the amino acids of the contact surface molecules of the first antibody replace large side chains (e.g. tyrosine or tryptophan). On the contact surface molecules of the second antibody create compensatory "cavities" of identical or smaller relative to the large side chain(s) by replacing the large side chains of amino acids smaller (e.g., alanine or threonine). This provides a mechanism to increase the output of heterodimer compared to other unwanted end-products such as d is a dimer.

Bespecifically antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate may be associated with Avidya, and the other with Biotin. Such antibodies are proposed, for example, for immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be obtained using a suitable method of cross-linking. Suitable connecting means well known in the field and described in U.S. patent No. 4676980 together with a number of methods of cross-linking.

Methods of obtaining bespecifically antibodies, fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained using chemical binding.

In article Brennan et al., Science, 229: 81 (1985)described a technique where the intact antibody proteoliticeski process of obtaining fragments F(ab')2. These fragments regenerate in the presence of detialing complex agent sodium arsenite to stabilize neighboring diols and prevent the formation of disulfide bonds. The Fab fragments' are formed after conversion to dinitrobenzoate (TNB) derivatives. One of the derived Fab'-TNB then converted into Fab'-thiol by restoring mercaptan what is designed and mixed with equimolar amounts of the other derived Fab'-TNB education especifismo antibodies. Received bespecifically antibodies can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of fragments, Fab'-SH, which can chemically bind with the formation bespecifically antibodies, fromE. coli. In Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe how to get a fully humanized molecules especifismo antibody (Fab')2. Each fragment Fab' separately secretively fromE. coliand subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies. Thus formed bespecifically antibody was able to contact the cells, sverkhekspressiya the HER2 receptor, and normal T-cells, and to run the lytic activity of cytotoxic lymphocytes against targets breast cancer person.

Also describes the various methods of obtaining and allocating fragments bespecifically antibodies directly from a culture of recombinant cells. For example, bespecifically antibodies were obtained using latinovich lightning. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). Peptides latinovich lightning from proteins Fos and Jun bound to Fab fragments' of two different antibodies by gene fusion. Homodimeric antibodies were restored in the hinge region with the formation of monomers, asetem again oxidized with the formation of heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. Technology "datel"described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)has provided an alternative mechanism for obtaining fragments bespecifically antibodies. The fragments contain the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) by a linker that is too short to allow linking the two domains on the same chain. Thus, the domains VH and VL of one fragment are forced to pair with complementary domains VL and VH of another fragment, thereby forming two antigenspecific plot. It was also reported another strategy to obtain fragments bespecifically antibodies through the use of dimers of single-chain Fv (sFv). Cm. Gruber et al., J. Immunol., 152: 5368 (1994).

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

6. Multivalent antibodies

The cell expressing the antigen bound to the antibody may be internalized (and/or metabolismrelated) polyvalent antibody faster than bivalent antibody. Antibodies of the present invention may be a polyvalent antibodies (which differ from IgM) with three or more antigensvyazyvayushchaya (for example, tetravalent antibodies), which can easily be obtained by recombinant expression of a nucleic acid that encodes a polypeptide chain of the antibody. Multivalent antibody may contain a dimerization domain, and three or more antigenspecific plot. In certain embodiments of the implementation of the dimerization domain contains (or consists of) Fc-region or a hinge region. In this embodiment, the antibody contains a Fc region, and three or more antigenspecific plot from N-Terminus to the Fc-region. In certain embodiments of the implementation of the polyvalent antibody contains (or consists of) three to about eight antigenspecific areas. In one such embodiment, the polyvalent antibody contains (or consists of) four antigenspecific plot. Polyvalent antibody contains at least one polypeptide chain (for example, two polypeptide, 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 polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n represents 0 or 1. For example, the polypeptide chain(s) may contain chain V-CH1-flexible linker-VH-CH1-Fc region or chain VH-CH1-VH-CH1-Fc region. Polyvalent antibody according to the present document may optionally contain at least two (e.g., four) of the polypeptide of variable domains of the light chain. Polyvalent antibody according to the present document may, for example, contain from about two to about eight polypeptides, variable domains of the light chain. Discussed in this section polypeptides, variable domains of the light chains contain a variable domain light chain and, optionally, further contain a CL domain.

7. Single-domain antibodies

In some embodiments, the implementation of the antibody according to the invention is a single domain antibody. Single-domain antibody is a chain of a single polypeptide containing the variable domain of the heavy chain or part of or the entire variable domain light chain, or portion of antibodies. In certain embodiments of the implementation of the single domain antibody is a single domain antibody man (Domantis, Inc., Waltham, MA; for example, see U.S. patent No. 6248516 B1). In one embodiment, the implementation of the single domain antibody comprises all or part of the variable domain of the heavy chain of the antibody.

8. Variants of antibodies

In some embodiments, the implementation provided by the modification(s) the amino acid sequence of the antibody, opisyvaet the traveler in this document. For example, it may be desirable to improve the binding affinity of and/or other biological properties of the antibody. Variants of the amino acid sequence of the antibody may be obtained by appropriate modification of the nucleotide sequence that encodes the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues from amino acid sequences, and/or insertion of amino acid residues in the sequence, and/or replacement of residues in the amino acid sequences of the antibodies. For the final design, you can implement any combination of deletions, insertions and substitutions, provided that the final construct possesses the desired characteristics. Changes of amino acids can be performed in a specified amino acid sequences of antibodies during retrieval sequence.

A suitable method for the identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "mutagenesis to alanine scanning", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this way to influence the interaction of amino acids with antigen define a residue or group is scheduled residues (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negative is a charged amino acid (e.g., alanine or polyalanine). Then those provisions of amino acids, demonstrating functional sensitivity to the substitutions improve through the introduction of additional or other options in the areas of replacement or for the parts replaced. Thus, although the plot of the implementation of the changes in amino acid sequence is predetermined, to predetermine the nature of the mutation is essentially not necessary. For example, for analysis of the properties of mutations in a given area, the target codon or the scheduled areas spend ala scanning or random mutagenesis and expressed immunoglobulins are screened for the desired activity.

Insertion in the amino acid sequence include the N - and/or C-terminal fusion in length from one residue to polypeptides containing a hundred or more residues, as well as insertion of one or more amino acid residues within the sequence. Examples of terminal insertions include an antibody with an N-terminal methionine residue. Other variants of antibody molecules with indels include the fusion to the N - or C-Terminus of an antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the half-life of antibodies in the serum.

In certain embodiments of the implementation of the antibody according to the invention to change to increase or decrease the degree of glycosylation of antibodies. Glyco is iliriana polypeptides, as a rule, is either N-linked or O-linked. N-linked refers to the attachment of a molecule of carbohydrate to the side chain of aspartic residue. Recognized sequences for enzymatic attachment molecule of carbohydrate to the side chain of asparagine are Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate usually serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.

The addition or deletion of sites of glycosylation of antibodies is convenient to carry out, changing the amino acid sequence so as to create or delete one or more of the above-described Tripeptide sequences (for N-linked glycosylation sites). Change can be realized by adding, removing or changing the sequence of the original antibody (for O-linked glycosylation sites) one or more residues of serine or threonine.

When titulo contains a Fc region, you can change the associated carbohydrate. For example, in patent application U.S. No. US 2003/0157108 (Presta, L.) described antibodies with a Mature carbohydrate structure that are not attached to the Fc region of antibodies fucose. Also see US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). In WO 2003/011878, Jean-Mairet et al., and U.S. patent No. 6602684, Umana et al., indicated antibodies with dividing in half N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of antibodies. In WO 1997/30087, Patel et al., reported antibodies, at least one galactose residue in the oligosaccharide attached to the Fc region of antibodies. Regarding antibodies with altered carbohydrate attached to its Fc-region, see WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.). About antigenspecific molecules with modified glycosylation see also US 2005/0123546 (Umana et al.).

In certain embodiments of the implementation of the variant glycosylation includes Fc-region, where the structure of the carbohydrate attached to an Fc region lacks fucose. Such variants have enhanced ADCC function. Optional Fc-region additionally contains one or more amino acid substitutions, which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (residue numbering according to Eu). Examples of publications related to "deforsirovannym" or "photostability" antibodies include US 2003/0157108; WO 2000/61739; WO 2001/29246; S 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing deoksigenirovanii antibodies include cells Lec13 CHO lack furosemideusedto protein (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); patent application U.S. No. US 2003/0157108 A1, Presta, L. and WO 2004/056312 A1, Adams et al., especially at example 11), and cell lines with knockout, such as CHO cells with knockout of the gene alpha-1,6-fucosyltransferase, FUT8 (Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614(2004)).

In one of the embodiments the antibody change to improve the time half-life in serum. To extend the half-life of antibodies in serum antibody (especially in a fragment of antibody), you can embed one epitope binding receptor of salvation, as described, for example, in U.S. patent 5739277. As used herein, the term "epitope-binding receptor salvation" refers to an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the half-life of the IgG molecule in the serum ofin vivo(U.S. patent 2003/0190311, US6821505; U.S. patent 6165745; U.S. patent 5624821; U.S. patent 5648260; U.S. patent 6165745; U.S. patent 5834597).

Another type of variant is to replace the variant amino acids. These variants contain at least one amino acid residue in front of the OLE antibodies substituted by another residue. Of interest for substitutional mutagenesis include the hypervariable regions, but also provides for changes to FR. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions". If such substitutions result in a desirable change in biological activity, then you can make a more substantial changes, indicated in table 1 as "illustrative replacement", or as further described below with respect to classes of amino acids, and screening products.

Table 1
The original balanceIllustrative replacementPreferred replacement
Ala (A)Val; Leu; IleVal
Arg (R)Lys; Gln; AsnLys
Asn (N)Gln; His; Asp, Lys; ArgGln
Asp (D)Glu; AsnGlu
Cys (C)Ser; AlaSer
Gln (Q)Asn; GluAsn
Glu (E)Asp; GlnAsp
Gly (G)AlaAla
His (H)Asn; Gln; Lys; ArgArg
Ile (I)Leu; Val; Met; Ala; Phe; NorleucineLeu
Leu (L)Norleucine; Ile; Val; Met; Ala; PheIle
Lys (K)Arg; Gln; AsnArg
Met (M)Leu; Phe; IleLeu
Phe (F)Trp; Leu; Val; Ile; Ala; TyrTyr
Pro (P)AlaAla
Ser (S)ThrThr
Thr (T)Val; SerSer
Trp (W)Tyr; PheTyr
Tyr (Y)Tp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe; Ala; NorleucineLeu

Significant modification of the biological properties of the antibody is carried out by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the frame of the polypeptide in the area of the substitution, for example, the conformation in the form of a layer or a spiral, (b) the charge or hydrophobicity of the molecule at the site of the target or (c) the size of the side chain. Amino acids can be grouped according to similarity of the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A)Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged polar: Gly (G), Ser (S)Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) acidic: Asp (D)Glu (E)

(4) basic: Lys (K), Arg (R), His (H).

Alternative naturally occurring residues may be divided into groups based on common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitution leading to the replacement of the representative of one of these classes for another class. Such substituted residues can also be entered in the conservative areas C the men or in the remaining (non-conservative) areas.

One of the types of substituted variants include replacement of one or more residues of the hypervariable region of the initial antibody (for example, gumanitarnogo antibodies or human antibodies). Typically, the received option(s)selected for further refinement, contains modified (for example, improved biological properties relative to the original antibody, from which they receive. A convenient way of obtaining such substituted variants include affinity maturation using phage display. In brief several sections of hypervariable regions (e.g., 6-7 sites) are matirovanie with obtaining all possible amino acid substitutions at each site. Thus obtained antibodies exhibit on the particles of filamentous phage in the form of mergers, at least part of the envelope protein of a phage (e.g., the product of the gene III M13), Packed in each particle. Then exposed on the phage variants is subjected to screening for their biological activity (e.g. binding affinity of). To identify areas candidate hypervariable regions for modification can be performed scanning mutagenesis (e.g., alanine scanning) to determine residues of the hypervariable region, making a significant contribution to the binding to the antigen. Alternative sludge is optionally may be beneficial to analyze a crystal structure of the complex of antigen-antibody to determine the points of contact between the antibody and antigen. Such contact residues and neighboring residues are candidates for replacement known in the field of ways, including the methods presented in this document. Upon receipt of such options panel of variants is subjected to screening using known in the field of methods, including the methods described herein, and antibodies with improved properties in one or more relevant assays may be selected for further refinement.

The nucleic acid molecules encoding amino acid sequence variants of antibodies, produced by many known in the field of methods. These methods include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence variants) or getting through mediated by oligonucleotides (or site-specific) mutagenesis, PCR mutagenesis, and cassette mutagenesis of the previously received version or betweenthey version antibodies.

It may be desirable to perform one or more modifications of amino acids in the Fc-region of an antibody according to the invention, receiving, as a rule, a variant Fc region. The variant Fc region may contain a sequence of Fc-region of a person (e.g., Fc region of IgG1, IgG2, IgG3, or IgG4 human), stereoselectivity modification (for example, a substitution) at one or more positions of the amino acids, including the position of the cysteine in the hinge.

In accordance with the description and methods in this field is provided that in some embodiments, the implementation of the antibody according to the invention may contain one or more changes compared to the corresponding antibody wild-type, for example, in the Fc-region. However, these antibodies will remain essentially the same characteristics required for therapeutic use, compared to their analogous to wild-type. For example, suppose that in the Fc-region can be some changes that will lead to altered (either increased or decreased) binding of C1q and/or caused by complement cytotoxicity (CDC), for example as described in WO 99/51642. Relative to other examples of variants of the Fc-region see also Duncan & Winter Nature 322: 738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821 and WO 94/29351. In WO 00/42072 (Presta) and WO 2004/056312 (Lowman) described variants of the antibodies with enhanced or reduced binding to FcR. The content of these patent publications are incorporated herein by reference in full. See also Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). In US 2005/0014934A1 (Hinton et al.) described antibodies with increased half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyr et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), as described in US2005/0014934A1 (Hinton et al.). These antibodies contain the Fc region with one or more substitutions that improve the binding of the Fc region with FcRn. In U.S. patent No. 6194551B1, WO 99/51642 described variants of polypeptides with altered amino acid sequences of the Fc-region and the ability to enhanced or reduced binding to C1q. The content of these patent publications are incorporated herein by reference in full. Also see Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

In one aspect the invention relates to antibodies that contain modifications at the surface of contact of the Fc polypeptides containing the Fc region, where modifications to facilitate and/or encourage heterodimerization. These modifications include the introduction of the ledge in the first Fc polypeptide and the cavity of the second Fc polypeptide, where the protrusion is located in the cavity so as to encourage the formation of a complex of first and second Fc polypeptides. Methods for producing antibodies with these modifications known in this field, for example as described in U.S. patent No. 5731168.

9. Derived antibodies

Antibodies of the present invention can be further modified to contain additional non-protein groups, which are well known in the field and readily available. Preferably groups suitable for derivatization of the antibody, are water-soluble polymers. Non-limiting examples of water-soluble polymers include as non-limiting examples of polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (or homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, copolymers of polypropyleneoxide/ethylene oxide, polyoxyethylene polyols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. Propionic aldehyde of the polyethylene glycol may have advantages in production due to its stability in water. The polymer can be any molecular weight and can be branched or unbranched. The number attached to the antibody of the polymers can vary, and if you attach more than one polymer, they may be the same or different molecules. Typically, the number and/or type used for derivatization of polymers can be determined on the basis of the submissions, including as non-limiting examples of specific properties and functions of antibodies to improve, whether derived antibody to use the La therapy under certain conditions, etc.

In another embodiment, the provided conjugates of the antibody and the protein molecules can be selectively heated by radiation. In one embodiment, the implementation of non-protein molecule is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)). The radiation can be any waves and includes, as non-limiting examples of wavelengths that do not harm normal cells, but which are heated non-protein molecule to a temperature at which the cells near the antibody is a non-protein molecule perish.

Some methods of obtaining antibodies

1. Some are based on the hybridomas ways

Monoclonal antibodies to CD22 according to the invention can be obtained hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be obtained by means of recombinant DNA (U.S. patent No. 4816567).

In the hybridoma method, a mouse or other suitable animal host, such as a hamster, subjected to immunization to stimulate lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Antibodies to CD22, generally, induce in animals by multiple subcutaneous (sc) or intraperitoneally (ip) injection CD22 and adjuvant. CD22 can be well-known in the field of ways, not the quiet of which is also described in this document. For example, CD22 can be obtained by recombinant means. In one of the embodiments of animals subjected to immunization derived CD22, which contains the extracellular part of CD22, fused with the Fc-part of the heavy chain of the immunoglobulin. In one of the embodiments of animals subjected to immunization fused protein CD22-IgG1. In one of the embodiments of animals subjected to immunization immunogenic derivatives of CD22 in solution with monophosphorylation A (MPL)/dicenomicon trehalose (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT) and the solution is injected intradermally in several areas. Two weeks later, animals repeatedly subjected to immunization. Over the period from seven to fourteen days in animals take blood and serum will be analyzed by the titer of antibodies to CD22. Animals continue to immunize before the release of the title on the plateau.

Alternatively, the lymphocytes can givein vitro. Then lymphocytes merge with myeloma cells using a suitable tool to merge, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)).

Thus obtained hybridoma cells were seeded and grown in a suitable medium for culturing, for example, a medium containing one or more substances that inhibit the growth or survival Nikitich source myeloma cells. For example, if the original myeloma CL is located lacked the enzyme hypoxanthineguanine (HGPRT or HPRT), the medium for culturing the hybridomas typically will contain gipoksantin, aminopterin and thymidine (Wednesday HAT), which prevent the growth of cells with deficiency of HGPRT.

In certain embodiments of the implementation of the myeloma cells are cells that effectively merge, support stable high-level production of antibody producing selected antibody cells and sensitive to the environment, such as environment HAT. Illustrative myeloma cells include as non-limiting examples of the myeloma line of mice, such as lines derived from tumors of mice MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California USA, cells and SP-2 or X63-Ag8-653, available from the American Type Culture Collection, Rockville, Maryland USA. To obtain monoclonal antibodies person described cell lines of human myeloma and heteromyinae cell line mouse-human (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

Medium for cultivation, which grow hybridoma cells, to analyze the production of monoclonal antibodies that bind to CD22. Preferably, the binding specificity produced by hybridoma cells for monoclonal antibodies thus determined by or through analysis of the binding ofin vitrosuch as radioimmunoassay EN is Liz (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of monoclonal antibodies can be determined by analysis of Scatchard from Munson et al., Anal. Biochem., 107: 220 (1980).

After determining that hybridoma cells produce antibodies of the desired specificity, affinity and/or activity, the clones can be subclinical through serial dilutions and raise the standard methods (Goding, Monoclonal Antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)). Suitable media for culturing for this purpose include, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells can be grownin vivoas ascitic tumors in an animal. Monoclonal antibodies secreted by the subclones, the appropriate way isolated from the environment for cultivation, ascitic fluid or serum conventional methods of purification of immunoglobulins, for example, such as protein A-sepharose, chromatography on hydroxyapatite, the electrophoresis gel, dialysis, or affinity chromatography.

2. Some methods of screening libraries

Antibodies to CD22 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 many ways to generate libraries of phage display and screening such libraries for antibodies with the desired characteristics of the mi-binding. Such methods are described mainly in Hoogenboom et al., (2001) in Methods in Molecular Biology 178: 1-37 (O'brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments implement in Lee et al., (2004) J. Mol. Biol. 340: 1073-1093.

In principle, synthetic clones of antibodies chosen by screening phage libraries containing phage exhibiting various fragments of variable regions (Fv) antibody, fused with the protein shell of the phage. Such phage library is subjected to affinity chromatography with the desired antigen. Clones expressing the fragments Fv, the ability to communicate with the desired antigen, adsorbed on the antigen and, thus, separated from nesvezhije clones in the library. Then bind the clones elute with antigen and can be enriched by additional cycles of adsorption of the antigen/elution. Any of the antibodies to CD22 according to the invention can be obtained by developing a suitable method of screening for the selection of interest phage clone with the subsequent construction of clone full-length antibodies to CD22 using the Fv sequences from the interest of phage clone and the appropriate sequence of a constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

In certain embodiments of the implementation antigennegative domain antibodies which are square of two variable (V) regions of approximately 110 amino acids, one from each light (VL) and heavy (VH) chains, where both are three hypervariable loops (HVR) or complementarity determining region (CDR). Variable domains can be functionally displayed on the phage in the form of single-chain Fv fragments (scFv), in which VH and VL are covalently bound via a short, flexible peptide, or as Fab fragments, in which each of them fused with the constant domain, and they interact ecovalence, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, encoding scFv phage clones and encoding Fab phage clones collectively referred to as "phage clones Fv or Fv clones".

The repertoire of genes of VH and VL can be individually cloned polymerase chain reaction (PCR) and randomly recombine in phage libraries, which you can then search antigennegative clones, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to immunogenum without having to construct a hybrid. Alternatively, you can clone the original repertoire to provide a single source of human antibodies for a wide range of non-native and native antigens without any immunization, as described in Griffiths et al., EMBO J 12: 725-734 (1993). Finally, the original library also is you can get synthetically, cloning naprestolnye segments V-genes from stem cells, and using PCR primers containing random sequence to encode vysokopribylnyj CDR3 regions and to facilitate the rearrangement ofin vitroas described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments of the implementation for exhibiting fragments of antibodies by fusion with the minor protein shell pIII use thread-like phage. Antibody fragments can be exhibited in the form of single-chain Fv fragments, in which the domains VH and VL are connected on the same polypeptide chain flexible polypeptide spacer, for example, 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 the other is secreted in periplasm bacterial host cell, where the Assembly structure of the Fab-protein shell, which is exhibited on the surface of phage by replacing some of envelope proteins of wild-type, for example, as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

Typically, nucleic acids encoding fragments of antibody genes, derived from immune cells obtained from humans or animals. If desired library, aimed at obtaining clones to CD22, a subject subjected to immunization CD22 with call answer antibodies and for constructing libraries allocate cells Selezen the and and/or circulating B-cells, other peripheral blood lymphocytes (PBL). In a preferred embodiment, a library of gene fragments of human antibodies directed at obtaining clones to CD22, get generating a response by antibodies to CD22 in transgenic mice carrying a functional matrix of immunoglobulin genes of human (and with no functioning system for the production of endogenous antibodies) so that immunization CD22 is a source of B-cells producing human antibodies to CD22. Obtaining producing human antibodies of mice described below.

Additional enrichment groups reacting to CD22 cells can be performed using a suitable screening method for the selection of B-cells expressing specific for membrane-bound CD22 antibody, for example, by separating cells using affinity chromatography with CD22 or adsorption of cells labeled with fluorochromes CD22 followed-activated flow sorting of cells (FACS).

On the other hand, the use of spleen cells and/or B cells or other PBL from an unimmunized donor provides a better representation of the possible repertoire of antibodies, and also allows you to construct a library of antibodies using any animal (human or non-human), which CD22 is not antigenic. For libraries, the soda is gene construct containing antibodies in vitro, the subject of taking stem cells to obtain nucleic acids encoding nepristoinye gene segments antibodies. Interest of the immune cells can be obtained from many species of animals, species such as human, mouse, rat, lagomorphs, wolf, dog, cat, pig, cow, horse and poultry etc.

Nucleic acid encoding the gene segments of the variable domains of antibodies (including segments of the VH and VL), isolated from the interest of cells and amplified. In the case of gene libraries rebuilt genes of VH and VL of the desired DNA can be obtained by allocating the genomic DNA or mRNA from lymphocytes with subsequent polymerase chain reaction (PCR) with primers matching the 5' and 3'ends rebuilt genes VH and VL, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), and thus, the diversity of the repertoire of V genes for expression. V-genes can amplify from cDNA and genomic DNA with a reverse primer at the 5'end of the exon encoding the Mature V-domain, and direct primer located within the J-segment, as described in Orlandi et al., (1989) and Ward et al., Nature, 341: 544-546 (1989). However, amplification with cDNA reverse primers can be located in the first exon, as described in Jones et al., Biotechnol., 9: 88-89 (1991), and direct the primers may be located in the constant region, as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-572 (1989). To maximize complementarities in the primers, you can create a degeneracy as described in Orlandi et al., (1989) or Sastry et al., (1989). In certain embodiments of the implementation of diversity libraries maximize by using PCR primers directed to each family of V-genes for amplification of all possible arrangement of VH and VL are present in the sample nucleic acid immune cells, for example, as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as described in method Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of amplified DNA in expressing vectors in PCR primer, you can make the rare restriction sites as a marker at one end, as described in Orlandi et al., (1989), or by additional amplification PCR with labeled primer as described in Clackson et al., Nature, 352: 624-628 (1991).

Repertoire synthetically reconstructed V-genes can be obtainedin vitrosegment V-genes. Most of the segments VH-human genes cloned and sequenced (published in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (published in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all major conformation of loop H1 and H2) can be used to obtain diversity of the repertoire of VH genes using PCR primers encoding the H3 loop with different sequence and length, campisano in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). The repertoire of VH can also be obtained in a variety of sequences collected in a long H3 loop the same length, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Segments Vκ and Vλ human cloned and sequenced (published in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to obtain the synthetic repertoire of light chains. The synthetic repertoire of V genes on the basis of a number of styling VH and VL and lengths L3 and H3 encodes the antibody with a significant variety of patterns. After amplification, the coding V-genes are DNA segments V-genes in the germ line can be movedin vitroway Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

The repertoire of the fragments of antibodies can be obtained by combining together the repertoire of genes of VH and VL in different ways. Each repertoire can be obtained in different vectors, and the vectors recombinein vitrofor example, as described in Hogrefe et al., Gene, 128: 119-126 (1993), orin vivothrough co-infection, for example, loxP system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). In the approach recombinationin vivoto overcome the limit of the size of the library, asked the efficiency of transformationE. coliuse the double-stranded nature of the Fab-fragments. The naive repertoire of VH and VL clone separately, one in fahmida, and the other in the phage vector. Then two libraries combined e the PTO ragovoy infections contain fahmida bacteria, to each cell contained a different combination, and the size of the library was limited only by the number of cells present (approximately 1012clones). Both vectors contain the signals of recombinationin vivoso that the genes of the VH and VL recombine into a single replicon together and packaged into phage virions. These huge libraries provide a wide variety of bodies with good affinity (Kd-1approximately 10-8M).

Alternatively, the repertoires can be cloned sequentially into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or to gather together by PCR and then cloned, for example, as described in Clackson et al., Nature, 352: 624-628 (1991). Assembly PCR can also be used to join DNA VH and VL DNA encoding a flexible peptide spacer with the formation of a repertoire of single-chain Fv (scFv). Another way is to use the "Assembly PCR in the cage for combining the genes of VH and VL in lymphocytes by PCR, and then cloned repertoires of linked genes, as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).

Antibodies produced by libraries from naive cells (natural or synthetic)can be the average affinity (Kd-1from approximately 106up to 107M-1), butin vitroyou can also simulate maturation is finesti by constructing and re-select from secondary libraries, as described in Winter et al., (1994), above. For example, you can randomly make mutationin vitrothrough the use of making mistakes polymerase (published in 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 way Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, you can implement the affinity maturation by random mutagenesis of one or more CDRs, for example, using PCR with primers carrying a random sequence, overlapping interest CDR, the selected individual clones Fv and be screened for the most affinity clones. In WO 9607754 (published on 14 March 1996) described a method of inducing mutagenesis in a complementarity determining region light chain immunoglobulin to obtain a library of light chain genes. Another effective approach is a recombination of domains VH or VL, selected by phage display repertoire of naturally occurring variants of the V-domains obtained from unimmunized donors and the screening of the greatest affinity in a few cycles of the permutation circuits, as described in Marks et al., Biotechnol., 10: 779-783 (1992). This method allows to obtain antibodies and antibody fragments with affiniscape 10-9M or less.

Screening of libraries you can pursue various well-known in this area SP is ways. For example, CD22 can be used to coat the wells of adsorption tablets, to Express on the cell-hosts attached to absorbent tablets or used in the sorting of cells, or konjugierte with Biotin for capture with the use of streptavidin coated granules, or use any other method for panning libraries phage display.

Samples ragovoy library is brought into contact with immobilized CD22 under conditions suitable for binding at least part of the phage particles with the adsorbent. As a rule, the conditions, including pH, ionic strength, temperature and the like, chosen to mimic physiological conditions. Phages associated with the solid phase, washed, and then elute the acid, for example, as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or alkali, for example, as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or through competition antigen CD22, for example, in the way similar to the way competition antigen Clackson et al., Nature, 352: 624-628 (1991). Phages may be enriched in the 20-1000 times per cycle of selection. In addition, the enriched phage can be grown in bacterial culture and subjected to additional cycles of selection.

The effectiveness of selection depends on many factors, including the kinetics of dissociation at wash, and whether a few fragments of antibodies on a single phage ignoreme is but to contact the antigen. Antibodies with fast kinetics of dissociation and low affiniscape binding) can be saved by using a short time of washing, polyvalent phage display and high density coating antigen in the solid phase. High density not only stabilizes the phage through multivalent interactions, but also conducive to the re-linking dissociative phage. The selection of antibodies with slow dissociation kinetics (and good affiniscape binding) can be performed through the use of a long time of washing 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).

For CD22 can be a choice between fagbemi antibodies with different affiniscape, even with affiniscape, which differ slightly. However, it is likely that random mutagenesis of the selected antibody (e.g., as performed in some methods of affinity maturation) will cause a lot of mutations, most of which are linked to the antigen, and the minority with greater affinity. With a limited number of CD22 rare phages with high affinity could compete. To save all mutants with high affinity phages can be incubated with excess biotinylated CD22, but with biotinylated CD22 in concentration the AI less both molarity, than the target molar affinity constant for CD22. Then the phages that bind with high affinity, you can capture streptavidin coated paramagnetic beads.

This "equilibrium capture" enables you to select antibodies in their affinely binding sensitivity, which allows you to isolate mutant clones with an affinity of only two times greater than that of a large excess of phages with lower affinity. You can also vary the conditions used at wash phages associated with the solid phase for separation on the basis of the kinetics of dissociation.

Clones against CD22 can be selected on the basis of activity. In certain embodiments implementing the invention relates to antibodies to CD22, which are associated with living cells, which in nature Express CD22. In one of the embodiments the invention relates to antibodies to CD22, which block the binding between a ligand of CD22 and CD22, but do not block the binding between a ligand of CD22 and other protein. The Fv clones corresponding to such antibodies to CD22, can be selected by (1) selection of clones against CD22 from ragovoy library, as described above, and optionally amplifying the selected population of phage clones by growing population in a suitable bacterial host; (2) the selection of CD22 and other protein against which the desired blocking and not the lock activity respectively; (3) adsorption of phage clones against CD22 on immobilized CD22; (4) use of an excess of another protein for the elution of any undesired clones that recognize CD22 binding determinants overlap or are shared with the binding determinants of the other protein; and (5) elution of clones that remained adsorbed after stage (4). Optional clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection process described in this section one or more times.

DNA encoding derived from a hybrid monoclonal antibodies or Fv clones of phage display technique according to the invention, it is easy to select and sequence the conventional methods (e.g., using oligonucleotide primers designed for specific amplification of interest regions encoding the heavy and light chains, with matrix hybridoma or ragovoy DNA). After DNA extraction can be placed in expressing vectors, which are then transferout in cell host, such as cellsE. colicells , COS monkey cells Chinese hamster ovary (CHO) or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant cell host. A review article on recombin nteu expression in bacteria coding DNA antibodies include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv-clones according to the invention can be combined with known DNA sequences encoding the constant region of the heavy chain and/or light chain (e.g., appropriate DNA sequence can be obtained by Kabat et al., above) to obtain clones encoding full-size and/or polnorazmernyi heavy and/or light chain. It is clear 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 and any kind of animals. As used herein, Fv-clone obtained from DNA variable domain of one species of animal (such as man), and then merged with the DNA of a constant region from another species of animal with obtaining the coding sequence(s) for "hybrid" full-size heavy chain and/or light chain is included in the definition of "chimeric" and "hybrid" antibody. In certain embodiments of the implementation of the Fv-clone obtained from DNA variable domain of the human merge with DNA constant region of the person receiving the coding sequence(s) full-size or polnorazmernyi heavy and/or light chains of a human.

DNA encoding the antibody to CD22 according to the invention, obtained from a hybrid, you can also mod vizirovat, for example, placing the coding sequence of the constant domains of the heavy and light chains man in place of the homologous murine sequences derived from the hybridoma clone (e.g. as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding obtained from hybrids or Fv-clones antibody or its fragment can be further modified by covalent binding to the coding sequence of the immunoglobulin whole or part of the coding sequence of the non-immunoglobulin polypeptide. Thus, you receive a "chimeric" or "hybrid" antibodies having binding specificity obtained from Fv-clone or hybrid antibodies according to the invention.

3. The vectors, cells of the host and recombinant methods

For recombinant generate antibodies according to the invention encoding their nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody, it is easy to select and sequence the conventional methods (e.g., using oligonucleotide primers capable of specific binding to genes encoding the heavy and light chains of the antibody). There are many vectors. The choice of vector depends partly on the used host cell. As the rule is, cell owners or prokaryotic, or eukaryotic (typically, mammalian cells) origin. It is clear 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 human or any species of animal.

Obtaining antibodies using prokaryotic host cells:

Construction of vectors

Polynucleotide sequences encoding polypeptide components of the antibodies according to the invention can be obtained by standard recombinant methods. The desired polynucleotide sequence can be extracted and sequenced from producing antibodies cells, such as hybridoma cells. Alternatively, polynucleotide can be synthesized using nucleotide synthesizer or PCR methods. After receiving encoding the polypeptide sequence is inserted into a recombinant vector capable of replication and expression of heterologous polynucleotides in prokaryotic hosts. For this purpose, the present of the present invention can use a variety of vectors are available and known in the field. The selection of the appropriate vector depends mainly on the size of the nucleic acid to be inserted into the vector and of the particular the year-the host for transformation vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide or both) and its compatibility with a particular cell of the host in which it is located. Vector components generally include, as non-limiting examples plot start replication, selective marker gene, the promoter, the binding site of the ribosome (RBS), signal sequence, the insertion of a heterologous nucleic acid and the sequence termination of transcription.

Basically, in relation to these hosts use plasmid vectors containing replicon and control sequences which are derived from species compatible with the host-cell. Vector, as a rule, is the site of replication, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example,E. coliusually transformed using pBR322, a plasmid derived from a species ofE. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and, thus, provides a simple method for the identification of transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or it can be modified so that it sod is neigh, promoters that can be used by the microbial organism for expression of endogenous proteins. Examples of derivatives of pBR322, used for the expression of specific antibodies, are described in detail in Carter et al., U.S. patent No. 5648237.

In addition, in relation to these owners as transforming vectors can be used phage vectors containing replicon and control sequences that are compatible with the microorganism host. For example, in obtaining a recombinant vector that can be used to transform suitable host cells, such asE. coliLE392, you can use the bacteriophage, such as λGEM.TM.-11.

Expressing the vector according to the invention may contain two or more pairs of the promoter-cistron encoding each of the components of the polypeptide. The promoter is a noncoding regulatory sequence above (5') cistron, which modulates its expression. Prokaryotic promoters, usually divided into two classes, inducible and constitutive. The inducible promoter is a promoter that initiates elevated levels of transcription controlled them cistron in response to changes in cultivation conditions, for example, the presence or absence of nutrients or temperature changes.

Well-known Bo is isoi a number of promoters, identifies a variety of potential host cells. The selected promoter can functionally bind to DNA cistron that encodes a light or heavy chain by removing the promoter from the DNA source by cleavage with restriction enzymes and inserting the selected promoter sequence in the vector according to the invention. To control the amplification and/or expression of the target genes can be used natural promoter sequence and many heterologous promoters. In some embodiments, the implementation of the use of heterologous promoters, as they typically provide greater transcription and higher yields downregulation of a target gene in comparison with the natural promoter of the target polypeptide.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp) and hybrid promoters such as the tac promoter or the trc. However, also other suitable promoters that function in bacteria (such as other known bacterial or phage promoters). Their nucleotide sequences are published, thus enabling the specialist to act ligera them with cisternae coding target light and heavy chains (Siebenlist et al., (1980) Cell 20 269), using linkers or adapters to add any required restriction sites.

In one aspect of the invention, each cistron in the recombinant vector contains a component of the secretory signal sequence, which directs the broadcast of the expressed polypeptide through the membrane. Typically, the signal sequence may be a component of the vector, or it may be part of the DNA of the target polypeptide, which is inserted into the vector. The signal sequence is selected for the purpose of this invention, should be a signal sequence that recognizes and processes (i.e. it signal peptidases) a host cell. For prokaryotic host cells that do not recognize and process the signal sequence found in nature with heterologous polypeptides, the signal sequence is substituted by the selected prokaryotic signal sequence, for example, from the group consisting of a leader sequence of alkaline phosphatase, penitsillinazy, Ipp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one of the embodiments of the invention the signal sequence used in both cistronic expressing systems represent a signal by which sledovatelnot STII or their variants.

In another aspect of the production of the immunoglobulins according to the invention can be carried out in the cytoplasm of the host cell and, thus, do not require the presence of secretory signal sequences in each cistron. Thus, the light and heavy chains of the immunoglobulins expressed, stacked and assembled with the formation of a functional immunoglobulin in the cytoplasm. Certain host strains (e.g., strains ofE. colitrxB) provide conditions in the cytoplasm, which favor the formation of disulfide bonds, thus ensuring the correct arrangement and Assembly of expressed protein subunits. Proba and Pluckthun Gene, 159: 203 (1995).

Antibodies according to the invention can also be obtained by using expressing the system in which the quantitative ratio expressed by the components of the polypeptide can be regulated to maximize output secreted and correctly assembled antibodies according to the invention. Such adjustment is carried out, at least partially, by simultaneous adjustment of the power of broadcast to the polypeptide components.

One way power adjustment broadcast described in Simmons et al., U.S. patent No. 5840523. It uses the options field of translation initiation (TIR) inside cistron. For this TIR you can get a number of variants of the sequence of amino acids, and nucleic acids with a range of forces broadcast, thus, providing a convenient means to adjust this factor to the desired level of expression in a particular circuit. Options TIR can be obtained by using common methods of mutagenesis, which lead to changes of codons that can change the amino acid sequence. In certain embodiments of the implementation of changes in the nucleotide sequence are silent. Changes in TIR, along with changes in the signal sequence may include, for example, changes in the amount or location of sequences Shine-Dalgarno. One way of obtaining mutant signal sequences is a receiving Bank "codons" in the beginning of the coding sequence that do not change amino acid sequence of the signal sequence (i.e. the changes are silent). This can be done by modifying the nucleotide in the third position of each codon; in addition, some amino acids such as leucine, serine and arginine, have several first and second positions, which can make the difficulty in obtaining Bank. This method of mutagenesis is described in detail in Yansura et al., (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

In one of the embodiments receive a set of vectors with a range of power TIR for each cistron in them. This limited n the Bor allows you to compare the levels of expression of each chain, also, the output of desired products antibodies with different combinations of force TIR. Force TIR can be determined by quantifying the expression level of the reporter gene, as described in Simmons et al., U.S. patent No. 5840523. On the basis of comparison of power broadcast select the desired individual TIR for the combination in constructions expressing vectors according to the invention.

Prokaryotic cells are the owners that are suitable for expression of the antibodies of the invention include Archaebacteria and Eubacteria, such as gram-negative or gram-positive organisms. Examples of suitable bacteria includeEscherichia(for example,E. coli),Bacilli(for example,B. subtilis),Enterobacteriathe speciesPseudomonas(for example,P. aeruginosa),Salmonella typhimurium,Serratia marcescans,Klebsiella,Proteus,Shigella,Rhizobia,VitreoscillaorParacoccus. In one of the embodiments use the gram-negative cells. In one of the embodiments as hosts for the invention to use cellsE. coli. Examples of strains ofE. coliinclude strain W3110 (Bachmann, Cellular and Molecular Biology, Vol.2 (Washington, D.C.: American Society for Microbiology, 1987), p.1190-1219; ATCC Deposit No. 27325) and its derivatives, including strain 33D3 with genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. patent No. 5639635). Also suitable for other strains and derivatives thereof, such asE. coli294 (ATCC 31446),E. coliB,E. coliλ 1776 (ATCC 31537), andE. coliRV38 (ATCC 31608). These examples are illustrative and not restrictive. Methods of obtaining derivatives of any of the above bacteria with defined genotypes known in this field and are described, for example, Bass et al., Proteins, 8: 309-314 (1990). As a rule, it is necessary to choose the appropriate bacteria, taking into account replenishement replicon cells of bacteria. For example, the speciesE. coli,SerratiaorSalmonellacan appropriately be used as a master, when to deliver replicon uses well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Typically, a host cell must secrete minimal amounts of proteolytic enzymes, and cell culture may be desirable to introduce additional protease inhibitors.

The production of antibodies

Cell owners transform described above expressing vectors and cultured in conventional nutrient media, if necessary, modified to ensure induction of promoters, selection of transformed cells and amplification of the genes encoding the desired sequences.

Transformation means introducing DNA prokaryotic host, so that the DNA is replicated in the form of an extrachromosomal element or integrated into the chromosome. Depending on the host cell transformation spend STD is bound ways, appropriate to such cells. For bacterial cells, which contain significant barriers in the form of cell walls, as a rule, apply the treatment with calcium using calcium chloride. In another method transformation using polyethylene glycol/DMSO. Another method is electroporation.

Prokaryotic cells used to obtain the polypeptides according to the invention are grown in environments known in the field and suitable for culturing a selected host cells. Examples of suitable media include Luria broth (LB) with the necessary nutritional supplements. In some embodiments, the implementation of the environment also contains a tool for selection, is selected on the basis of the design expressing vector for selective growth of prokaryotic cells containing expressing vector. For example, in environment for the growth of cells expressing the gene of resistance to ampicillin, add ampicillin.

In addition to the sources of carbon, nitrogen and inorganic phosphate, also you can add any necessary additives in suitable concentrations, alone or in mixture with other additives or environment, for example, with a complex nitrogen source. Optional media for cultivation may contain one or more reducing agents selected from the group consisting of g is utation, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated under suitable temperatures. In certain embodiments implement for the growth ofE. colitemperature growth range from approximately 20°C to about 39°C; from about 25°C to about 37°C or approximately 30°C. the pH of the medium may take any value in the range from about 5 to about 9, generally, depending on the host body. In certain embodiments of the implementation of the pH toE. coliis from about 6.8 to about 7.4 for, or approximately 7,0.

If expressing the vector according to the invention using inducible promoter, expression of the protein induce under conditions suitable for activation of the promoter. In one aspect of the invention for controlling transcription of the polypeptides used the PhoA promoter. Thus, the transformed cell hosts are cultivated in an environment with limited phosphate for induction. In certain embodiments of the implementation environment with limited phosphate is C.R.A.P. (for example, see Simmons et al., J. Immunol. Methods (2002), 263: 133-147). In accordance with the vector structure you can use various other inductors, as is well known in this field.

In one embodiment, the wasp is estline expressed polypeptides of the present invention are secreted into periplasm host cells and distinguish them from there. The selection of protein typically involves the destruction of the microorganism, as a rule, tools such as osmotic shock, sonication or lysis. Once the cells are destroyed, you can remove cell debris and whole cells by centrifugation or filtration. Proteins then can be cleaned, for example, by affinity chromatography on a resin. Alternatively, proteins can be transported in media for culturing and you can select from there. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of proteins produced. Then expressed polypeptides can be isolated and identified using well known methods such as polyacrylamide gel electrophoresis (PAGE) and analysis by Western blotting.

In one aspect of the invention, obtaining antibodies is carried out in large quantities by fermentation process. Available in various large-scale procedures semi-continuous fermentation for the production of recombinant proteins. Large-scale fermentation is conducted at least in tanks of 1000 liters, and in certain embodiments implement in tanks at approximately 1000 to 100,000 liters. In these fermenters are used blade mixer to distribute oxygen and nutrients, in particular, glucose (the preferred source of carbon/energy). Fermentation on a small scale, usually refers to fermentation in the fermenter, the volume of which is not more than approximately 100 liters and can vary from about 1 liter to about 100 liters.

In the fermentation process the induction of the expression of the protein, usually carried out after the cells are grown in suitable conditions, to the desired density, for example, to OD550 of approximately 180-220 when cells are in early stationary phase. You can use various inductors, in accordance with the applicable design vector, as is well known in this field and described above. Cells before induction can be grown for more than short periods of time. Cells usually induce approximately 12-50 hours, although you can use a longer or shorter induction time.

To improve the yield and quality of the polypeptides according to the invention can be modified in various fermentation conditions. For example, to improve the proper Assembly and folding of secreted polypeptides antibodies for co-transformation of prokaryotic host cells can be used for more vectors, sverkhekspressiya protein chaperones, for example, Dsb proteins (DsbA, DsbB, DsbC, DsbD and or Dsb) or FkpA (peptideprophet-CIS,TRANS-isomerase with chaperone activity). It is shown that protein chaperones facilitate proper folding and solubility of heterologous proteins produced in bacterial cells-hosts. Chen et al., (1999) J. Biol. 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. Environ. 39: 199-210.

To minimize proteolysis of expressed heterologous proteins (especially those that are sensitive to proteolysis), according to the present invention can be used by some strains hosts deficient in proteolytic enzymes. For example, strains of host cells can be modified with the implementation of the gene(s) mutation(s) in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and combinations thereof. Some strains ofE. coliscarce on the protease that are available and are described, for example, in Joly et al., (1998), above; Georgiou et al., in U.S. patent No. 5264365; Georgiou et al., in U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2: pp.63-72 (1996).

In one of the embodiments as host cells in expressing the system according to the invention using strains ofE. colithat is deficient in proteolytic enzymes and transformed plasmids, sverkhekspressiya one or more protein-chaperone.

Purification of antibodies

p> In one embodiment, the implementation produced according to the invention protein antibodies optionally purified to obtain essentially homogeneous preparations for further analyses and applications. You can use standard, well-known in the field methods protein purification. The following procedures are illustrative examples of suitable cleaning procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.

In one aspect for immunoaffinity cleaning products antibodies according to the invention are used protein A immobilized on a solid phase. Protein A is A protein of the cell wall mass of 41 kDa fromStaphylococcus aureuswith high affinity binds to Fc region of antibodies. Lindmark et al., (1983) J. Immunol. Meth. 62: 1-13. Solid phase on which immobilized protein A, can be a column containing the surface of glass or silicon dioxide, or a column of glass with controlled pore size or a column of silicic acid. In some embodiments, the application of the column cover with a reagent, such as glycerin, for a possible before the rotation nonspecific adhesion of impurities.

As the first stage of purification of the product obtained from the cell culture described above, can be placed on a solid phase with immobilized protein A, to ensure specific binding of the antibody of interest, with protein A. Then the solid phase is washed to remove nonspecific related impurities. Finally, of interest antibody is separated from the solid phase by elution.

Obtaining antibodies using eukaryotic host cells:

Vector for use in eukaryotic cells is the owner usually includes one or more of the following non-limiting components: a signal sequence, the plot of the beginning of replication, one or more marker genes, an enhancer element, a promoter and a sequence of termination of transcription.

Component signal sequence

Vector for use in eukaryotic cells-the owner may also contain a signal sequence or other polypeptide having a specific cleavage site with the N-Terminus of the Mature protein or polypeptide of interest. Heterologous signal sequence may be a sequence that is recognized and processed (i.e. cleaved signal peptidases) in the cell host. For expression in CL is de mammal available signal sequence mammals, as well as viral secretory leader sequence, for example, the signal of herpes simplex virus gD. DNA for this region of the protein precursor are ligated in reading frame to DNA that encodes the antibody.

Plot start replication

Generally, the plot of the beginning of the replication is not required in expressing vectors mammals. For example, the plot of the beginning of the SV40 replication, as a rule, can be used only because it contains the early promoter.

Component selective gene

Expressing and cloning vectors may contain selective gene, also known as selective marker. Typical selective genes encode proteins that (a) provide resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) compensate for auxotrophic deficiencies, if appropriate, or (c) provide the necessary nutrients not available from complex environment.

In one example schema selection use the drug to prevent the growth of the host cell. Those cells that are successfully transformed with a heterologous gene produce a protein that provides resistance to a drug and, thus, experience the mode selection. Examples of such dominant selection use Lek is stennie means neomycin, mycofenolate acid and hygromycin.

Another example of a suitable selective markers for mammalian cells are those that provide the identification of cells competent relative to the capture nucleic acid antibodies, for example, DHFR, thymidine kinase, metallothionein-I and-II, preferably genes metallothionein primates, adenoidectomies, ornithindecarboxilase, etc.

For example, in some embodiments, the implementation of cells transformed by selective DHFR gene, first identified by culturing all of transformed cells in the medium for cultivation that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, the implementation of a suitable cell host, which uses wild-type DHFR is the cell line of Chinese hamster ovary (CHO), deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or co-transformed with DNA sequences encoding the antibody, protein DHFR wild-type and the other is a selective marker such as aminoglycoside-3'-phosphotransferase (APH), can be subjected to selection by cultivation in a medium containing the tool selection for selective marker, such as antibiotic-aminoglyco the ID, for example, kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

Component promoter

Expressing and cloning vectors usually contain a promoter that is recognized by the host organism and is functionally linked to a nucleic acid that encodes an polypeptide (e.g. antibody). In eukaryotes known promoter sequences. For example, essentially all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases above the site of transcription initiation. Another sequence found 70 to 80 bases above the start of transcription of many genes is an area CNCAAT, where N can be any nucleotide. With the 3'end of most eukaryotic genes is the sequence AATAAA, which may be a signal adding poly-A-tail to the 3'-end of the coding sequence. In certain embodiments of the implementation in eukaryotic expressing vectors appropriately, you can embed any or all of the sequence data.

Transcription from vectors in the cells of the host mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, the virus of chicken pox, adenovirus (such as adenovirus 2), human papilloma virus big horn the cattle, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis B virus and the monkey virus 40 (SV40), from heterologous mammalian promoters, e.g. the actin promoter or promoter of the immunoglobulin from the promoters of heat shock proteins, provided that such promoters are compatible with the systems of the host cell.

Early and late promoters of SV40 virus are conveniently obtained as restriction fragments of SV40, which also contain the site of the beginning of replication of SV40 virus. Pretani the promoter of the human cytomegalovirus is conveniently obtained as a restriction fragment HindIII E. In U.S. patent No. 4419446 described system the expression of DNA-hosts-mammals using human papilloma virus of cattle as a vector. Modification of this system is described in U.S. patent No. 4601978. Cm. also Reyes et al., Nature 297: 598-601 (1982), which describes the expression of the cDNA of β-interferon person in mouse cells under the control of the promoter timedancing from herpes simplex virus. Alternatively, as a promoter, you can use the long terminal repeat of rous sarcoma virus.

Component enhancer

Transcription of DNA encoding the antibody according to this invention, in higher eukaryotes, often increase the embedding in vector enhancer sequence. Currently, there are many enhancer sequence is of elesta from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, as a rule, use the enhancer of virus eukaryotic cells. Examples include the SV40 enhancer late side of the point of initiation of replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyoma late side of the point of initiation of replication and enhancers of adenoviruses. Cm. also Yaniv, Nature 297: 17-18 (1982), which describes enhancer elements for activation of eukaryotic promoters. The enhancer can be incorporated into the vector at position 5' or 3' relative to a sequence that encodes a polypeptide antibodies, but, as a rule, it is located in the region from the 5'end of the promoter.

Component of the transcription termination

Expressing the vectors used in eukaryotic cells-the owners may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5'and, occasionally 3'-terminal untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA that encodes the antibody. One of the suitable components for termination of transcription is the region polyadenylation growth hormone in cattle. Cm. WO94/11026 about icandy in this application expressing vector.

Selection and transformation of host cells

Cell-hosts suitable for cloning or expression of DNA described in this document vectors include cells of higher eukaryotes, as described herein, including cells of vertebrate hosts. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable cell lines of mammalian hosts represent a line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); a line of embryonic human kidney (293 cells or 293 cells, subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); the cells of the Djungarian hamster kidney (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); cells Sertoli mouse (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); kidney cells of monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells, dog kidney (MDCK, ATCC CCL 34); liver cells buff rats (BRL 3A, ATCC CRL 1442); cells of the human lung (W138, ATCC CCL 75); the cells of the human kidney (Hep G2, HB 8065); tumor of the mammary gland of the mouse (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); the cells, MRC 5; FS4 cells and human hepatoma (Hep G2).

Cell owners transform described above expressing or cloning vectors is to obtain antibodies and cultured in conventional nutrient media modified if necessary for the induction of promoters, selection of transformed cells, or amplifying the genes encoding the desired sequences.

Culturing host cells

Cell owners used to obtain the antibodies according to this invention, can be grown in different environments. For culturing the host cells can be used commercially available medium such as Ham''s F10 (Sigma), minimal maintenance medium ((MEM), (Sigma), RPMI-1640 (Sigma) and modified by way of Dulbecco Wednesday Needle ((DMEM), Sigma). In addition, as media for culturing host cells you can use any medium 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; WO 90/03430; WO 87/00195; or U.S. patent Re. 30985. Any of these media may be supplemented as 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 GENTAMYCINTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or equivale tym source of energy. You can also include any other well-known experts in the field of additives in suitable concentrations. Culturing conditions, such as temperature, pH, etc. correspond to those used for host cells selected for expression, and, as a rule, they are known to specialists in this field.

Purification of antibodies

When using recombinant methods, the antibody can be produced intracellularly or it can directly secretariats on Wednesday. If the antibody is produced intracellularly, in the first stage removes debris from the particles, i.e. the cells of the host or lysed fragments, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, samples of the supernatant liquid such expression systems can be first concentrated using a commercially available filter for concentrating proteins, for example, devices for ultrafiltration Amicon or Millipore Pellicon. Any of the above stages, you can add a protease inhibitor such as PMSF, for the inhibition of proteolysis, and you can add antibiotics to prevent the growth side of the dirt.

Obtained from cell composition antibodies can be cleaned, for example, chromatography on hydroxyapatite, electrophoresis gel, dialysis and Finney chromatography with affinity chromatography, which is a convenient way. The possibility of using protein A as an affinity ligand depends on the species and isotype of immunoglobulin Fc domain that is present in the antibody. Protein A can be used for purification of antibodies, which are based on the heavy chain γ1, γ2, or γ4 human (Lindmark et al., J. Immunol. Methods 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for ø3 person (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which is attached an affine ligand can be a agarose, although available, and other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene, provide higher flow rates and lower processing time, compared with similar results obtained when using agarose. Where the antibody contains a CH3 domain, for cleaning uses resin Bakerbond ABX™ (J.T. Baker, Phillipsburg, NJ). Depending on the antibodies that need to get other available methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on SEPHAROSE™ heparin chromatography on anyone - or cation-exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE, and the deposition of sulfate and mania.

After any(s) phase(s) pre-treatment mixture containing an antibody and pollutants, can be subjected to further purification, for example by chromatography hydrophobic interactions with low pH using a buffer for elution with a pH of approximately in the range of 2.5 to 4.5, preferably carried out at low salt concentrations (e.g., approximately in the range of 0-0,25 M salt).

Generally, various methods for producing antibodies for use in research, testing and clinical application in this area is well developed, in accordance with the above described methods and/or the opinion of a specialist in this field for a specific interest antibodies.

Immunoconjugate

The invention also relates to immunoconjugates (interchangeably referred to as "the conjugates of the antibody-drug" or "ADC")that contain any antibodies to CD22 according to the invention conjugated to one or more cytotoxic means, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e radioconjugates).

In particular is the option exercise immunoconjugate contains antibody to CD22 and chemotherapeutic agent or other toxin. Chemotherapeutic agents suitable for receiving immunoconjugates described herein (e.g., above). You can also use enzymatically active toxins and fragments thereof, and they are described in this document.

In certain embodiments of the implementation immunoconjugate contains antibody to CD22 and one or more low molecular weight toxins, including as non-limiting examples of low molecular weight drugs, such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecin and CC1065, and the derivatives of these drugs, which have cytotoxic activity. Hereinafter discussed in detail, examples of such immunoconjugates.

1. Illustrative immunoconjugate - conjugates antibody-drug

Immunoconjugate (or conjugate antibody-drug" ("ADC")) according to the invention may be of formula I, below, where the antibody to CD22 conjugated (i.e. covalently associated with one or more groups of medicines (D) through an optional linker (L).

Ab-(L-D)pThe formula I

Thus, antibody to CD22 can be konjugierte with drug either directly or through a linker. In the form of the ule I p is the average number of groups of the drug to the antibody, which may be in the range of, for example, from approximately 1 to approximately 20 groups of the drug to the antibody, and in certain embodiments of the implement from 1 to about 8 groups of the drug to the antibody.

Illustrative linkers

Herein described illustrative linkers and groups of medicines. The linker may contain one or more linker components. Illustrative of the linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe), p-aminobenzeneboronic ("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate ("SMCC"), and N-Succinimidyl (4-iodates)aminobenzoate ("fairs are forthcoming-Siab"). In this area there are various linker components, some of which are described below.

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

In some embodiments, implementation of the linker is component may contain "extension", which binds to the antibody with a different linker component or molecule drugs. Illustrative extensions below (where the wavy line indicates the sites of covalent bonds with uniteam):

In some embodiments, implementation of the linker component may contain amino acid unit. In one such embodiment, the amino acid unit allows cleavage of the linker by the protease, thus facilitating the release of drugs from immunoconjugate when exposed to intracellular proteases, such as lysosomal enzymes. For example, see Doronina et al., (2003) Nat. Biotechnol. 21:778-784. Illustrative amino acid units include as non-limiting examples of 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). The amino acid unit can contain amino acid residues that occur in nature, as well as minor amino acids not found in nature similar amino acids such as citrulline. Amino acid units to stroyraboty and optimized in their selectivity for enzymatic cleavage by a specific enzyme, for example, associated with tumor proteases, cathepsin B, C and D or protease plasmin.

In some embodiments, implementation of the linker component may contain "spacer elements" unit, which antibody binds with a molecule drugs, either directly or through extension and/or amino acid units. Spacer elements unit can be "self-destructive" or "nesporazumi". "Resumeresume" spacer elements unit represents a spacer elements unit, remaining associated with the molecule medicines enzymatic (e.g., proteolytic) the splitting of the ADC. Examples resumeresume spacer elements units include as non-limiting examples of glycine spacer elements unit and spacer elements unit glycine-glycine. Also consider other combinations of peptide spacers suitable for the specific sequence of enzymatic degradation. For example, enzymatic cleavage ADC containing spacer elements unit glycine-glycine through associated with tumor cells protease, leads to the release of the group glycine-glycine-molecule drugs from the remainder. In one such embodiment, the group of glycine-glycine-molecule drugs then subjected to a separate hydrolysis step in cancer cells, so the way spacer elements separating unit glycine-glycine spacer elements unit from molecule drugs.

"Auto-disable" spacer elements unit provides the release of a molecule drugs without a separate hydrolysis step. In certain embodiments of the implementation of the spacer elements unit linker contains p-aminobenzyl unit. In one such embodiment, to an amino acid unit via amide linkages attached p-aminobenzoyl alcohol, and between benzyl alcohol and the cytotoxic agent is formed carbamate, methylcarbamate or carbonate. For example, see Hamann et al., (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment, the implementation of the spacer elements unit is a p-aminobenzeneboronic (PAB). In certain embodiments of the implementation fenelonov part of the p-aminoaniline units substituted Qmwhere Q represents-C1-C8-alkyl, -O-(C1-C8-alkyl), -halogen,- nitro or-cyano; and m is an integer in the range 0-4. Examples of auto spacer elements units additionally include as non-limiting examples of aromatic compounds that are electronically similar to the p-aminobenzyl alcohol (for example, see U.S. patent 2005/0256030 A1), such as derivatives of 2-aminoimidazole-5-methanol (Hay et al., (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para-aminobenzoate. You can use the spacers that are undergoing education cycle when the hydrolysis of the amide bond, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted ring system is bicyclo[2.2.1] bicyclo[2.2.2] (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). Examples of auto spacers suitable for the ADC, is the elimination of amines containing medicines, substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447).

In one embodiment, the implementation of the spacer elements unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, as described below, which can be used for embedding and release of multiple drugs.

where Q represents-C1-C8-alkyl, -O-(C1-C8-alkyl), -halogen, -nitro or-cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges from 1 to about 20.

The linker may contain any one or more of the above linker components. In certain embodiments of the implementation of the linker is as indicated in parentheses in the following formula II ADC

Ab-([Aa-Ww-Yy]-D)pFormula II/td>

where A represents an extension cord, and a is an integer from 0 to 1; W is an amino acid unit, w is an integer from 0 to 12; Y is a spacer elements unit, and y is 0, 1 or 2; and Ab, D, and p are defined as above for formula I. Illustrative embodiments of such linkers are described in U.S. patent 20050238649 A1, which is incorporated herein by reference in full.

Illustrative of the linker components and their combinations are shown below in the context of formula II ADC

Linker components, including the extension, spacer and amino acid units, it is possible to synthesize well-known in this field means, such as the methods described in U.S. patent 2005-0238649 A1.

Illustrative group of medicines

Mitanin and maytansinoid

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

Molecules maytansinoids medicines are an attractive group of medicines for the conjugates of the antibody-drug, as they are (i) relatively affordable to get through fermentation or chemical modification or derivatization products of fermentation, (ii) amenable to derivatization of functional groups suitable for conjugation with antibodies through not by a disulfide linkers, (iii) stable in plasma, and (iv) effective against many lines of tumor cells.

Connection maytansine suitable for use in the form of molecules maytansinoid drugs, well known in this field and they can be isolated from natural sources by known methods or obtained by means of genetic engineering (see Yu et al., (2002) PNAS 99:7968-7973). Maytansines and analogues maytansine can also be obtained synthetically by known methods.

Illustrative embodiments of the groups maytansinoids medicines include DM1; DM3 and DM4, as described in the present who eat the document.

Auristatin and dolastatin

In some embodiments, the implementation immunoconjugate contains the antibody of the invention conjugated to dolastatins or a peptide analog or derivative, for example, auristatin (U.S. patent No. 5635483; 5780588). It is shown that dolastatin and auristatin hinder the dynamics of microtubules, the hydrolysis of GTP and division of nuclei and cells (Woyke et al., (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have antitumor (U.S. patent No. 5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965). Molecule drug dolastatin or auristatin can be attached to the antibody at the N-(amino)end or the C(carboxyl)the end of the peptide molecule drugs (WO 02/088172).

Illustrative embodiments of auristatin include attached at the N-end group of medicines monomethylaniline DE and DF, described in Senter et al., Proceedings of the American Association for Cancer Research, volume 45, abstract No. 623, presented March 28, 2004, the description of which is incorporated into this description by reference in full.

The peptide molecule drugs can be selected from the formulas DEand DFbelow:

where the wavy line DEand DFindicates the site of covalent joining the antibody or the antibody-linker is omponent, and independently, at each position

R2selected from H and C1-C8-alkyl;

R3selected from H, C1-C8-alkyl, C3-C8-carbocycle, aryl, C1-C8-alkylaryl, C1-C8-alkyl-(C3-C8-carbocycle), C3-C8-heterocycle, and C1-C8-alkyl-(C3-C8-heterocycle);

R4selected from H, C1-C8-alkyl, C3-C8-carbocycle, aryl, C1-C8-alkylaryl, C1-C8-alkyl-(C3-C8-carbocycle), C3-C8-heterocycle, and C1-C8-alkyl-(C3-C8-heterocycle);

R5selected from H and methyl;

or R4and R5together form a carbocyclic ring system and have the formula -(CRaRb)n-where Raand Rbindependently selected from H, C1-C8-alkyl and C3-C8-carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R6selected from H and C1-C8-alkyl;

R7selected from H, C1-C8-alkyl, C3-C8-carbocycle, aryl, C1-C8-alkylaryl, C1-C8-alkyl-(C3-C8-carbocycle), C3-C8-heterocycle, and C1-C8-alkyl-(C3-C8-heterocycle);

each R8independently selected from H, OH, C1-C8-alkyl, C3-C8-carbocycle and O-(C1-C8-al the sludge);

R9selected from H and C1-C8-alkyl;

R10selected from aryl or C3-C8-heterocycle;

Z represents O, S, NH, or NR12where R12represents a C1-C8-alkyl;

R11selected from H, C1-C20of alkyl, aryl, C3-C8-heterocycle, -(R13O)m-R14or -(R13O)m-CH(R15)2;

m is an integer in the range 1-1000;

R13represents a C2-C8-alkyl;

R14represents H or C1-C8-alkyl;

each occurrence of R15independently represents H, COOH, -(CH2)n-N(R16)2, -(CH2)n-SO3H or -(CH2)n-SO3-C1-C8-alkyl;

each occurrence of R16independently represents H, C1-C8-alkyl or -(CH2)n-COOH;

R18selected from-C(R8)2-C(R8)2-aryl, -C(R8)2-C(R8)2-(C3-C8-heterocycle), and-C(R8)2-C(R8)2-(C3-C8-carbocycle); and

n is an integer in the range from 0 to 6.

In one embodiment, the implementation of R3, R4and R7independently represent an isopropyl or sec-butyl, and R5represents-H or methyl. In the illustrative embodiment, the wasp is estline each of R 3and R4represent isopropyl, R5represents-H, and R7represents a sec-butyl.

In another embodiment, each of R2and R6represent methyl, and R9is a-H.

In another embodiment, each occurrence of R8represents-OCH3.

In the illustrative embodiment, each of R3and R4represent isopropyl, each of R2and R6represent methyl, R5represents-H, R7represents sec-butyl, each occurrence of R8represents-OCH3and R9is a-H.

In one of the embodiments Z represents-O - or-NH-.

In one embodiment, the implementation of R10represents aryl.

In the illustrative embodiment, R10represents phenyl.

In the illustrative embodiment, when Z represents-O-, R11represents-H, methyl ortert-butyl.

In one of the embodiments, when Z represents-NH, R11represents a-CH(R15)2where R15represents -(CH2)n-N(R16)2and R16represents a C1-C8-alkyl or -(CH2)n-COOH.

In another embodiment, the wasp is estline, when Z represents-NH, R11represents a-CH(R15)2where R15represents -(CH2)n-SO3H.

Illustrative variant implementation of auristatin formula DErepresents MMAE, where the wavy line indicates the covalent bond of the conjugate antibody-drug linker (L):

Illustrative variant implementation of auristatin formula DFrepresents MMAF, where the wavy line indicates the covalent bond of the conjugate antibody-drug linker (L) (see U.S. patent 2005/0238649 and Doronina et al., (2006) Bioconjugate Chem. 17:114-124):

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

and

In one aspect of the molecule drugs in R11you can attach hydrophilic group, including as non-limiting examples of esters of triethylene glycol (TEG), as shown above. Without regard to any particular theory of the hydrophilic group help in INTA is the similar and prevent agglomeration of the molecules of the drug.

Illustrative embodiments of the ADC of formula I containing auristatin/dolastatin or their derivatives, described in U.S. patent 2005-0238649 A1 and Doronina et al., (2006) Bioconjugate Chem. 17:114-124, which are incorporated herein by reference 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 represents from 1 to about 8, "Val-Cit" is a dipeptide valine-citrulline; and "S" is a sulfur atom:

Illustrative embodiments of the ADC of formula I containing MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, it is shown that immunoconjugate containing MMAF, attached to the antibody through a linker, which proteoliticeski does not split, have activity comparable to immunoconjugate containing MMAF, attached to the antibody through proteoliticeski biodegradable linker. See, Doronina et al., (2006) Bioconjugate Chem. 17:114-124. In such instances, it is believed that the release of drug acts by destroying antibodies in the cell. Id.

Typically, groups of peptide drugs can be obtained by forming a p is pidou the relationship between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, by the method of liquid-phase synthesis (see E. Schröder and K. Lübke, "The Peptides", volume 1, p.76-136, 1965, Academic Press)that is well known in the field of peptide chemistry. Group of medicines auristatin/dolastatin can be obtained by the methods of U.S. patent 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, the group of medicines auristatin/dolastatin formula DFsuch as MMAF and their derivatives, can be obtained by methods described in U.S. patent 2005-0238649 A1 and Doronina et al., (2006) Bioconjugate Chem. 17: 114-124. Group of medicines auristatin/dolastatin formula DEsuch as MMAE and their derivatives, can be obtained by 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 easy to synthesize by conventional means, for example, as described in Doronina et al., (2003) Nat. Biotech. 21:778-784, and the publication of patent application no US 2005/0238649 A1, and then konjugierte with interest the antibody.

Load drug

Load the medicinal product is designated as p, and it represents the average number of molecules of the drug on the anti-Christ. ate in a molecule of formula I. Load the medicinal product may be in the range from 1 to 20 molecules of the drug (D) antibody. The ADC of formula I include the collection of antibodies conjugated with a group of molecules of the drug, from 1 to 20. The average number of molecules of the drug to the antibody in preparations of the ADC after conjugation reactions may be characterized by conventional methods such as mass spectroscopy, ELISA and HPLC. It is also possible to determine the quantitative distribution of the ADC from the point of view p. In some instances, separation, purification, and characterization of homogeneous ADC, where p is a certain value from another ADC with another load of the medicinal product can be performed by means of reversed-phase HPLC or electrophoresis.

For some conjugates of the antibody-drug p may be limited by the number of lots attach to the antibody. For example, where the join occurs on cysteine thiol, as in illustrative embodiments, the implementation presented above, the antibody may carry only one or a few tylnej groups of cysteine, or it can carry only one or more reactive enough tylnej groups, through which you can attach the linker. In certain embodiments the higher the load Lakers the public means, for example, p>5, can lead to aggregation, insoluble state, toxicity or loss of cell permeability of certain conjugates of the antibody-drug. In certain embodiments of the exercise load of the medicinal product for the ADC according to the invention is in the range from 1 to about 8; from about 2 to about 6, from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3,9; from about 3.2 to about 3,8; from about 3.2 to about 3,7; from about 3.2 to about 3.6; from about 3.3 to about 3,8; or from about 3.3 to about 3.7V. In fact, it was shown that for certain ADC optimal ratio of molecules of the drug to the antibody may be less than 8, and may be from about 2 to about 5. Cm. U.S. patent 2005-0238649 A1 (incorporated herein by reference in full).

In certain embodiments of the implementation during the reaction of conjugation with the antibody conjugated to the number of molecules of drugs, less than theoretical maximum of the molecules of the drug. For example, the antibody may contain lysine residues, which do not react with the intermediate connection is of a drug-linker or linker reagent, as is discussed below. Only the most reactive group of lysine can react with a linker reagent, reacting with the amine. Typically, the antibody does not contain many free and reactive tylnej groups of cysteine, which can be contacted with a molecule drugs; in fact, most tylnej cysteine residues are antibodies as disulfide bridges. In certain embodiments of the implementation of the antibody can be recovered with the reducing agent, such as dithiothreitol (DTT) or tricarbonylchromium (TCEP), in conditions of partial or full recovery, obtaining reactive tylnej groups of cysteine. In certain embodiments of the implementation of the antibody are subjected to conditions of denaturation for detection of reactive nucleophilic groups, such as lysine or cysteine.

The load ratio (drug/antibody) of the ADC can be controlled in various ways, for example by (i) limiting molar excess of the intermediate drug-linker or linker reagent relative to the antibody, (ii) time limit reactions of conjugation or temperature, (iii) partial or limited recovery for modification of the thiol of cysteine, (iv) constructing a recombinant method is mi amino acid sequence of the antibody so, to modify the number and position of cysteine residues to control the amount and/or provision of connections linker-drug (such as thioMab or thioFab obtained as described herein and in WO 2006/034488 (incorporated herein by reference in full)).

It should be understood that when the intermediate connection of a drug-linker or linker reagent reacts more than one nucleophilic group, followed by the reagent molecule drugs, the resulting product is a mixture of compounds of the ADC with the distribution of one or more drug molecules attached to the antibody. The average number of drugs per antibody can be calculated in a mixture by ELISA with two antibodies specific for antibodies specific for the drug. Individual molecules of the ADC can be identified in the mixture by mass spectroscopy and divide by HPLC, for example, by hydrophobic interaction chromatography (for example, see Hamblett, K.J., et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al., "Controlling the location of drug attachment in antibody-drug conjugates", Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments of the implementation of a mixture of conjugates can be distinguished homogeneous ADC with a single load value by electrophoresis or chromatography.

Some ways of getting immunoconjugates

The ADC of formula I can be obtained in several ways, using reaction conditions and reagents in 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 molecule drugs D; and (2) reaction of a nucleophilic group of a molecule drugs with a bivalent linker reagent with the formation of 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 last method is described in U.S. patent 20050238649 A1, which is incorporated herein by reference in full.

Nucleophilic groups on the antibodies include as non-limiting examples of (i) the N-terminal amino group, (ii) amino side chains, for example, lysine, (iii) tirinya groups of the side chains, for example, cysteine, and (iv) a hydroxyl or amino sugars, when the antibody is glycosylated. Amino group, tirinya and hydroxyl groups I have 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, esters, HOBt, halogenfree and acid halides; (ii) alkyl and benzyl halides such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimido group. Some antibodies have recoverable megamachine disulphide, i.e. cysteine bridges. Antibodies can be converted into reactive for conjugation with linker reagents by treatment with reducing agent such as DTT (dithiothreitol) or tricarbonylchromium (TCEP), so that the antibody has been fully or partially restored. Thus, each cysteine bridge will, theoretically, to form two reactive tylnej of the nucleophile. Alternatively, sulfhydryl groups can be introduced in antibodies by modifying lysine residues, for example, by the reaction of lysine residues with 2-aminothiophenol (reagent Troth), leading to the transformation of the amine in the thiol. Reactive tirinya group can be introduced into the antibody by introducing one, two, three, four or more cysteine residues (for example, by obtaining variants of antibodies containing one or more non-naturally occurring amino acid cysteine residues).

Conjugates of the antibody-drug according to the invention also monopolizing reaction between the electrophilic group on the antibody, such as carbonyl aldehyde or ketone group, with the nucleophilic group on the linker reagent or drug. Suitable nucleophilic groups on the linker reagent include as non-limiting examples of the hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and originated. In one of the embodiments the antibody is modified to introduce electrophilic groups capable of reacting with nucleophilic substituents on the linker reagent or drug. In another embodiment, sugar glycosylated antibodies can oxidize, for example, by oxidizing reagents based periodate with the formation of aldehyde or ketone groups that can react with the amino group of the linker reagents or molecules of the drug. The obtained imine group of the Schiff's base can form a stable connection or they can be recovered, for example, borhydride the reagents to form stable amine linkages. In one embodiment, the implementation of the reaction of the carbohydrate portion of a glycosylated antibody with either galactosialidosis or metaperiodate sodium can result in carbonyl group (aldehyde and ketone) antibody that can react with appropriate groups on the drug is the first tool (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies that contain N-terminal residues of serine or threonine, can be subjected to reaction with metaperiodate sodium, leading to the formation of aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. patent 5362852). This aldehyde can be subjected to reaction with a molecule drugs or nucleophile linker.

Nucleophilic groups on the molecule drugs include as non-limiting examples of amino groups, tirinya, hydroxyl, hydrazide, Aksinya, hydrazine, thiosemicarbazone group, carboxylate group of the hydrazine and arylhydrazines that can react with electrophilic groups on linker groups and linker reagents with the formation of covalent bonds, including (i) active esters such as NHS esters, esters, HOBt, halogenfree and acid halides; (ii) alkyl and benzyl halides such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimido group.

Compounds according to the invention specifically include as non-limiting examples of the ADC obtained with the following cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinils the background)benzoate), which are commercially available (e.g., 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 a protein, 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 -, page (such as bis(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, the immunotoxin of ricin can be obtained as described in Vitetta et al., Science 238:1098 (1987). Illustrative chelating agent for conjugation of the radionuclide to the antibody is labelled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA). Cm. WO94/11026.

Alternatively, you can get protein containing the antibody and cytotoxic agent, for example, by recombinant methods or peptide synthesis. Recombine the single DNA molecule can contain plots, encoding the antibody and the cytotoxic portion of the conjugate, located next to each other or separated by plot, encoding the linker peptide which does not violate the desired properties of the conjugate.

In another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for use in pre-targeting the tumor, where the conjugate of the antibody-receptor is administered to the patient, followed by removal is not bound peroxidase conjugate from the blood flow with the use of tools for removal and the subsequent introduction of a "ligand" (e.g., avidin), which anywhereman with a cytotoxic agent (e.g. a radionuclide).

2. Illustrative immunoconjugate - Conjugated cienciala-drug

Receiving modified with cysteine antibodies to CD22

DNA encoding variant amino acid sequence modified by cysteine antibodies to CD22 and the original antibody to CD22 according to the invention, receive a variety of ways that include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence variants), obtaining site-specific (or mediated by oligonucleotides) mutagenesis (Carter (1985) et al., Nucleic Acids Res. 13: 4431-4443; Ho et al., (1989) Gene (Amst.) 77: 51-59; Kunkel et al., (1987) Proc. Natl. Acad. Si. USA 82: 488; Liu et al., (1998) J. Biol. Chem. 273: 20252-20260), PCR mutagenesis (Higuchi, (1990)PCR Protocols, pp.177-183, Academic Press; Ito et al., (1991) Gene 102: 67-70; Bernhard et al., (1994) Bioconjugate Chem. 5: 126-132; and Vallette et al., (1989) Nuc. Acids Res. 17: 723-733) and 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, for example, QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations also get mediated by oligonucleotides by mutagenesis using double-stranded plasmid DNA as template through mutagenesis by PCR (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al., (1983) Methods Enzymol. 100: 468-500; and Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res. 10: 6487-6500). Variants of recombinant antibodies can be designed by manipulating fragments of the enzyme or by overlap extension PCR using synthetic oligonucleotides. Mutagenic primers encode the replacement(s) of cysteine codon. To obtain DNA that encodes a mutant-modified cysteine antibodies, it is possible to use standard 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 to CD22 and fragments of antibodiesin vitrofrom a set of genes of variable (V) domain of them is noglobulin from unimmunized donors, you can use the technology of phage display (McCafferty et al., (1990) Nature 348: 552-553). In accordance with this method, genes V-domain antibody clone in frame reading in the primary gene or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and exhibit as functional fragments of the antibodies on the surface ragovoy particles. Because filamentous particle contains a single-stranded copy of DNA of phage genome, selection on the basis of functional properties of antibodies also leads to the selection of the gene encoding the antibody possessing these 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; U.S. patent 5565332; U.S. patent 5573905; U.S. patent 5567610; U.S. patent 5229275).

Antibodies to CD22 can be synthesized chemically using well-known methods for the synthesis of oligopeptides or you can get them and clean using recombinant technology. Suitable amino acid sequence or part thereof can be obtained by direct peptide synthesis using solid-phase methods (Stewart et al., Solid-Phase Peptide Synthesis, (1969) W.H. Freeman Co., San Francisco, CA; Merrifield, (1963) J. Am. Chem. Soc, 85:2149-2154). The synthesis of proteinsin vitrocan be performed using manual methods or by using machines. Automated solid-phase synthesis can be performed, for example, and the use is protected by t-BOC or Fmoc amino acids with the use of the device for the synthesis of peptides Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Different parts of the antibody to CD22 or CD22 polypeptide can be synthesized chemically separately and combined using chemical or enzymatic methods of obtaining the desired antibodies to CD22 or CD22 polypeptide.

To obtain fragments of antibodies developed different ways. Typically, these fragments were obtained by proteolytic cleavage of the original antibody (Morimoto et al., (1992) Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al., (1985) Science, 229:81) or produced directly by recombinant host cells. Fragments Fab, Fv and ScFv antibody to CD22 can be Express inE. coliand to secrete fromE. colithus, providing a light receiving large amounts of these fragments. Antibody fragments can be isolated from phage libraries of antibodies discussed herein. Alternatively, fragments, Fab'-SH can be directly distinguished fromE. coliand chemically bind with the formation of fragments (Fab')2(Carter et al., (1992) Bio/Technology 10:163-167) or select directly from a culture of the recombinant host cells. Antibody to CD22 can be a single-chain Fv fragment (scFv) (WO 93/16185; U.S. patent 5571894; U.S. patent 5587458). Fragment antibodies to CD22 may also be a "linear antibody" (U.S. patent 5641870). These linear fragments of antibodies could the t to be monospecific or bespecifically.

The following description refers mainly to the receipt of antibodies to CD22 by culturing cells transformed or transfected with a vector containing nucleic acid encoding the antibody to CD22. DNA encoding the antibodies to CD22, can be obtained from a cDNA library derived from a tissue suspected of containing mRNA antibodies to CD22 and expressing it on detektiruya level. Thus, DNA antibodies to CD22 or CD22 polypeptide person can conveniently be obtained from a cDNA library derived from human tissues. The gene encoding the antibody to CD22, also can be obtained from a genomic library or by known methods of synthesis (e.g., automated nucleic acid synthesis).

Methods design, selection and receiving according to the invention allow to obtain a modified cysteine antibodies to CD22 with electrophilic groups that are reactive. These methods additionally allow you to get a connection conjugates of antibodies, such as connections conjugate antibody-drug (ADC), with the molecules of the drug in specified, designed, selected sites. Reactive cysteine residues on the surface antibodies provide specific conjugation of the molecule drugs possessioneeeeeeeee thiol group, such as maleimide or halogenoacetyl. Nucleophilic reactivity of thiol functional group of the Cys residue with a group of maleimide is about 1000 more than any other functional group of the amino acids in the protein, such as amino group of lysine residues or N-terminal amino group. Specific for thiol functional group in iodization and maleimide reagents may react with the amine groups, but require a higher pH (>9,0) and a longer reaction time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The number of free thiols in the protein can be determined by standard analysis of Ellman. The example linked by a disulfide bond of pentamer is immunoglobulin M, while an example of a protein with an internal disulfide bridges linking together subunit is immunoglobulin G. In proteins such as these, for the formation of reactive thiol you want to restore the disulfide bond with a reagent such as dithiothreitol (DTT) or selenol (Singh et al., (2002) Anal. Biochem. 304:147-156). This approach can lead to the loss of tertiary structure of the antibody and the binding specificity of the antigen.

Analysis Pheselector (Phage ELISA for Selection of Reactive Thiols) allows for screening of reactive groups of cysteine in antibodies in phage ELISA format, thereby, design modified with cysteine antibodies (WO 2006/034488). Modified cysteine antibody cover the surface of the hole, and then incubated with fagbemi particles add HRP labeled secondary antibody and spend the definition of absorption. Mutant proteins exposed on the phage, can be subjected to screening is fast, reliable and high-performance way. You can get a library of modified cysteine antibodies and expose their breeding binding using the same approach to identify the inclusion of appropriately reactive centers of free Cys from random libraries of antibody protein-phage or other proteins. This method comprises carrying out the reaction of cysteine mutant proteins expressed on the phage, affinity reagent or reporter group, which is also capable of reacting with a thiol.

Analysis PHESELECTOR allows the screening of reactive tylnej groups in antibodies. Identification of options A121C this method is illustrative. To identify a wider range of choices ThioFab with reactive thiol groups can effectively see a Fab molecule. To identify and quantify the accessibility of solvent to the amino acid residues in the polypeptide used parameter fractional surface accessibility. Poverhnostno the availability can be expressed as surface area (Å 2), which can be contacted with a molecule of solvent, e.g. water. The space occupied by water, is approximately a sphere with a radius of 1.4 Å. The software is freely available or licensed (Secretary CCP4, Daresbury Laboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825 or on the Internet: www.ccp4.ac.uk/dist/html/TNDEX.html) as CCP4 Suite of crystallographic programs that use algorithms to calculate the surface accessibility of each amino acid protein with known rentgenotomograficheskie obtained coordinates ("The CCP4 Suite: Programs for Protein Crystallography" (1994) Acta. Cryst. D50:760-763). Two illustrative software module performing the calculations of surface accessibility, are "AREAIMOL" and "SURFACE", on the basis of algorithms B. Lee and F.M. Richards (1971) J. Mol. Biol. 55: 379-400. AREAIMOL defines solvent-accessible surface of the protein as the locus of points of the center of the measuring sphere (representing a solvent molecule) at rolling it on the van der Waals surface of the protein. AREAIMOL calculates the solvent-accessible surface area through the creation of surface points on the extended each atom in the field (at a distance from the center of the atom is equal to the sum of the radii of the atom and meter) and removing those which lie lie in equivalent areas associated with the neighboring atoms. AREIMOL finds the solvent-accessible area of atoms in the PDB file with coordinates and summarizes the available space on the remaining, on circuits and for the whole molecule. Available area or differing areas) for individual atoms can be written in the output pseudo-PDB file. AREAIMOL involves one radius for each element and recognizes only a limited number of different elements.

AREAIMOL and give us the absolute SURFACE distance, i.e. the number of angstroms (Å) in the square. Fractional surface accessibility calculated relative to the standard state, typical for amino acids in the polypeptide. The standard condition is a Tripeptide Gly-X-Gly, where X represents the interest amino acid, and standard status must be "stretched out" conformation, i.e. similar to the conformation of the beta-layer. The stretched conformation maximizes the availability X. Calculated the available area divided by the area available in the standard condition in the Tripeptide Gly-X-Gly and gain coefficient, which represents the fractional availability. The percentage of availability is a fractional 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 fractional accessibility of amino acid residue for the water sector on the basis of the x-ray coordinates of the polypeptide. Fractional poverhnostno the accessibility of each amino acid in the antibody can be calculated using the information about the available crystal structures (Eigenbrot et al., (1993) J. Mol. Biol. 229: 969-995).

DNA encoding modified cysteine antibodies, it is easy to select and sequence the traditional ways (e.g., by using oligonucleotide probes that are capable of specific binding to genes encoding the heavy and light chains of murine antibodies). As a source of such DNA are hybridoma cells. After DNA extraction can be placed in expressing vectors, which are then transferout in cell host, such as cellsE. colicells , COS monkey cells Chinese hamster ovary (CHO) or other cells of the host mammal, such as myeloma cells (U.S. patent 5807715; U.S. patent 2005/0048572; U.S. patent 2004/0229310)that would otherwise not produce protein antibodies, to obtain the synthesis of monoclonal antibodies in the recombinant cell host.

After the design and selection of modified cysteine antibodies, for example, ThioFab, with integrated, highly reactive, unpaired Cys residues, can be obtained by (i) expression in bacteria, for example,E. coli(Skerra et al., (1993) Curr. Opinion in Immunol. 5:256-262; Pluckthun (1992) Immunol. Revs. 130:151-188) or the system of culture of mammalian cells (WO 01/00245), for example, cells of the Chinese hamster (CHO); and (ii) purification using conventional methods of protein purification (Lowman et al., (1991) J. Biol. Chem. 266(17):10982-10988).

Built tirinya groups Cys takes the Ute in the reaction with electrophilic linker reagents and intermediate compounds, the drug-linker with the formation of the modified cysteine conjugates antibody-drug and other labeled modified cysteine antibodies. Residues Cys-modified cysteine antibodies and are in the original antibody, which are paired and form mesapotamia and interchain disulfide bonds, have no reactive tylnej groups (if not treated with reducing agent) and do not react with electrophilic linker reagents or intermediate compounds, the drug-linker. Newly built Cys residue may remain unpaired and able to response, i.e. konjugierte with 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 in accordance with the serial numbering system. This serial numbering system correlates with the numbering system according to Kabat (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), starting from N-end, different from the numbering system according to Kabat (bottom row) inserts, marked a, b, c. Using the numbering system according to Kabat actual linear amino acid sequence may contain fewer or additional s is nakilat in accordance with the shortening, insert in FR or CDR of the variable domain. Options sites-modified cysteine heavy chains indicate systems sequential numbering and numbering according to Kabat.

In one embodiment, the implementation of the modified cysteine antibody to CD22 get method, including

(a) replacing one or more amino acid residues of the original antibody to CD22-cysteine and

(b) determining the reactivity of thiol groups modified with cysteine antibodies to CD22 by reaction of modified cysteine antibody to react with the thiol reagent.

Modified cysteine antibody may be more reactive with the reactive thiol reagent than the original antibody.

Amino acid residues of free cysteine can be heavy or light chains or constant or variable domains. Fragments of antibodies, for example Fab, you can also modify one or more amino acids cysteine, replacing amino acid fragment of the antibody with obtaining the modified cysteine fragments of antibodies.

Another variant embodiment of the invention relates to a method of obtaining (a) a modified cysteine antibodies to CD22, including

(a) introducing one or more amino acid cysteine in the original antibody to CD22 to obtain the mod is fitiavana the cysteine antibodies to CD22 and

(b) determining the reactivity of thiol groups modified with cysteine antibodies to CD22 by reaction of modified antibodies with cysteine-reactive thiol reagent;

where modified cysteine antibody is more reactive with the reactive thiol reagent than the original antibody.

Stage (a) of the method of obtaining modified with cysteine antibodies may include

(i) carrying out mutagenesis nucleic acid sequence that encodes a modified cysteine antibody;

(ii) the expression of the modified cysteine antibodies and

(iii) isolation and purification of modified cysteine antibodies.

Stage (b) method of obtaining modified with cysteine antibodies may include the expression of the modified cysteine antibodies on viral particle selected from ragovoy or fahmideh particles.

Stage (b) method of obtaining modified with cysteine antibodies can also include

(i) reaction of modified antibodies with cysteine-reactive thiol affinity reagent to obtain affine labeled modified by cysteine antibodies; and

(ii) measuring the binding affinity labeled modified by cysteine antibodies with breathtaking environments.

Another variant embodiment of the invention represents the manual screening-modified cysteine antibodies reactive, unpaired amino acids cysteine on the reactivity of thiol groups, including

(a) introducing one or more amino acid cysteine in the original antibody to obtain a modified cysteine antibodies;

(b) reaction of the modified antibodies with cysteine-reactive thiols affinity reagent to obtain affine labeled modified by cysteine antibodies; and

(c) measuring the binding affinity labeled modified by cysteine antibodies with breathtaking environments; and

(d) determining the reactivity of thiol groups of modified antibodies with cysteine-reactive thiol reagent.

Stage (a) of the method of screening of modified cysteine antibodies may include

(i) carrying out mutagenesis nucleic acid sequence that encodes a modified cysteine antibody;

(ii) the expression of the modified cysteine antibodies and

(iii) isolation and purification of modified cysteine antibodies.

Stage (b) of the method of screening the modified cysteine antibodies may include the expression of the modified cysteine antibodies on viral particle selected from ragovoy or fahmideh particles.

Stage (b) of the method of screening the modified cysteine antibodies can also include

(i) reaction of the modified cysteine EN is the body with reactive thiol affinity reagent to obtain affine labeled, modified by cysteine antibodies; and

(ii) measuring the binding affinity labeled modified by cysteine antibodies with breathtaking environments.

Modification options IgG 10F4 to CD22-cysteine

Cysteine was introduced in section 118 (EU numbering) (equivalent to position 121 of heavy chain sequential numbering) of the heavy chain full-length, chimeric original monoclonal antibodies to CD22 ways of modification of the cysteine described in this document.

The original antibody, "std Anti-CD22 Hu 10F4v3 Fc (sequence of the heavy chain: SEQ ID NO: 88, a sequence of light chain: SEQ ID NO: 87, pigv) modified with cysteine to receipt A118C thio hu anti-CD22 10F4v3" (sequence of the heavy chain: SEQ ID NO: 92, the sequence of the light chain: SEQ ID NO: 91, Fig), "S400C thio hu anti-CD22 10F4v3" (sequence of the heavy chain: SEQ ID NO: 93, a sequence of light chain: SEQ ID NO: 91, Fig), or "V205C thio-anti-CD22 10F4v3" (sequence of the heavy chain: SEQ ID NO: 88, a sequence of light chain: SEQ ID NO: 91, figv and 17).

These modified cysteine monoclonal antibodies expressed in CHO cells (Chinese hamster) short-term fermentation in media containing 1 mm cysteine.

Labeled modified cysteine antibodies to CD22

Modified cysteine antibodies to CD22 can be site-specifically and efficiently bind to react with them with the thiol reagent. Reacting with a thiol reagent can be a multifunctional linker reagent, exciting, i.e. affine, aiming reagent (for example, the reagent in the form of bioteknologi linker), detektiruya label (for example, fluorophoric reagent), vehicle immobilization on a solid phase (e.g., SEPHAROSE™, polystyrene or glass) or the intermediate connection of a drug-linker. One example of reacting with the thiol reagent is an N-ethylmaleimide (NEM). In the illustrative embodiment, the reaction ThioFab with reagent Biotin-linker gives biotinylated ThioFab, whereby it is possible to detect and measure the presence and reactivity of engineered cysteine residue. The reaction ThioFab with multifunctional linker reagent gives ThioFab with a functional linker, which can further react with the reagent molecule drugs or other label. The reaction ThioFab with intermediate connection of a drug-linker gives the conjugate ThioFab-drug.

Illustrative methods described herein can be applied mainly to identify and generate antibodies, and more generally to other proteins through the application of design and stages screening described in this document.

This approach can be applied in order to change for the conjugation of other reacts with thiol reagents, in which the reactive group is, for example, maleimide, todatetime, pyridyldithio, or other reacts with thiols of the conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp.40-55, 643-671). Reacting with a thiol reagent can be a molecule drugs, fluorophores, such as a fluorescent dye such as fluorescein or rhodamine, a chelating agent for imaging or radiotherapy metal, peptide or ones label or detektiruya label or modifying the clearance of the tool, such as various isomers of polyethylene glycol, a peptide that communicates with the third component, or another carbohydrate or a lipophilic agent.

The use of modified cysteine antibodies to CD22

Modified cysteine antibodies to CD22 and their conjugates can be used as a therapeutic and/or diagnostic tools. The present invention additionally relates to methods of prevention, control, treat and / or alleviate one or more symptoms associated with the associated with B-cell disorder. In particular the present invention relates to FPIC is BAM prevention, regulation of treating or alleviating one or more symptoms associated with a cell proliferative disorder, such as a malignant tumor, for example, lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell. The present invention additionally relates to methods of diagnosis associated with CD22 disorders or a predisposition to develop such disorders, as well as to methods of identifying antibodies and antigenspecific fragments of antibodies, which are preferably associated with associated with B-cell polypeptides CD22.

Another variant of implementation of the present invention refers to the use of modified cysteine antibodies to CD22 to obtain drugs suitable for treatment of a condition that is responsible for associated with B-cell disorder.

The conjugates of modified cysteine antibody-drug (conjugated cienciala-drug)

Another aspect of the invention is a compound conjugate antibody-drug containing modify the new-cysteine antibody to CD22 (Ab) and the molecule drugs (D) auristatin, where modified cysteine antibody through a linker group (L) is connected by one or more free amino acids cysteine with D; where the compound has the formula I:

Ab-(L-D)pI

where p represents 1, 2, 3 or 4; and where the modified cysteine antibody produced by the method comprising substituting one or more amino acid residues of the original antibody to CD22 one or more free amino acids cysteine.

Figure 10 shows embodiments of modified cysteine conjugates of the antibody to CD22-drug (ADC), where the molecule drug auristatin related to the functional group resulting from the design of cysteine in the light chain (LC-ADC); the heavy chain (HC-ADC) and Fc region (Fc-ADC).

The potential benefits of modified cysteine conjugates of the antibody to CD22-drug include improved safety (greater therapeutic index), improved PK parameters are stored megamachine disulfide bonds of the antibody, which can stabilize the conjugate and keep it active binding conformation, defined areas conjugation of drugs and obtaining the modified what Steinem conjugates antibody-drug via conjugation modified with cysteine antibody reagent drug-linker leads to a more homogeneous product.

Linkers

"Linker", "linker unit" or "link" means a chemical molecule that contains covalent bond or a chain of atoms that covalently bind the antibody to the molecule drugs. In various embodiments, the implementation of a linker is specified as L. "Linker" (L) represents a bifunctional or polyfunctional molecule, which can be used to associate one or more molecules of the drug (D) and antibodies (Ab) to obtain the conjugates of the antibody-drug (ADC) of formula I. the Conjugates of the antibody-drug (ADC) can be conveniently obtained by use of a linker with a reactive functional group for binding with drug and antibody. The thiol of the cysteine-modified cysteine antibody (Ab) can form a bond with an electrophilic functional group of the linker reagent molecule drug or an intermediate connection of a drug-linker.

In one aspect, the linker contains a reactive center, which contains an electrophilic group which can react with the nucleophilic cysteine, located on the antibody. The cysteine thiol of the antibody reacts with an electrophilic group on the linker and forms a covalent bond with the linker. Suitable electrophilic groups include in to the number of non-limiting examples maleimido and halogenated group.

Linkers include bivalent radical, such as alkerdeel, Allen, heteroaryl, groups such as -(CR2)nO(CR2)n-repeating unit alkyloxy (for example, polietilene, PEG, polymethylenes), alkylamino (for example, polyethylenimine, Jeffamine™); and an ester of dibasic acids and amides including succinate, succinamide, diglycolate, malonate and caproamide.

Modified cysteine antibodies react with linker reagents or intermediate compounds, the drug-linker, with electrophilic functional groups such as maleimide or α-halogencarbonic, in accordance with the method of conjugation on page 766 in Klussman, et al., (2004), Bioconjugate Chemistry 15(4):765-773, and in accordance with the Protocol of example x.

The linker may consist of one or more linker components. Illustrative of the linker components include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af'), p-aminobenzeneboronic ("PAB"), N-Succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC), N-Succinimidyl-(4-iodates)aminobenzoate ("fairs are forthcoming-Siab), ethylenoxy-CH2CH2O - in the form of one or more recurring units ("EO" or "PEO"). For more linker components known in this area is, and some are described in this document.

In one of the embodiments, the linker L ADC has the formula:

-Aa-Ww-Yy-

where

-A - a extension cable, covalently linked to a cysteine thiol of the antibody (Ab);

a represents 0 or 1;

each W independently represents an amino acid unit;

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

-Y - is a spacer elements unit covalently linked to a molecule drugs; and

y represents 0, 1 or 2.

Extension

Extension (-A-), when present, is capable of binding of the antibody with the amino acid unit (-W-). When this antibody (Ab) is a functional group that can form a bond with the functional group of the extension. Suitable functional groups that may be present on the antibody in nature or via chemical manipulation include, as non-limiting examples of sulfhydryl (-SH), amino, hydroxyl, carboxy, anomeric hydroxyl group of a carbohydrate and carboxyl. In one aspect of the functional groups of antibodies are sulfhydryl or amino groups. Sulfhydryl groups can be obtained by reduction of the intramolecular disulfide bond anticelulitis, sulfhydryl groups can be obtained by reaction of an amino group of a lysine residue of the antibody with the use of 2-aminothieno (reagent Troth) or other giving sulfhydryl reagent. In one of the embodiments the antibody (Ab) is free Tilney group of cysteine, which can form a bond with an electrophilic functional group of the extension. Illustrative cords in the conjugates of formula I is depicted by formulas II and III, where Ab-, -W-, -Y-, -D, w and y are as defined above, and R17is a bivalent radical selected from (CH2)rC3-C8-carbocycle, O-(CH2)rarylene, (CH2)rarylene, Allen-(CH2)r-, (CH2)r-(C3-C8-carbocycle), (C3-C8-carbazolyl)-(CH2)rC3-C8-heterocyclyl, (CH2)r-(C3-C8-heterocyclyl), -(C3-C8-heterocyclyl)-(CH2)r-, -(CH2)rC(O)NRb(CH2)r-, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-, -(CH2)rC(O)NRb(CH2CH2O)r-, -(CH2)rC(O)NRb(CH2CH2O)r-CH2-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-CH2-and -(CH 2CH2O)rC(O)NRb(CH2)r-; where Rbrepresents H, C1-C6-alkyl, phenyl or benzyl; and r independently represents an integer in the range 1-10.

Aralen includes bivalent aromatic hydrocarbon radical of 6-20 carbon atoms derived by removing two hydrogen atoms from an aromatic cyclic system. Typical allenbyi groups include as non-limiting examples of radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc.

Heterocyclic groups include cyclic system in which one or more cyclic atoms are a heteroatom, such as nitrogen, oxygen and sulfur. Heterocyclic radical contains from 1 to 20 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S. the Heterocycle may be a monocycle with the number of members in the loop from 3 to 7 (from 2 to 6 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S) or Bicycle with a number of circular members from 7 to 10 (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example, system bicyclo[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, from 1950 up to the present time), specifically in volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include, as examples, not limitations pyridyl, dihydropyridin, tetrahydropyranyl (piperidyl), thiazolyl, tetrahydrothiophene, oxidized grey tetrahydrothiophene, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianeptine, indolyl, indolinyl, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydropyranyl, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6H-1,2,5-tedisamil, 2H,6H-1,5,2-detainer, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine, 2H-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, honokalani, hintline, cinnoline, pteridine, 4Ah-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, pyrazinyl, phenoxazines, isopropanol, bromanil, imidazolidinyl, imidazolyl, pyrazolidine, pyrazoline, piperazinil, indolinyl, isoindolyl, hinokitiol, morpholinyl, oxazolidin is l, benzotriazolyl, benzisoxazole, oxindoles, benzoxazolyl and satanail.

Carbocyclic groups include saturated or unsaturated cyclic system with the number of carbon atoms from 3 to 7 in the form of a monocycle or from 7 to 12 in the form of Bicycle. Monocyclic carbocycle contain from 3 to 6 cyclic atoms, more usually 5 or 6 cyclic atoms. Bicyclic carbocycle contain from 7 to 12 cyclic atoms, for example, arranged in the form of bicyclo[4,5], [5,5], [5,6] or [6,6], or 9 or 10 cyclic atoms arranged in the form of bicyclo [5,6] or [6,6]. Examples of monocyclic carbon cyclic systems 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.

Based on all of the illustrative embodiments the ADC of formula I, such as II-VI, it should be understood that even when not explicitly specified, the antibody is linked from 1 to 4 molecules of the drug (p=1-4), depending on the number of internal cysteine residues.

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

Illustrative extension of the formula II, p is obtained from maleimido-propanol (MP), where R17represents -(CH2)2is:

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

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

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

In another embodiment, the extension associated with the modified cysteine antibody to CD22 through disulfide bond built between the sulfur atom of the cysteine antibodies and sulfur atom extension. Representative extension by this variant of the invention represented by formula IV, where R17, Ab-, -W-, -Y-, -D, w and y are as defined above.

In another embodiment, the reactive group of the extension contains a reactive thiol functional group that can form a bond with the free cysteine thiol of the antibody. Examples of reactive thiols functional groups include as non-limiting upon the development of maleimide, α-halogenoacetyl, activated esters, such as operations esters, 4-nitroaniline esters, pentafluoroaniline esters, tetrafluoroaniline esters, anhydrides, acid chlorides, sulphonylchloride, isocyanates and isothiocyanates. Representative cords in this variant implementation of the presented formulas Va and Vb, where-R17-, Ab-, -W-, -Y-, -D, w and y are as defined above;

In another embodiment, the linker may be a branched linker type for covalent joining more than one antibody molecule drugs through a branched polyfunctional linker molecule (Sun et al., (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al., (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) Tetrahedron Letters 43:1987-1990). Branched linkers can increase the molar ratio of drug to antibody, i.e. the load that is associated with the efficiency of the ADC. Thus, when the modified cysteine antibody carries only one reactive Tilney group of cysteine, many drug molecules can be attached via a branched linker.

Amino acid unit

The linker may contain amino acid residues. The amino acid the percentage unit (-W w-), when present, links the antibody (Ab) molecule drugs (D), modified by cysteine conjugate antibody-drug (ADC) according to the invention.

-Ww- is a dipeptide, Tripeptide, tetrapeptide, pentapeptidnogo, Hexapeptide, heptapeptide, octapeptide, nonapeptide, Decapeptide, undecapeptide or dodecapeptide unit. Amino acid residues comprising the amino acid unit include the remains, found in nature, as well as minor amino acids not found in nature similar amino acids such as citrulline. Each unit-W - is independently has the following in square brackets in the formula, w represents an integer in the range from 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)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH22CH2CH(OH)CH2NH22-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl.

When R19different from hydrogen, the carbon atom is linked to R19is chiral. Each carbon atom is attached to R19independently is (S)- or (R)-configuration, or racemic mixture. Thus, the amino acid unit can be enantiomerically pure, racemic or diastereomeric.

Illustrative amino acid units,- Ww- include a dipeptide, Tripeptide, tetrapeptide or 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 comprise amino acid linker component, include the remains, found in nature, as well as minor amino acids not found in nature similar amino acids such as citrulline.

The amino acid unit can enzymatically cleave one or more enzymes, including associated with tumors of the protease, with the release of a molecule drugs (-D), in one of the embodiments, after the release ofin vivoprotonated with receipt is m drug (D). Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by specific enzymes, for example, associated with tumors with protease, cathepsin B, C and D or the protease plasmin.

Spacer elements unit

Spacer elements unit (-Yy-)when it is present (y=1 or 2), binds to the amino acid unit (-Ww-molecule drugs (D), when the amino acid unit is present (w=1-12). Alternatively, the spacer elements unit associates the extension with the molecule drugs, when the amino acid unit is missing. Spacer elements unit also links molecule drugs with unit antibodies, when amino acid unit and the extension element is missing (w, y=0). Spacer elements units come in two basic types: samoudalyayushcheisya and not samoudalyayushcheisya. Not smodlaka spacer elements unit represents a spacer elements unit in which the spacer elements unit or all of spacer elements, the unit remains in a bound state with one molecule drugs after removal, in particular enzyme, amino acid element from the conjugate antibody-drug or molecule drug-linker. When the ADC containing a glycine-glycine spacer elements unit and the and glycine spacer elements unit undergoes enzymatic cleavage associated with tumor cells by protease associated with malignant tumor protease or associated with lymphocyte protease, a glycine-glycine-molecule drugs or glycine-molecule drugs hatshepsuts Ab-Aa-Ww-. In one embodiment, the implementation in the target cell is independent hydrolysis reaction that breaks down communication glycine-molecule drugs and release the drug.

In another embodiment,- Yy- represents the p-aminobenzoylamino (PAB) unit, which fenelonov part replaced with Qmwhere Q represents-C1-C8-alkyl, -O-(C1-C8-alkyl), -halogen, -nitro or-cyano; and m is a number in the range from 0-4.

Illustrative embodiments of not smodlaka spacer elements unit (-Y-) are: -Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one of the embodiments given molecule drug-linker or ADC, in which the spacer elements unit is absent (y=0), or their pharmaceutically acceptable salt or MES.

Alternatively, the ADC containing smodlaka spacer elements unit can liberate-D. In one embodiment, the implementation of the-Y - is a group of PAB, to the which is connected with the-W wthrough the nitrogen atom of the amino in the PAB group, and connected directly to-D via a carbonate, urethane group or a group of simple ether, where the ADC is illustrative structure:

where Q represents-C1-C8-alkyl, -O-(C1-C8-alkyl), -halogen, -nitro or-cyano; m is an integer ranging from 0-4; and p ranges from 1 to 4.

Other examples smodlaka spacers include as non-limiting examples of 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 analogues PAB (U.S. patent 2005/0256030), beta-glucuronide (WO 2007/011968), and ortho - or paraaminosalicylic. You can use the spacers that are subjected to cyclization by the hydrolysis of amide linkages, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., (1995) Chemistry Biology 2:223), appropriately substituted ring system is bicyclo[2.2.1] bicyclo[2.2.2] (Storm et al., (1972) J. Amer. Chem. Soc. 94:5815) and amides of 2-aminophenylamino acid (Amsberry et al., (1990) J. Org. Chem. 55:5867). Deleting containing amine drugs, which are substituted by glycine (Kingsbury et al., (1984) J. Med. Chem. 27:1447), is also an example of camouglage spacer, suitable ADC.

Illustration the e spacer elements unit (-Y y-) correspond to the formulas X-XII:

Branched linkers

In another embodiment, the linker L may be a branched linker type for covalent joining more than one antibody molecule drugs through a branched polyfunctional linker group (Sun et al., (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al., (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Branched linkers can increase the molar ratio of drugs and antibodies, i.e. the load that is associated with the efficiency of the ADC. Thus, when the modified cysteine antibody has only one reactive Tilney group of cysteine, via a branched linker, you can attach a large number of molecules of the drug. Illustrative embodiments of branched branched linkers include 2,6-bis(hydroxymethyl)-p-crenoline and 2,4,6-Tris(hydroxymethyl)phenol 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, the implementation of the spacer elements unit is a branched bis(hydroxymethyl)styrene (BHMS), which can be used to activate and release of many drugs, on ladydi structure:

containing 2-(4-aminobenzylidene)propane-1,3-volovoy branching unit (WO 2004/043493; de Groot et al., (2003) Angew. Chem. Int. Ed. 42:4490-4494), where Q represents-C1-C8-alkyl, -O-(C1-C8-alkyl), -halogen, -nitro or-cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges from 1 to 4.

Illustrative embodiments of the compounds of the conjugates of the antibody-drug 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 conjugates of the antibody-drug formula Ia include XIVa-e:

where X represents:

Y represents:

and R independently represents H or C1-C6-alkyl; and n is from 1 to 12.

In another embodiment, the linker has a reactive functional group, which has a nucleophilic group that reacts with an electrophilic group present on the antibody. Suitable electrophilic groups on the antibody include as non-limiting examples carbonyl the diversified group of aldehydes and ketones. Nucleophilic heteroatom group of the linker may react with an electrophilic group on the antibody and form a covalent bond with a unit of antibodies. Suitable nucleophilic groups on the linker include as non-limiting examples of the hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and originated. Electrophilic group on the antibody provides a convenient site for binding to the linker.

Typically, the peptide linkers type can be obtained by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, in accordance with the method of synthesis in the liquid phase (E. Schröder and K. Lubke (1965) "The peptides", volume 1, pp 76-136, Academic Press)that is well known in the field of peptide chemistry. The intermediate linker can be collected through any combination or sequence of reactions, including spacer elements extending and amino acid units. Spacer elements extending and amino acid units may use reactive functional groups are electrophilic, nucleophilic or free radical. Reactive functional groups include as non-limiting examples of carboxy, hydroxyl, para-nitrophenylarsonic, isothiocyanate and the ear is Asia group, such as O-mesyl, O-tosyl, -Cl, -Br, -I; or maleimide.

In another embodiment, the linker may be substituted by groups that modulate the solubility or reactivity. For example, charged the Deputy, such as sulfonate (-SO3-) or ammonium, may increase water solubility of the reagent and facilitate the reaction of attaching a linker reagent to the antibody or the molecule drugs, or to facilitate the reaction of binding of Ab-L (intermediate compound of the antibody-linker) with D, or D-L (intermediate compound drug-linker) with Ab, depending on the synthesis method used to obtain the ADC.

Linker reagents

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

Conjugates of the antibody-drug can also be obtained with linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate), and including bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3and BM(PEO)4that are commercially available from Pierce Biotechnology, Inc., Customer Service Department, P.O. Box 117, Rockford, TL. 61105 U.S.A, U.S.A 1-800-874-3723, International +815-968-0747. Bis-maleimide reagents allow connection Tilney group-modified cysteine antibody containing thiol molecule drugs, label, or intermediate coupling of the linker, sequentially or simultaneously. Other functional groups, in addition to maleimide that react with Tilney group-modified cysteine antibody molecule drug, a label or an intermediate connection of the linker include todatetime, bromoacetamide, vinylpyridine, disulfide, pyridyldithio, isocyanate and isothiocyanate.

Suitable linker reagents can also be obtained from other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO), or 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. 7:180-186; U.S. patent 614345; WO 02/088172; U.S. patent 2003130189; US 2003096743; WO 03/026577; WO 03/043583 and WO 04/032828.

The extension of the formula (IIIa) can be introduced into the linker the following reaction of the linker reagent with N-end amino acid unit:

where n is a number ranging from 1-10 and T represents-H or-SO3Na;

where n is a number in the range 0-3;

and

Extensions can be introduced into the linker reaction following bifunctional reagent N-end amino acid unit:

where X represents Br or I.

The extension of a formula can also be entered in the linker reaction following bifunctional reagent N-end amino acid unit:

Illustrative dipeptide linker reagent valine-citrulline (val-cit or vc)containing maleimide extension cable and pair-aminobenzeneboronic (PAB) samoudalyayushcheisya the spacer has the structure:

Illustrative dipeptide linker reagent phe-lys (Mtr, mono-4-methoxytrityl)containing maleimide extension and smodlaka spacer elements unit PAB, you can get at Dubowchik, et al., (1997) Tetrahedron Lettrs, 38:5257-60, and it has the structure:

Illustrative compounds conjugate antibody-drug according to the invention include:

where Val is a valine; Cit is citrulline; p represents 1, 2, 3 or 4; and Ab is a modified cysteine antibody to CD22.

Receiving modified with cysteine conjugates of the antibody to CD22-drug

The ADC of formula I can be obtained in several ways, using reaction conditions and reagents in organic chemistry, known to specialists in this area, including (1) the reaction of a group of cysteine-modified cysteine antibody with a linker reagent, with the formation of intermediate compounds of the antibody-linker Ab-L, via a covalent bond, followed by reaction with an activated molecule drugs D; and (2) reaction of a nucleophilic group of a molecule drugs with a linker reagent, with the formation of intermediate compounds, the drug-linker D-L, via a covalent bond, followed by reaction with a group of cysteine modified the cysteine antibodies. Methods of conjugation (1) and (2) can be used for a variety of modified cysteine antibodies, molecules of medicinal medium spans the VA and linkers with obtaining conjugates antibody-drug formula I.

Tirinya group of cysteine antibodies are nucleophilic and capable of reacting with the formation of covalent bonds with electrophilic groups on linker compounds and intermediate compounds, the drug-linker, including (i) active esters such as NHS esters, esters, HOBt, halogenfree and acid halides; (ii) alkyl and benzyl halides such as halogenated; (iii) aldehydes, ketones, carboxylic and maleimido group; and (iv) disulfides, including pyridylsulfonyl by sulfide metabolism. Nucleophilic groups on the molecule drugs include as non-limiting examples of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazines group capable of reacting with the formation of covalent bonds with electrophilic groups on linker groups and linker reagents.

Modified cysteine antibodies can be converted into reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (reagent Cleland, dithiothreitol) or TCEP (Tris hydrochloride(2-carboxyethyl)phosphine; Getz et al., (1999) Anal. Biochem. Vol.273: 73-80; Soltec Ventures, Beverly, MA), followed by re-oxidation to re-education miaocheng and interchain disulfide with the ides (example x). For example, full-length, modified cysteine monoclonal antibodies (timeby)expressed in CHO cells recover by approximately 50-fold excess of TCEP for 3 h at 37°C to restore the disulfide bonds in the cysteine adducts, which may be formed between the newly introduced cysteine residues and the cysteine present in the media for cultivation. Restored Tioman diluted and applied on the column HiTrap S in 10 mm sodium acetate, pH 5 and elute through PBS containing 0.3 M sodium chloride. Disulfide bond was re-established between cysteine residues present in the original Mab diluted (200 nm) of aqueous copper sulfate (CuSO4) at room temperature over night. Alternatively, dehydroascorbic acid (DHAA) is an effective oxidant to re-establish miaocheng disulfide groups modified with cysteine antibodies after reductive cleavage of cysteine adducts. You can use other oxidizing agents, i.e. oxidizing agents, and the conditions of oxidation, which is known in this field. Also an effective oxidation by atmospheric air. This stage soft partial re-oxidation leads to effective education miaocheng disulfides with high reliability isochronal tirinya group newly introduced cysteine residues. Approximately 10-fold excess of the intermediate drug-linker, for example, MC-vc-PAB-MMAE was added, stirred and left to stand for approximately one hour at room temperature to achieve conjugation and formation of conjugate antibody to CD22 10F4v3-drug. Mixture for conjugation was subjected to gel filtration and put it in column HiTrap S and suirable through it to remove excess intermediate drug-linker and other impurities.

On Fig shows the General process for obtaining modified with cysteine antibodies expressed from a cell culture, for conjugation. When the cellular environment for culturing contain cysteine, between the newly introduced cysteine amino acid and cysteine from the medium can form disulfide adducts. These cysteine adducts, depicted as a circle in the illustrative tiombe (left) on Fig, you must restore to get reactive modified with cysteine antibodies for conjugation. Cysteine adducts, presumably in conjunction with various megamachine disulfide bonds, subject to recovery of cleavage with getting restored form antibodies, by means of reducing agents, such as TCEP. Megamachine disulfide bond m is waiting for forming a pair of cysteine residues re-formed in the partial oxidation of copper sulfate, DHAA, or exposure to atmospheric oxygen. Newly made, converted by means of engineering and not formed a pair of cysteine residues remain available for reaction with linker reagents or intermediate compounds, the drug-linker with the formation of conjugates of antibodies according to the invention. Timeby expressed in cell lines mammals, lead to internal anywherefrom the Cys adduct with the introduced methods engineering Cys through education connection-S-S-. Thus, peeled timeby treated by the methods of recovery and re-oxidation as described in example x, to obtain the reactive tomarow. These timeby used for conjugation with maleimide containing cytotoxic drugs, fluorophores and other labels.

Methods of screening

Another variant of implementation of the present invention relates to a method of determining the presence of a CD22 polypeptide in a sample suspected of containing the CD22 polypeptide, where the method includes the impact on the sample modified with cysteine antibodies to CD22 or its conjugate antibody-drug that binds to the CD22 polypeptide and determining binding of modified cysteine antibodies to CD22, or its conjugate antibody-drug with a CD22 polypeptide in the sample where the presence of such binding indicates the presence of a CD22 polypeptide in the sample. Optionally, the sample may contain cells (which can be a malignant cell), presumably expressing CD22 polypeptide. Modified cysteine antibody to CD22 or its conjugate antibody-drug, used in the method is optional, you can mark amenable to detection label to attach to a solid substrate, or similar.

Another variant of implementation of the present invention relates to a method of diagnosing the presence of a tumor in a mammal, where the method comprises (a) bringing into contact the test sample containing tissue cells obtained from the mammal with a modified cysteine antibody to CD22 or its conjugate antibody-drug that are associated with the CD22 polypeptide, and (b) detection of complex formation between the modified cysteine antibody to CD22, or its conjugate antibody-drug, and CD22 polypeptide in the test sample, where the formation of the complex indicates the presence of mammalian tumors. Optional modified cysteine antibody to CD22 or its conjugate antibody-drug subject to labeling amenable to detection label, attached to a solid substrate, or similar, and/or the test sample of tissue cells receive from the individual, pedological is but with malignancy.

Metabolites are conjugates of the antibody-drug

In the scope of the present invention are metabolic productsin vivocompounds of the ADC described in this document, to the extent that the products are new and non-obvious compared to the prior art. Such products may be due to, for example, oxidation, recovery, hydrolysis, amidation, esterification, enzymatic cleavage, and the like, input connections. Thus, the invention includes a new and unobvious compounds produced by a process comprising bringing into contact connection according to this invention with a mammal for a period of time sufficient to obtain its metabolic product.

Metabolic products, as a rule, identify obtaining labeled with a radioactive label (e.g.,14C or3H) ADC, the introduction of parenteral in quantifiable detection dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, Guinea pig, monkey, or to man, waiting a sufficient period of time to pass metabolism (typically from about 30 seconds to 30 hours) and recovery of the products of its transformation from urine, blood or other biological samples. These products are easy to distinguish, is since they are labeled (other secrete antibody, capable of binding epitopes conserved in the metabolite). Patterns of metabolites determined in a traditional way, for example, through analysis of MS, LC/MS or NMR. Basically, the analysis of metabolites carried out in the same way as traditional studies of the metabolism of drugs, known to experts in this field. The products of conversion, provided that they otherwise fail to meetin vivosuitable for diagnostic assays for therapeutic dosing of the compounds of the ADC according to the invention.

Pharmaceuticals

Introduction conjugates antibody-drug, including conjugates thio-antibody-drug

Conjugates of the antibody-drug (ADC), comprising the conjugates thio-antibody-drug (TDC), according to the invention can be entered by any means suitable for the condition to be treated. ADC, as a rule, will enter parenteral, i.e. infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally and epidurally.

For the treatment of these malignant tumors, in one of the embodiments, the conjugate is an antibody-drug is administered by intravenous infusion. Dosage, administered by infusion, is in the range from about 1 μg/m2up rough is about 10000 g/m 2the dose is generally one dose per week with only one, two, three or four doses. Alternatively, the range of dosage is from about 1 μg/m2to about 1000 mg/m2from about 1 μg/m2to about 800 g/m2from about 1 μg/m2up to approximately 600 mg/m2from about 1 μg/m2up to approximately 400 mg/m2from approximately 10 mg/m2to about 500 mg/m2from approximately 10 mg/m2to about 300 mg/m2from approximately 10 mg/m2to about 200 mg/m2and from about 1 μg/m2to about 200 mg/m2. Dose can be administered once a day, once a week, several times per week but less than once a day, several times per month but less than once a day, several times per month but less than once a week, once a month or periodically to facilitate or mitigate the symptoms of the disease. Introduction you can continue using any of the described intervals until remission of the tumor or symptoms of lymphoma, leukemia to be treated. Introduction you can continue after remission or relief of symptoms, where such remission or relief of symptoms prolong this continuing introduction.

Also from Britanie relates to a method of alleviating autoimmune diseases, including the introduction of a patient suffering from an autoimmune disease, a therapeutically effective amount of the conjugate humanitariannet antibody 10F4-drug according to any one of the preceding embodiments. In preferred embodiments, the implementation of the antibody is administered intravenously or subcutaneously. Conjugate antibody-drug is administered intravenously at a dosage in the range from about 1 μg/m2to about 100 mg/m2on the dose and in a specific embodiment, the dosage is between 1 mg/m2to about 500 mg/m2. Dose can be administered once a day, once a week, several times per week but less than once a day, several times per month but less than once a day, several times per month but less than once a week, once a month or periodically to facilitate or mitigate the symptoms of the disease. Introduction you can continue with any of the above intervals to facilitate or mitigate the symptoms of autoimmune disease to be treated. Introduction you can continue after reaching facilitate or mitigate symptoms, where such relief or mitigation of symptoms prolong this continuing introduction.

The invention relates to a method for the treatment of B-cell disorders, including the introduction of patie the one suffering from B-cell disorder, such as B-cell proliferative disorder (including, as non-limiting examples, lymphoma and leukemia), or an autoimmune disease, a therapeutically effective amount gumanitarnogo antibody 10F4 according to any one of the preceding embodiments, and this antibody is not conjugated with a cytotoxic molecule or amenable to detection molecule. The antibody typically will enter at a dosage in the range from about 1 μg/m2to about 1000 mg/m2.

In one aspect of the invention additionally relates to pharmaceutical preparations containing at least one antibody to CD22 according to the invention and/or at least one immunoconjugate, and/or at least one conjugate antibody to CD22-drug according to the invention. In some embodiments, the implementation of the pharmaceutical preparation contains 1) antibody to CD22 and/or its conjugate antibody to CD22-drug, and/or immunoconjugate, and 2) a pharmaceutically acceptable carrier. In some embodiments, the implementation of the pharmaceutical preparation contains 1) antibody to CD22 and/or its immunoconjugate, and optional, 2) at least one additional therapeutic agent.

Pharmaceutical preparations containing the antibody or immunoconjugate is according to the invention or a conjugate of the antibody-drug according to the invention, receive for storage by mixing the antibody or conjugate antibody-drug having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions or lyophilized or other dried products. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane; 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 about 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; sugars such as agarose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; complex compounds with metals (for example, complexes of Zn-protein); and/or nonionic surface-active agents such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The pharmaceutical preparations to be used for the introduction ofin vivoas a rule, are sterile. This is easily achieved by filtration through sterile filtration membranes.

The active ingredients can also be enclosed in a microcapsule obtained, for example, ways koatservatsii or interphase polymerization, for example, a microcapsule-based hydroxymethylcellulose or gelatin and a microcapsule-based poly(methylmethacrylate), respectively, in colloidal systems for delivery of a drug (for example, liposomes, microspheres based on albumin, microemulsions, nanoparticles and nanocapsules), or in the microemulsions. Such methods are described in theRemington's Pharmaceutical Sciences,16th edition, Oslo, 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 matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of matrices with a slow release VK is ucaut polyesters, hydrogels (for example, poly-2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem ethylene-vinyl acetate, degradiruete copolymers of lactic acid and 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-hydroxybutiric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid provides the release of molecules within 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denaturing or to form aggregates as a result of exposure to moisture at 37°C, leading to loss of biological activity and possible changes in immunogenicity. For stabilization, you can develop appropriate strategies, depending on the mechanism involved. For example, if it is revealed that the mechanism of aggregation is an intermolecular bond formation S-S through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling article the penalties humidity, using appropriate additives, and developing specific polymer compositions of the matrix.

The treatment of conjugate antibody-drug

It is assumed that the conjugates of the antibody-drug (ADC) according to the present invention can be used to treat various diseases or disorders, for example, characterized by the overexpression of tumor antigen. Illustrative state or hyperproliferative disorders include benign or malignant tumors; leukemia and lymphoid malignancies. Other violations include neuronal, glial, astrocytic, hypothalamic, glandular, macrophagal, epithelial, stromal, associated with blastocele, inflammatory, angiogenic and immunologic, including autoimmune disorders.

Connect the ADC, which have been identified in animal models and cellular assays can be further tested with the tumor of higher primates and in clinical trials in humans. Clinical trials in humans can be designed to test the effectiveness of monoclonal antibodies to CD22 or immunoconjugate according to the invention in patients with B-cell proliferative disorder, including as non-limiting examples of lymphoma, Nehaj the warrior's lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell. A clinical trial may be designed to assess the effectiveness of the ADC in combination with a known treatment such as radiotherapy and/or chemotherapy involving known chemotherapeutic and/or cytotoxic tools.

As a rule, the disease or disorder to be treated is a hyperproliferative disease, such as B-cell proliferative disorder, and/or B-cell malignancy. Examples of malignant tumors to be treated by the present document include, as non-limiting examples, B-cell proliferative violation, selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell.

A malignant tumor may contain expressio the existing CD22 cells, so the ADC of the present invention is able to contact with malignant cells. To determine the expression of CD22 in malignant tumors available in various diagnostic/prognostic methods of analysis. In one embodiment, the implementation of the overexpression of CD22 can be analyzed by IHC. Immersed in paraffin tissue sections from biopsy of the tumor can be subjected to IHC analysis and agree with the criteria in the intensity of staining of protein CD22 in relation to the degree of staining and the proportion of the investigated tumor cells.

For the prevention or treatment of diseases suitable dosage ADC will depend on the type of disease to be treated, as defined above, the severity and course of the disease, preventive or therapeutic purposes, the introduction of molecules, previous treatment, the clinical history of the patient and response to the antibody, and from the decision of the attending physician. The molecule is advisable to enter the patient once or over a series of injections. Depending on the type and severity of the disease, the dose from about 1 μg/kg to 50 mg/kg (for example, 0.1 to 20 mg/kg) molecules is the source of possible dose for administration to a patient, for example, either by one or by several separate injections, or by continuous infusion. Typical daily dose the transportation can vary from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Illustrative dosage ADC, subject to the introduction of the patient, is in the range from about 0.1 to about 10 mg/kg weight of the patient.

In the case of repeated injections over several days or more, depending on the condition, treatment continues until a desired suppression of disease symptoms. Illustrative diagram of dispensing includes the introduction of the initial loading dose of approximately 4 mg/kg with subsequent weekly support a dose of approximately 2 mg/kg of antibody to ErbB2. Can be fit other dosing regimens. The monitoring of this treatment easy to perform common methods and analysis.

Combined treatment

Conjugate antibody-drug (ADC) according to the invention can be combined in a pharmaceutical combination preparation or dosing regimen as combination therapy, with a second compound having properties against malignant tumors. The second compound of the pharmaceutical combination of the drug or dosing regimen preferably has the form of activity, complement ADC combination, so that they do not have any adverse effect on each other.

A second connection may be a chemotherapeutic agent, ototoksicescoe means, cytokine, growth inhibitory agent, protivogelmintnoe tool and/or cardioprotector. It is advisable that such molecules are present in combination in amounts that are effective for the intended purpose. The pharmaceutical composition containing the ADC according to the invention may also possess a therapeutically effective amount of a chemotherapeutic drug, such as an inhibitor of the formation of tubulin, a topoisomerase inhibitor, or a DNA binding agent.

In one aspect, the first connection is an ADC to CD22 according to the invention and a second compound is an antibody to CD20 (either a simple antibody or ADC). In one of the embodiments, the second compound is an antibody to CD20 rituximab (Rituxan®) or 2H7 (Genentech, Inc., South San Francisco, CA). Other antibodies suitable for combination immunotherapy through ADC to CD22 according to the invention, include, but are not limited to, antibody to VEGF (e.g., Avastin®).

With an anti-cancer treatment identified in accordance with this invention, can be combined with other treatment regimens, including as a non-limiting example, radiation therapy, and/or bone marrow transplantation and peripheral blood, and/or cytotoxic agent, chemotherapeutic agent or a growth inhibitory agent. One is the first of such embodiments chemotherapeutic agent is a tool or combination of tools, for example, such as cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapy, such as antibody to CD20 (e.g., Rituxan®) or antibody to VEGF (e.g., Avastin®). Combined treatment can be carried out as a simultaneous or alternate mode. The sequential introduction of the combination can be entered for two or more injections. The combined introduction includes co-administration, using separate drugs or a single pharmaceutical preparation, and consistent introduction in any order, preferably where there is a period of time during which both (or all) active agents along exert their biological activity.

In one embodiment, implementation of the treatment by the ADC involves the combined introduction of anti-cancer tools identified in this document, and one or more chemotherapeutic agents or inhibiting the growth of funds, including the simultaneous introduction of "blends" of various chemotherapeutic agents. Chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics. Drugs and dosing schedules for such chemotherapy drugs can be used to instrument the operations of the manufacturer or as empirically determine a qualified technician. Drugs and dosing schedules for such chemotherapy are also described in Chemotherapy Service" (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

Stable dosages for any of the above in conjunction input means are the dosages currently used, and can be reduced due to the combined action (synergy) of the newly identified tools and other chemotherapeutic agents or treatments.

Combined treatment can provide "synergy" and show "synergistic" effect, i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects of using the compounds separately. A synergistic effect may be attained when the active ingredients are (1) produced jointly and simultaneously introduced or delivered in the combined standard drug composition; (2) delivered serially or in parallel as separate formulations; or (3) any other mode. Upon delivery with alternate treatment a synergistic effect can be achieved when the compound is administered or delivered sequentially, for example, by different injections in separate syringes. Basically, during alternate treatment effective dosage of each active ingredient is administered sequentially, i.e. the series, while at the comb is Nirvana treatment effective dosages of two or more active ingredients are administered together.

Metabolites are conjugates of the antibody-drug

In the scope of the present invention are metabolic productsin vivocompounds of the ADC described in this document, to the extent that the products are new and non-obvious compared to the prior art. Such products may be due to, for example, oxidation, recovery, hydrolysis, amidation, esterification, enzymatic cleavage, and the like, input connections. Thus, the invention includes a new and unobvious compounds produced by a process comprising bringing into contact connection according to this invention with a mammal for a period of time sufficient to obtain its metabolic product.

Metabolic products, as a rule, identify obtaining labeled with a radioactive label (e.g.,14C or3H) ADC, the introduction of parenteral in quantifiable detection dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, Guinea pig, monkey, or to man, waiting a sufficient period of time to pass metabolism (typically from about 30 seconds to 30 hours) and recovery of the products of its transformation from urine, blood or other biological samples. These products are easy to distinguish because they are labeled (other secrete antibody, capable of binding epitopes conserved in the metabolite). Patterns of metabolites determined in a traditional way, for example, through analysis of MS, LC/MS or NMR. Basically, the analysis of metabolites carried out in the same way as traditional studies of the metabolism of drugs, known to experts in this field. The products of conversion, provided that they otherwise fail to meetin vivosuitable for diagnostic assays for therapeutic dosing of the compounds of the ADC according to the invention.

ADDITIONAL uses for ANTIBODIES TO CD22 AND IMMUNOCONJUGATES

Diagnostic methods and methods of detection

In one aspect of the antibodies to CD22 and immunoconjugate according to the invention is suitable for detecting the presence of CD22 in a biological sample. The term "detection"as used herein covers the quantitative or qualitative detection. In certain embodiments of the implementation of the biological sample contains a cell or tissue. In certain embodiments of the exercise of such fabrics include normal and/or malignant tissues that Express CD22 at higher levels relative to other tissues, for example, B-cells and/or associated with B-cells of the tissue.

In one aspect the invention relates to a method of detecting the presence of CD22 in biology is Eskom sample. In certain embodiments of implementing the method comprises bringing into contact the biological sample with the antibody to CD22 under conditions that allow the binding of antibody to CD22 with CD22, and detection, is the formation of a complex between the antibody to CD22 and CD22.

In one aspect the invention relates to a method for the diagnosis of disorders associated with increased expression of CD22. In certain embodiments of implementing the method comprises bringing into contact the test cells with the antibody to CD22; determining the level of expression (either quantitatively or qualitatively) CD22 test cell by detecting binding of the antibody to CD22 with CD22; and comparing the level of expression of CD22 test cell with the level of expression of CD22 control cell (e.g., a normal cell, the source of which is the same fabric, or a cell that expresses CD22 at levels comparable to such a normal cell), where a higher level of expression of CD22 in the test cell compared to the control cell indicates the presence of disorders associated with increased expression of CD22. In certain embodiments of the implementation of the test cell is obtained from an individual suspected of having disorder associated with increased expression of CD22. In certain embodiments of the implementation of the violation presents yet 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 disorder, and/or B-cell proliferative violation, including as non-limiting examples, lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell.

In certain embodiments implement a method of diagnosis or detection, such as described above enables the detection of binding antibodies to CD22 with CD22 expressed on the cell surface or in the preparation of membranes derived from cells expressing CD22 on the surface. In certain embodiments of implementing the method comprises bringing into contact of the cells with antibody to CD22 under conditions that allow the binding of antibody to CD22 with CD22 and detection, whether formed complex between the antibody to CD22 and CD22 on the cell surface. Illustrative analysis for detection of binding antibodies to CD22 with CD22 expressed on the cell surface, is an analysis of the "FACS".

For detection has been the project of antibodies to CD22 with CD22 you can use some other ways. Such methods include, as non-limiting examples, analyses of binding antigen, which is well known in this area, such as Western blotting, radioimmunological assays, ELISA (enzyme linked immunosorbent assay), immunological sandwich assays, analyses thus, fluorescent immunological assays, immunological assays with protein A and immunohistochemistry (IHC).

In certain embodiments of the implementation of antibodies to CD22 are labeled. Labels include as non-limiting examples, a label or group, which detects directly (such as fluorescence chromophore, electroplate, chemiluminescent, and radioactive labels), as well as groups such as enzymes or ligands, which reveal indirect, for example by enzymatic reaction or molecular interaction. Illustrative labels include, as non-limiting examples of radioactive isotopes32P,14C,125I3H and131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, for example, luciferase of Svetlana and bacterial luciferase (U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, whether Azim, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and the xanthine oxidase-related 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 of the implementation of antibodies to CD22 immobilized on an insoluble matrix. Immobilization leads to the separation of antibodies to CD22 from any CD22, which remains free in solution. This is usually carried out either by the transfer of antibodies to CD22 in an insoluble form prior to analysis, for example, by adsorption at the water-insoluble matrix or surface (Bennich et al., U.S. 3720760)or covalent binding (e.g., using cross-linkage by glutaraldehyde), or by transferring antibodies to CD22 in an insoluble form after the formation of a complex between the antibody to CD22 and CD22, for example, by using thus.

Any of the above embodiments diagnosis or detection can be performed using immunoconjugate according to the invention instead of antibodies to CD22, or in addition to it.

Therapeutic methods

The antibody or immunology the gat according to the invention can be used, for example, in therapeutic waysin vitro,ex vivoandin vivo. In one aspect the invention relates to methods for inhibiting cell growth or proliferation, orin vivoorin vitromoreover , the method includes the impact on the cell antibodies to CD22 or immunoconjugate under conditions that allow binding immunoconjugate with CD22. "Inhibition of cell growth or proliferation" means the reduction of cell growth or proliferation, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% and includes the induction of cell death. In certain embodiments of the implementation of the cell is a tumor cell. In certain embodiments of the implementation of the cell is a B-cell. In certain embodiments of the implementation of the cell represents a xenograft, for example, as explained in this document.

In one aspect the antibody or immunoconjugate according to the invention is used to treat or prevent B-cell proliferative disorders. In certain embodiments of the implementation of cellular proliferative violation associated with increased expression and/or activity of CD22. For example, in certain embodiments of the implementation of B-cell proliferative violation associated with increased expression of CD22 on the surface of B-cells. In certain embodiments of the implementation Kletocnae proliferative violation is a tumor or a malignant tumor. Examples of B-cell proliferative disorders to be treated by the antibodies or immunoconjugates according to the invention include as non-limiting examples of lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell.

In one aspect the invention relates to methods for treating B-cell proliferative disorders, comprising the administration to the individual an effective amount of antibodies to CD22 or immunoconjugate. In certain embodiments implement a method of treating B-cell proliferative disorders includes an introduction to the individual an effective amount of a pharmaceutical preparation containing the antibody to CD22 or immunoconjugate to CD22 and, optionally, at least one additional therapeutic agent, such as tools, are presented below. In certain embodiments implement a method of treating cell proliferative disorders includes an introduction to the individual an effective amount of a pharmaceutical preparation containing 1) immunoconjugate containing antibody to CD22 and cytotoxic cf the rotary; and optional, 2) at least one additional therapeutic agent, such as tools below.

In one aspect, at least some of the antibodies or immunoconjugates according to the invention can bind CD22 from species other than human. Thus, antibodies or immunoconjugate according to the invention can be used to bind CD22, for example, in a cell culture containing CD22, humans, or other mammals with CD22, which antibody or immunoconjugate according to the invention comes into cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus macaques and rhesus, pig or mouse). In one embodiment, the implementation of the antibody to CD22 or immunoconjugate can be used for targeting CD22 on B cells by bringing into contact of the antibody or immunoconjugate and CD22 with the formation of antibodies or complex immunoconjugate-antigen, so that the conjugated cytotoxin immunoconjugate penetrates into the cell. In one embodiment, the implementation of CD22 CD22 is a person.

In one embodiment, the implementation of the antibody to CD22 or immunoconjugate can be used in the method of CD22 binding in an individual suffering from a disorder associated with increased expression and/or activity of CD22, and the method comprises the administration to the individual antibodies is a or immunoconjugate, so what happens binding of CD22 from the individual. In one embodiment, the implementation of the related antibody, or immunoconjugate internalized in B-cell expressing CD22. In one embodiment, the implementation of CD22 CD22 is a person, and the individual is a human. Alternatively, the individual can be a mammal expressing CD22 is associated with antibody to CD22. In addition, the individual may be a mammal, which was introduced CD22 (for example, through the introduction of CD22 or by the expression of the transgene encoding CD22).

Antibody to CD22 or immunoconjugate you can enter to man for therapeutic purposes. In addition, antibody to CD22 or immunoconjugate you can enter does not belong to human mammal expressing CD22, with which the antibody cross-reacts (e.g., a Primate, pig, rat or mouse) for veterinary purposes or as models of human disease in animals. In respect of the latter, such animal models may be suitable for assessing therapeutic efficacy of antibodies or immunoconjugates according to the invention (e.g., testing of dosages and mode of administration).

Antibodies or immunoconjugate according to the invention can be used in therapy either alone or in combination with other compositions. For example the EP, the antibody or immunoconjugate according to the invention can be entered together with at least one additional therapeutic agent and/or adjuvant. In certain embodiments of the implementation of the additional therapeutic agent is a cytotoxic agent, chemotherapeutic agent or a growth inhibitory agent. In one of these embodiments chemotherapeutic agent is a tool or combination of tools, such as cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubicin, vincristine (Oncovin™), prednisolone, CHOP, CVP, or COP, or immunotherapy, such as antibody to CD20 (e.g., Rituxan®) or antibody to VEGF (e.g., Avastin®), where the combined treatment is suitable in the treatment of malignant tumors and/or B-cell disorders, such as B-cell proliferative disorders including lymphoma, nahodkinskuju lymphoma (NHL), aggressive NHL, relapsing aggressive NHL, relapsing slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma mantle cell.

Such methods of combined treatment noted above encompass combined administration (where two or the more therapeutic agents are included in one or in different preparations) and a separate introduction, in the case of which the introduction of antibodies or immunoconjugate according to the invention can occur prior to, simultaneously and/or after administration of an additional therapeutic agent and/or adjuvant. Antibodies or immunoconjugate according to the invention can also be used in combination with radiation therapy.

The antibody or immunoconjugate according to the invention (and any additional therapeutic agent or adjuvant) can be entered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal introduction, and, if desired for local treatment, the introduction into the wound. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the antibody or immunoconjugate it is expedient to introduce pulse infusion, particularly with declining doses of the antibody or immunoconjugate. Dosing can be performed in any suitable way, 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, dose and type manner, consistent with good medical practice. Factors to be considered in this context include Conques is to maintain the violation, to be 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, the scheme of administration and other factors known to medical practitioners. The antibody or immunoconjugate should not be made, but not necessarily made with one or more of the means applied for the prevention or treatment of interest violations. The effective amount of such other funds depends on the amount of antibody or immunoconjugate present in the product, the type of disorder or treatment, and other factors discussed above. They usually are used in the same dosages and routes of administration described herein, or from about 1 to 99% of the dosages described herein, or in any dosage and through any method that is empirically/clinically determined, as appropriate.

For the prevention or treatment of diseases suitable dosage of the antibody or immunoconjugate according to the invention (when used alone or in combination with one or more other additional therapeutic means, such as chemotherapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunology the ATA, the severity and course of the disease, preventive or therapeutic purposes of introducing the antibodies or immunoconjugates, previous treatment, the clinical history of the patient and response to the antibody or immunoconjugate and from the decision of the attending physician. The antibody or immunoconjugate it is expedient to introduce the patient once or over a series of injections. Depending on the type and severity of the disease, the dose from about 1 μg/kg to 100 mg/kg (for example, 0.1 to 20 mg/kg) of antibody or immunoconjugate is the source of possible dose for administration to a patient, for example, either by one or by several separate injections, 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. In the case of repeated injections over several days or more, depending on the condition, treatment continues until a desired suppression of disease symptoms. One illustrative dosage of the antibody or immunoconjugate is in the range from about 0.05 mg/kg to about 10 mg/kg Thus, the patient can enter a single or multiple doses of the antibody or immunoconjugate constituting approximately 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination). Takyaduzy can be entered periodically, for example, every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody or immunoconjugate). You can enter the source of a higher loading dose, followed by one or more lower doses. Illustrative dispensing includes the introduction of the initial loading dose, a component of approximately 4 mg/kg, with subsequent weekly support dose comprising about 2 mg/kg of antibody. However, there may be other suitable dosing regimens. Monitoring the progress of treatment can easily be made by conventional methods and analysis.

Analyses

Antibodies to CD22 and immunoconjugate according to the invention can be characterized for their physical/chemical properties and/or biological activity of the various methods of analysis known in this field.

Analyses of activity

In one aspect provided by the tests to identify antibodies to CD22 or immunoconjugates possessing biological activity. Biological activity may include, for example, the ability to inhibit cell growth or proliferation (e.g., activity against the "destruction of cells"), or the ability to induce cell death, including requested summirovaniyu cell death (apoptosis). Also provides antibodies or immunoconjugate having such biological activityin vivoand/orin vitro.

In certain embodiments of the implementation of the antibody to CD22 or immunoconjugate tested with respect to its ability to inhibit cell growth or proliferationin vitro. Analysis of inhibition of cell growth or proliferation are well known in this field. In some analyses of cell proliferation, examples of which are studies of "destruction of the cells described herein to measure cell viability. One such assay is a luminescent analysis of the viability of the cells CellTiter-Glo™ Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). In this analysis, we determine the number of viable cells in culture based on quantification of ATP present, which is an indicator of metabolically active cells. Cm. Crouch et al., (1993) J. Immunol. Meth. 160: 81-88, U.S. patent No. 6602677. The assay can be performed in 96 - or 384-well format, which ensures its suitability for automated high-throughput screening (HTS). Cm. Cree et al., (1995) AntiCancer Drugs 6: 398-404. The method of analysis involves adding a single reagent (reagent CellTiter-Glo®) directly to cultured cells. This leads to cell lysis and the formation of luminesce the private signal, produced by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be registered by means of a luminometer or device for imaging with CCD camera. The output luminescence is expressed as relative light units (RLU).

Another analysis of cell proliferation is the analysis of the "MTT", colorimetric analysis, which measures the oxidation of bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium to formazan mitochondrial reductase. Like the analysis of CellTiter-Glo™, this analysis shows the number of metabolically active cells present in the cell culture. For example, see Mosmann (1983) J. Immunol. Meth. 65: 55 to 63, and Zhang et al., (2005) Cancer Res. 65: 3877-3882.

In one aspect the antibody to CD22 tested with respect to its ability to induce cell deathin vitro. Tests for the induction of cell death is well known in this field. In some embodiments, the implementation of such analyses is measured, for example, the loss of integrity of the membranes, as indicated by the capture of iodide of propecia (PI), Trypanosoma blue (see Moore et al., (1995) Cytotechnology, 17:1-11) or 7AAD. Illustrative analysis capture PI cells cultured in the modified Dulbecco environment the Needle (D-MEM:F-12 ham (50:50)supplemented with 10% in aktivirovannoi heating FBS (Hyclone) and 2 mm L-glutamine. Thus, the analysis carried out in the absence of complement and immune effector cells. Cells were seeded with a density of 3×106a Cup of 100×20 mm Cup and allow to attach overnight. The medium removed and replaced with fresh medium alone or medium containing various concentrations of the antibody or immunoconjugate. Cells are incubated for 3 days. After processing, the monolayers are washed with PBS and choose to unpin it by trypsinization. The cells are then centrifuged at 1200 rpm for 5 minutes at 4°C, the precipitate resuspended in 3 ml cold binding of Ca2+buffer (10 mm Hepes, pH of 7.4, 140 mm NaCl, 2.5 mm CaCl2) and divided into aliquots covered in 35-mm grid 12×75 mm tubes (1 ml per tube, 3 tubes for the treated group) for removal of cell clumps. Then in a test tube add PI (10 μg/ml). Samples analyzed using the flow cytometer FACSCAN™ and software FACSCONVERT™ CellQuest (Becton Dickinson). Thus, identify antibodies or immunoconjugate that induce statistically significant levels of cell death as determined by the capture of PI.

In one aspect the antibody to CD22 or immunoconjugate tested for their ability to induce apoptosis (programmed cell death)in vitro. Illustrative way analysis of antibodies or immunoconjugates that indusiry the apoptosis, is the analysis of the binding of annexin. Illustrative analysis of binding of annexin cells are cultivated and sown in the Cup, as discussed in the previous paragraph. The medium removed and replaced with fresh medium alone or medium containing from 0.001 to 10 μg/ml antibody or immunoconjugate. After incubation for three days the monolayers washed with PBS and choose to unpin it by trypsinization. The cells are then centrifuged, resuspended in binding Ca2+buffer and divided into aliquots in test tubes, as discussed in the previous paragraph. Then in a test tube add annexin (e.g., annexin V-FITC) (1 μg/ml). Samples analyzed using the flow cytometer FACSCAN™ and software FACSCONVERT™ CellQuest (Becton Dickinson). Thus, identify antibodies or immunoconjugate that induce statistically significant levels of binding of annexin relative to control. Other illustrative analysis with respect to the antibodies or immunoconjugates, which induce apoptosis, is a colorimetric analysis of the DNA-histone ELISA for detection mineclearance degradation of genomic DNA. Such analysis can be performed using, for example, set the Cell Death Detection ELISA (Roche, Palo Alto, CA).

Cells for use in any of the above testsin vitroinclude cells or cell lines, which in nature Express CD2, or which were constructed for the expression of CD22. Such cells include tumor cells that sverkhekspressiya CD22 relative to normal cells, the source of which is the same fabric. Such cells include cell lines (including lines of tumor cells), which Express CD22, and cell lines that do not normally Express CD22, but were transliterowany nucleic acid that encodes a CD22.

In one aspect the antibody to CD22 or immunoconjugate tested for their ability to inhibit cell growth or proliferationin vivo. In certain embodiments of the implementation of the antibody to CD22 or immunoconjugate tested for their ability to inhibit tumor growthin vivo. For such testing, you can use a model systemin vivosuch as model xenografts. Illustrative systems with xenografts of tumor cells injected does not belong to man the animal with a suitable way weakened immune system, for example, the 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 implementation of the above system with xenografts tumor cells are tumor cells from patient man. Finally the cells, suitable for models xenografts include cell lines of leukemia and lymphoma rights, which include, as non-limiting examples, cells of BJAB-luc (EBV-negative cell line lymphoma Burkitt, transfusiona luciferase reporter gene), Ramos cells (ATCC, Manassas, VA, CRL-1923), Raji cells (ATCC, Manassas, VA, CCL-86), 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 of the implementation of the tumor cells injected does not belong to man the animal with a suitable way immunosuppressed by subcutaneous injection or by transplantation in a suitable area, such as fatty tissue of the breast.

Analyses linking and other tests

In one aspect the antibody to CD22 tested with respect to its antigennegative activity. For example, in certain embodiments of the implementation of the antibody to CD22 tested with respect to its ability to bind to CD22 expressed on the cell surface. For this test, you can use the FACS analysis.

In one aspect of to identify a monoclonal antibody that competes with antibody 10F4.4.1 mouse, humanized antibody 10F4v1, humanized antibody 10F4v3 is/or antibody 5E8.1.8 mouse for binding to CD22, you can use competitive analysis. In certain embodiments of the implementation of such a competing antibody binds to the same epitope (e.g., linear or conformational epitope)that is bound antibody 10F4.4.1 mouse humanitariannet antibody 10F4v1, humanitariannet antibody 10F4v3 and/or antibody 5E8.1.8 mouse. Illustrative competitive analyses include as non-limiting examples of conventional tests, such as presented in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed illustrative ways of mapping the epitope to which antibody binds presented in Morris (1996) "an epitope Mapping Protocols", Methods in Molecular Biology Vol.66 (Humana Press, Totowa, NJ). They say that the two antibodies bind to a single epitope if each of them is blocking the binding of the other by 50% or more.

In the illustrative competitive analysis of immobilized CD22 incubated in the solution containing the first labeled antibody to bind to CD22 (e.g., antibody 10F4.4.1 mouse humanitariannet antibody 10F4v1, humanitariannet antibody 10F4v3 and/or antibody 5E8.1.8 mouse) and a second unlabeled antibody that is tested against its ability to compete with the first antibody for binding to CD22. The second antibody may be in the supernatant of hybridoma. As a control, immobilized CD22 incubated in RA the creators, contains the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CD22, excess unbound antibody is removed, and measure the amount of label associated with immobilized CD22. If the amount of label associated with immobilized CD22 significantly 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 CD22. In certain embodiments of the implementation of the immobilized CD22 is located on the cell surface or in the preparation of membranes derived from cells expressing CD22 on the surface.

In one aspect of the purified antibodies to CD22 can be further characterized by a series of analyses, including, as non-limiting examples of N-terminal sequencing, amino acids analysis, sedentarius exclusion high-performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain cleavage.

In one of the embodiments the invention relates to a modified antibody that possesses some but not all effector functions, which makes it a desirable candidate for many applications in which the half-life ofin vivoantibodies are important, but are necessary and or harmful certain effector functions (such as complement and ADCC). In certain embodiments of the implementing measure the activity Fc antibodies in order to ensure that these are desirable properties. Analysis of cytotoxicityin vitroand/orin vivocan be performed to confirm the reduction/elimination of CDC activity and/or ADCC. For example, analyses of the binding of Fc-receptor (FcR) can be performed to verify that the antibody not binding to FcγR (thus, probably no ADCC activity), but retained the ability to bind FcRn. The main cells mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). Example of analysis ofin vitroto assess ADCC activity of interest molecules described in U.S. patent No. 5500362 or 5821337. Suitable effector cells for such assays include mononuclear cells of peripheral blood (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of interest molecules can be estimatedin vivofor example, in animal models, such as the model described in Clynes et al., PNAS (USA) 95:652-656 (1998). Analyses of the binding of C1q can also be carried out to confirm that the antibody is unable to bind C1q and thus deprived of CDC activity. To assess activation of complement can be analyzed by the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). The FcRn binding definition and elimination/half-life ofin vivoit is also possible to use known in the field of methods.

EXAMPLES

The following are examples of the methods and compositions according to the invention. It is clear that in practice it is possible to apply various other variants of implementation, given the General description above.

Example 1: Getting mouse monoclonal antibodies to CD22 person

Got mouse monoclonal antibodies able to specifically bind CD22. Female BALB/c mice at the age of six weeks, were immunized in their paw pads purified extracellular domain of CD22 human labeled his-8, deprived of domains 3 and 4 (SEQ ID NO: 30 (ECD) plus the sequence GRAHHHHHHHH C-end), or the extracellular domain of CD22, labeled his-8, containing domains 1-7 (SEQ ID NO: 28 (ECD) plus the above label of the sequence His) in Freund Ribi. Subsequent injections were performed in the same way after one and three weeks after the initial immunization. Three days after the final injection inguinal and popliteal lymph nodes were removed and combined, and obtained a suspension of individual cells, passing the fabric through the steel mesh. Cells were exposed to the merger in the ratio of 4:1 is myeloma mouse such as P3X63-Ag8.653 (ATCC CRL 1580) in the medium with high glucose (DMEM)containing 50 wt%./about. polyethylene glycol 4000. Then fused cells were planted in 96-well plates to cultivating the tissue with a density of 2×105on the hole. After 24 hours was added to the selective environment HAT (gipoksantin/aminopterin/thymidine, Sigma, #H0262). Fifteen days after the merger supernatant growing cells tested in the presence of antibodies specific for human CD22, using enzyme-linked immunosorbent assay (ELISA).

For subsequent studies were selected mouse monoclonal antibodies to human CD22 10F4.4.1 (mu10F4) and 5E8.1.8 (mu5E8), based on cellular assays and analyses in tablets, which showed that antibodies specifically associated with human CD22. These analyses are described in the following paragraphs.

Tests based on ELISA: Screening for antibodies to CD22 by ELISA carried out as follows, with all the incubation was performed at room temperature. On the test plates (Nunc Immunoplate) applied within 2 hours of cleared CD22 in 50 mm sodium carbonate buffer, pH 9,6, then blocked with 0.5% bovine serum albumin in phosphate-buffered saline (PBS) for 30 minutes and then washed four times by PBS containing 0.05% Tween 20 (PBST). Add supernanny with a test antibody and incubated for two hours at which Strahovanie, then washed four times by PBST. Tablets are by adding 100 μl/well of a solution containing 10 mg of the dihydrochloride o-phenylenediamine (Sigma, #P8287) and 10 μl of 30% hydrogen peroxide in 25 ml of phosphate-citrate buffer, pH 5.0, and incubated for 15 minutes. The reaction is stopped by adding 100 μl/well of 2.5 M sulfuric acid. Data obtained by reading the tablets in an automatic reader tablets ELISA absorption 490 nm.

Example 2: Analysis based on FACS for the study of monoclonal antibodies (MAb) to human CD22

The CHO cells expressing human CD22 on the surface, incubated with the supernatant of hybridoma with antibodies to CD22 in 100 μl FACS buffer (0.1% of BSA, 10 mm sodium azide in PBS, pH 7,4) for 30 minutes at 4°C, followed by one wash with FACS buffer. The amount of binding of the antibody to CD22 was determined by incubation of aliquots of the antibody/cell mixture with polyclonal conjugated with FITC IgG goat or rabbit antibodies against mouse (Accurate Chem. Co., Westbury, NY) (tested for antibodies mouse) or IgG goat or rabbit antibodies against human (for humanized antibodies) for 30 minutes at 4°C followed by three washes with FACS buffer.

Example 3: Obtaining humanized antibodies to CD22

Received humanized 10F4 antibodies, where amino acid residues hypervariable region (HVR) (mutual is replaceable, referred to as complementarity determining region or CDR) was modified by site-specific mutagenesis (Kunkel et al., Methods Enzymol. (1987), 154:367-382) obtaining two options, gumanitarnogo 10F4v1 and gumanitarnogo 10F4v2 (also denoted herein as "10F4v1", "hu10F4v1", "10F4v2" or "hu10F4v2", respectively). The third option, humanitariannet 10F4v3 ("10F4v3" or "hu10Fv3")used in some studies, described herein, has the same amino acid sequences of light and heavy chains of the Mature protein, as hu10F4v2, however, contains a different signal sequence in the vector used for expression of the protein.

Humanization of the antibody 10F4 mice were performed as described in this document. In summary, a hypervariable region of the light and heavy chains 10F4 mouse cloned into modified consensus framework sequence with obtaining the amino acid sequences of the variable regions of light and heavy chains presented on figa and 2B. Alternative frame sequence light and heavy chains, which can be used as frame sequences of the antibodies according to the invention, represented in figure 3 and 4.

For the humanization of the antibody 10F4 used monovalent vector (pV0350-2B) fahmida for display Fab-g3 having two open reading frames under control of the promoter of phoAbasically, as described in Leeet al., J. Mol. Biol. 340:1073-93 (2004). The first open reading frame contained the signal sequence of thermostable STIIE. colifor secretion of the protein, fused to domains VL and CH1 acceptor sequence of the light chain. The second open reading frame contained the signal sequence of STII, merged with the domains VH and CH1 acceptor sequence of the heavy chain, followed by a shorter minor protein P3 shell phage.

Domains VH and VL of 10F4 mouse (SEQ ID NO: 89 and 90, respectively) were aligned with the consensus domain of human VH III subgroup (huIII) (SEQ ID NO: 24) and a consensual domain of a human Kappa I (huK1) (SEQ ID NO: 25), respectively. Amino acid sequence hypervariable regions (HVR interchangeable denoted herein as complementarity determining region (CDR)mouse MAb against human CD22 10F4 embedded in the consensus framework sequence as follows. HVR light chains (HVR-L1 (Kabat position 24-34), HVR-L2 (Kabat position 50-56) and HVR-L3 (Kabat position 89-97) antibodies mu10F4 embedded in the frame of human antibodies with the consensus sequence of the Kappa I (huKI) to obtain the humanized light chain 10F4v1 (SEQ ID NO: 17, pigv). HVR heavy chain (HVR-H1 (Kabat position 26-35), HVR-H2 (Kabat position 49-65) and HVR-H3 (Kabat position 95-102) antibodies mu10F4 was built in modifitsirovannyi consensual domain of human VH subgroup III (humIII), which differs from the sequence humIII three provisions: R71A, N73T and L78A (see Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992)), to obtain the humanized variable regions of the heavy chain 10F4v1 (SEQ ID NO: 16, figa). Genetic embedding HVR in the acceptor frameworks were carried out by Kunkel mutagenesis using a separate oligonucleotide for each hypervariable region. The sequence of each clone was determined by standard methods of DNA sequencing. Hypervariable region and a frame region, shown in figa and 2B, numbered according to the Kabat numbering (Kabat et al., (1991), above). Light and heavy chain sequence and amino acid sequence of the variable regions (including the HVR and frame work region (FR)) huKI, humIII, 10F4 mouse gumanitarnogo 10F4v1 and gumanitarnogo 10F4v2 presented on figa and 2B. Humanitariannet antibody 10F4v3 has an amino acid sequence identical to 10F4v2.

The nucleic acid molecules encoding amino acid sequence variants of the antibody, antibody fragment, domain, or VL domain, VH, is produced by many known in the field of methods. These methods include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence variants) or receiving mediated by oligonucleotides (what if the site-specific) mutagenesis, PCR mutagenesis, and cassette mutagenesis previously received a modified or unmodified variant antibody, antibody fragment, domain, VL or VH domain. For example, you can create libraries targeting available amino acid position in the VL, VH, and optionally one or more CDR for replacement of amino acids differing amino acids using the method of Kunkel. See, for example, Kunkel et al., Methods Enzymol. (1987), 154:367-382 and examples in it. Obtaining randomized sequences is also described below in the examples.

The sequence of oligonucleotides includes one or more of the provided sets of codons for a particular position in a CDR (HVR) or FR-region polypeptide according to the invention. The set of codons is a set of different nucleotide triplet sequences used to encode the required variant amino acids. Sets of codons can be represented using symbols to indicate specific nucleotides or equimolar mixtures of nucleotides, as presented below in accordance with the IUB code.

CODES IUB

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

(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)

N (A or C or G or T)

For example, to set the codons DVK, D can represent the nucleotides A or G or T; V can be a A or G or C; and K can be a G or T. This set of codons can represent 18 different codanova 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, for example, solid-phase synthesis of sequences that correspond to all possible combinations of nucleotide triplets provided by the set of codons, and which will encode the desired group of amino acids. The synthesis of oligonucleotides selected "degeneracy" of the nucleotides at specific positions is well known in this field. These sets of nucleotides that have specific sets of codons, can be synthesized using commercial devices for nucleic acid synthesis (available, for example, from Applied Biosystems, Foster City, CA) or can be obtained commercially (for example from Life Technologies, Rockville, MD). Thus, a set of synthesized oligonucleotides with a specific set of codons, as a rule, will include many of oligonucleotides with different sequences, and differences are determined by the set of codons within the entire sequence. Oligon cleotide, used according to the invention have sequences that allow hybridization with the matrix nucleic acid of the variable domain, and may also include sites for a restriction enzyme for cloning purposes.

In one method, the nucleic acid sequence encoding variant amino acids, you can create oligonucleotide-mediated by mutagenesis. This method is well known in this field, as described by Zoller et al., 1987, Nucleic Acids Res. 10:6487-6504. In summary, the sequence of nucleic acids encoding variant amino acids, created by hybridization of a set of oligonucleotides encoding the desired set of codons with DNA-matrix, where the matrix is a single-stranded form of a plasmid containing the matrix nucleic acid sequence of the variable region. After hybridization using DNA polymerase to synthesize an entire second complementary chain matrix, which, therefore, will incorporate the oligonucleotide primer, and will contain sets of codons, provide a set of oligonucleotides.

As a rule, use oligonucleotides with a length of at least 25 nucleotides. Optimal oligonucleotide will have 12 to 15 nucleotides that is fully complementary to the matrix on each side of the nucleotide(s)encoding the mutation(s). Uh what about the need for adequate hybridization of the oligonucleotide with single-stranded DNA molecule-matrix. The oligonucleotides are readily synthesized using known in the field of methods, such as methods described by Crea et al., Proc. NAT'l. Acad. Sci. USA, 75:5765 (1978).

DNA-matrix is to be made either by vectors, which are formed from the vectors bacteriophage M13 (suitable commercially available vectors M13mp18 and M13mp19)or through vectors that contain a single-stranded phage plot beginning of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is subject to mutation, you can embed one of these vectors in order to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 Sambrook et al., above.

To change the native DNA sequence of the oligonucleotide hybridized stranded matrix in a suitable hybridization conditions. Then add the enzyme polymerization of DNA, usually DNA polymerase T7 or piece maple DNA polymerase I, for synthesis of a complementary chain of a matrix using the oligonucleotide as a primer for synthesis. Thus, there is heteroduplex molecule, so that one strand of DNA encodes the mutated form of the gene 1, and the other strand (the original matrix) encodes the native, unmodified sequence of gene 1. Then this heteroduplex molecule transform in a suitable cell host, usually in prokaryotic is tapped, such asE. coliJM101. After cultivation of the cells placed on the agarose tablets and subjected to screening using oligonucleotide primers, radioactively labeled with 32-phosphate, to identify the bacterial colonies that contain the mutated DNA.

The method described immediately above, can be modified to create homoduplexes molecules, where both strands of the plasmid containing the mutation(s). Modifications are as follows: single-stranded oligonucleotide is annealed to single-stranded matrix, as described above. A mixture of three deoxyribonucleotides, desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTT), together with a modified timezonebias called dCTP-(aS) (which can be obtained from Amersham). This mixture was added to the complex matrix-oligonucleotide. Adding to this mix DNA polymerase produces DNA strand that is identical to the matrix, except for the mutant bases. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves for protection from cleavage with restriction endonucleases. After cutting the matrix circuit of the double-stranded heteroduplex using the appropriate restriction enzyme matrix circuit can be split by ExoIII nuclease or other suitable nuclease after the region that contains the site(sites), pagliusi the mutagenesis. Then the reaction is stopped, so that there is a molecule, which is only partially single-stranded. Then form a complete double-stranded homoduplex DNA using DNA polymerase in the presence of all four deoxyribonucleotides, ATP and DNA ligase. Then this homoduplex molecule can be transformed in a suitable cell host.

As indicated previously, the dialing sequence of the oligonucleotide is of sufficient length for hybridization with the matrix nucleic acid and may also, but not necessarily, contain the restriction sites. DNA-matrix can be obtained either by vectors, which are formed from the vectors of bacteriophage M13 or through vectors that contain a single-stranded phage plot beginning of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is subject to mutation, it is necessary to embed one of these vectors in order to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 Sambrook et al., above.

In accordance with another way the library can be obtained by providing sets of upstream and downstream oligonucleotides, and each set has a lot of oligonucleotides with different sequences and different sequences are determined, set the AMI codons, presented in the sequence of the oligonucleotides. Sets the top and bottom oligonucleotides, together with a matrix sequence of the nucleic acid of the variable domain, can be used in polymerase chain reactions to create a "library" of PCR products. The PCR products can be labeled as "cassette nucleic acids"because they can be merged with other related or unrelated sequences of nucleic acid, for example, viral coat proteins and dimerization domains, using known methods of molecular biology.

Sets of oligonucleotides can be used in polymerase chain reactions using matrix nucleic acid sequence of the variable domain of the matrix to create tapes of nucleic acids. The matrix nucleic acid sequence of the variable domain can be any of the heavy chains of immunoglobulins containing the sequence of the nucleic acid target (i.e. nucleic acid sequence encoding amino acids designed to replace). The matrix nucleic acid sequence of the variable region is part of a double-stranded DNA molecule having a first chain nucleic acid and complementarities chain nucleic acids. The matrix nucleic acid sequence of the variable domain comprises at least a portion of the variable domain, and has at least one CDR. In some cases, the matrix nucleic acid sequence of the variable region contains more than one CDR. The upstream part and downstream part of the matrix nucleic acid sequence of the variable region can be used for hybridization with the members of the set of the upstream oligonucleotide and the set of downstream oligonucleotides.

The first oligonucleotide set upstream primers can gibridizatsiya with the first chain nucleic acid and the second oligonucleotide set downstream primers can gibridizatsiya with the second chain nucleic acids. Oligonucleotide primers can include one or more sets of codons, and they can be designed for hybridization with a part of the matrix nucleic acid sequence of the variable region. The use of these oligonucleotides can make two or more sets of codons in the PCR product (i.e. the cassette nucleic acid) after PCR. Oligonucleotide primer, which hybridizes with portions of the nucleic acid sequence that encodes a variable domain antibodies, includes areas that code the display remains CDR, designed for replacement of amino acids.

The sets of upstream and downstream oligonucleotides can also be synthesized to include restriction sites in the sequence of the oligonucleotide. These restriction sites can facilitate embedding cassettes nucleic acid (i.e. the product of PCR reaction) in expressing vector with the additional sequence of the antibody. In one embodiment, the implementation of the restriction sites are intended to facilitate the cloning of nucleic acid cassettes without the introduction of extraneous sequences of nucleic acids or removing the source of the nucleic acid sequences of CDR or framework.

Cassette nucleic acid can be cloned into any suitable vector for the expression of part or the entire sequence of the light or heavy chain that contains the planned replacement of amino acids obtained by PCR reaction. In accordance with the methods described for this invention, the cassette nucleic acid clone in the vector, allowing the production of parts or the complete sequence of the light or heavy chain, merged with all the protein shell of a virus or part of it (i.e. creating protein), and reproduce on the surface of the particles or cells. Despite the fact that available and can be used for skin is to practice this invention, several types of vectors, famiglie vectors are the preferred vectors for use in the present document, since it is relatively easy to design and easy to amplify. Famiglie vectors usually contain a variety of components, including promoters, signal sequences, phenotypic selective genes, the plots start replication and other necessary components, known to specialists in this field.

When expression is subject to a specific combination of variant amino acids, cassette nucleic acid contains a sequence capable of encoding the entire variable domain of a heavy or light chain or a part of it and is able to encode combinations of variant amino acids. To obtain antibodies containing these variant amino acids or combinations of variant amino acids, as in the case of libraries, tapes nucleic acids can be embedded in expressing a vector containing an additional sequence of the antibody, for example, all or part of the variable or constant domains of the variable regions of light and heavy chains. These additional sequences of the antibodies can also be merged with other sequences of nucleic acids, such as sequences that encode proteins of the virus envelope and, thus, produziu the protein.

Example 4: Determination of the sequence of the variable region

Nucleic acid sequences and amino acid sequences of monoclonal antibody 10F4 mouse and humanized monoclonal antibody 10F4 were determined by standard methods. From hybridoma cells were extracted total RNA, producing mouse monoclonal antibodies against CD22 human 10F4.4.1 using RNeasy® Mini Kit (Qiagen, Germany). The variable domains of the light chain (VL) and variable domains of the heavy chain (VH) amplified using RT-PCR with degenerate primers. Direct the primers were specific for the amino acid sequences of the N-end areas VL and VH of the antibody. Accordingly, the reverse primers for the light chain and heavy chain were designed to anneal with the plot of the constant domain of the light chain (CL) and constant domain 1 heavy chain (CH1), which are highly conserved among species. Amplificatoare VH and VL were cloned in expressing vector pRK mammalian cells (Shields et al., J. Biol. Chem. 276:659-04 (2000)). Polynucleotide sequence of the inserts was determined using conventional sequencing methods. Amino acid sequences of the variable regions of the light and heavy chains of murine chimeric 10F4 and gumanitarnogo 10F4v1 and gumanitarnogo 10F4v2 presented at F. GA and 2B.

Humanitariannet 10F4v1 further modified in position 28 HVR-L1 (N28) (SEQ ID NO: 9) (see figv). The asparagine residue at this position is replaced by a valine residue (N28V) to obtain the HVR-L1 (SEQ ID NO: 10) for options hu10F4v2 and hu10F4v3, which showed high affinity binding. These options contain the same sequence of variable and constant domains of the Mature antibody and differ only in the signal sequence, which is not found in the Mature antibody according to the invention.

Conducted additional modifications of amino acid sequence in one or both of the amino acids Asn28 (N28) and/or Asn30 (N30) hypervariable region HVR-L1 (see figv) hu10F4v1. Because N28 and N30 are possible areas for deamination, tested amino acid substitutions at these sites. For example, the asparagine at position 28 (N28) was replaced with alternative A, Q, S, D, V or I, and asparagine at position 30 (N30) was replaced by alternative A or Q. Changes in amino acid sequence in the domain HVR-L1 according to the invention are presented in table 2 together with their binding affinity of tested competitive analysis in the phage ELISA (IC50) using standard methods.

Table 2
Options replacement in antibodies is hu10F4v1
Amino acid substitution in HVR-L1 FIGUHVR-L1 is SEQ ID NOThe affinity of binding (nm)
Without replacement (N28, N30)98
N28A, N30198
N28Q, N30207,3
N28S, N302112
N28D, N302212
N28V, N30107,3
N28I, N30239,8
N28, N30A327,7
N28, N30Q3310

For more options gumanitarnogo antibody 10F4 with a full-sized human IgG1 heavy and light chain subcloning separately in the previously described plasmid pRK (Gorman, C.M. et al., (1990), DNA Protein Eng. Tech. 2:3). The corresponding plasmids for the heavy and light chains (depending on the requirement which has been created change(s) sequence) cotransfection transformed by adenovirus cell line of embryonic human kidney, known as 293 (Graham, F.L. et al. (1977), J. Gen. Virol. 36: 59), using a highly effective method (Graham et al., above & Gorman, C.M., Science 221: 551). The medium replaced with serum-free and collect every day up to 5 days. Antibodies purified from United supernatants using CL-4B protein A-Sepharose (Pharmacia). Aliremove antibody is subjected to replacement in PBS buffer by gel filtration G25, concentrated by ultrafiltration using a Centriprep-30 or Method-100 (Millipore) and stored at 4°C. the Concentration of antibody was determined using ELISA with linking of total IgG.

Illustrative constant domains of the heavy chain IgG1 according to the invention presents on figa. Illustrative human constant domain κ light chain contains, for example,

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHDVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 37). Full amino acid sequence h10F4v2 presented on FIGU, in which the constant region light and heavy chains are indicated by underlining. Antibodies h10F4v1, v2 and v3 represent the antibodies of isotype IgG1.

Characterization of antibodies to CD22

Example 5: Mapping of epitopes:

CD22 epitopes that bind antibodies 10F4.4.1 and 5E8.1.8, was determined in the following ways. Sequence CD22, deprived of a variety of seven immunoglobulin-like domains, the main isoform of CD22 (CD22), Clonie is ovale and transformed into cells for stable expression. For example, variants of CD22, devoid, domain 1 (Δ1), domain 2 (Δ2) or domains 3 and 4 (Δ3,4), cloned, transformed into CHO cells and expressed on the cells. Control cells expressed CD22. Deletions was performed using a set of reagents Stratagene QuikChange XL™. A deletion of domain 1 was performed by deletions of amino acids 22-138; a deletion of domain 2 was performed by deletions of amino acids 139-242; and deletion of domains 3 and 4 was available as a minor isoforms CD22 (deletion of amino acids 241-417). All rooms amino acids refer to the numbering of the full-sized predecessor CD22, Wilson, G.L. et al., (see figure 1 in Wilson, G.L. et al., J. Exp. Med. 173: 137-146 (1991)). On Fig presents chart deletirovannykh domains. Binding was performed by flow cytometry using izotopicheskogo control. Linking 10F4.4.1 were detected using goat IgG antibodies against mouse Alexa 488. Detection of binding 5E8.1.8 was performed using biotinylated IgG goat antibodies against mouse plus streptavidin PE. Adverse effect on the binding of the antibody 10F4.4.1 mouse or 5E8.1.8 mouse in the absence of specific domains of ECD showed that the antibody binds to these domains. 10F4.4.1 and 5E8.1.8 mouse showed similar binding characteristics under these conditions. None of them was bound CD22, devoid, domain 1 or domain 2, and both of them were bound CD22, the content is of ASI 1 and 2, but devoid of domains 3 and 4. Using this method it was determined that 10F4.4.1 and 5E8.1.8 associated with domains 1 and 2 CD22 person, in sequence from amino acid 22 to amino acid 240 SEQ ID NO: 27 (see Wilson, G.L. et al., (1991), supra).

Example 6: Characterization of the affinity of binding with soluble antigen

The binding affinity of the antibody 10F4 mouse and gumanitarnogo antibody 10F4 with the soluble extracellular domain of CD22 (ECD) was determined using the measurements by surface plasmon resonance using the BIACORE® 3000 (Biacore, Inc., Piscataway, NJ). In summary, the biosensor chip based on carboxymethylamino dextran (CM5, Biacore Inc.) activated hydrochloride N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. These activated chips inflicted IgG1 antibody 10F4 to CD22 (mouse or humanitariannet) breeding to 5 μg/ml 10 mm sodium acetate, pH of 4.8, with the subsequent injection of a flow rate of 5 μl/minute to achieve approximately 500 response units (RU) attached antibodies. Next were injected with 1M ethanolamine to block unreacted groups. To measure the kinetics of a twofold serial dilution of human soluble antigen CD22-beta, labeled ECD-His (from approximately 500 nm to approximately 7.8 nm), inj is provoked in PBS with 0,05% Tween 20 at 25°C with a flow rate of 30 µl/min The rate of Association (konand the rate of dissociation (koff) was calculated using a simple one-to-one model binding Langmuir (Software BIAevaluation 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio of koff/kon. As control was used antibody to CD22, RFB4 (Chemicon International, Inc. Temecula, CA, catalog no. CBL147). The results of this experiment are presented in table 2, below.

TABLE 2
The binding affinity of antibodies with soluble CD22 CD22 human analysis (BIACORE®)
Clonekon/105koff/10-4Kd (nm)
10F4 mouse0,192,815
Chimeric 10F40,264,216
Humanitariannet 10F4v10,183,519
Humanitariannet 10F4v2 0,322,57,8
Control RFB40,331,44,2

Example 7: Characterization of the affinity of binding to the antigen on the cell surface

The binding affinity of 10F4.4.1 mouse and gumanitarnogo 10F4v1 and 10F4v2 with CD22 human and cynomolgus macaques (cyno), expressed on the surface of CHO cells, was investigated using competitive analysis. Briefly, CHO cells stably expressed full-CD22 human (SEQ ID NO: 27) or CD22 cynomolgus macaques (cyno) (SEQ ID NO: 31). Antibody to CD22 (10F4v1 or v2 mouse or humanitariannet 10F4v1 or v2) has codiroli the Iodogen reagent® [125I] to a specific activity of approximately 10 µci/µg. Cellular analysis of competitive binding was performed using serially diluted unlabeled antibodies to CD22. The antibody was allowed to contact with the cells for 4 hours at 4°C. the Affinity of binding, Kd, antibodies were determined by standard analysis of Scatchard conducted using the program to handle non-linear curve (for example, see Munson et al., Anal. Biochem., 107: 220-239, 1980). The results of this experiment are presented in table 3 below.

TAB THE ITZA 3
The binding affinity of MAb 10F4 with CD22 human and Cyno
AntibodyKd human CD22 (nm)Kd CD22 Cyno (nm)
Mu10F4.4.12,42,3
Hu 10F4v1*1,1, 1,71,4, 1,8
Hu 10F4v21,62,1
*Repeated the tests.

The results indicate that 10F4 mouse and humanitariannet 10F4 binds CD22 human and cyno, expressed on the surface of CHO cells with approximately equivalent affinity.

Example 8: Obtaining conjugates of the antibody to CD22-drug

ADC to CD22 was obtained by conjugation of antibodies to CD22 RFB4, 5E8 mouse, 10F4 mouse gumanitarnogo 10F4v1, gumanitarnogo timba 10F4v1 (thio-10F4v1), gumanitarnogo 10F4v2 and gumanitarnogo 10F4v3 with the following groups of drug-linker: spp-DM1, smcc-DM1, MC-vc-PAB-MMAE; MC-vc-PAB-MMAF; MC-MMAE and MC-MMAF, and these groups of drugs and linkers described herein and in WO 2004/010957, published on 5 February 2004, and WO 2006/034488 published 9 September 2005 (sun which of these patent applications are incorporated herein as reference in full). Before conjugation of antibodies was partially restored by TCEP using standard methods in accordance with the technology described in WO 2004/010957. Partially restored antibodies conjugatively with the above groups, the drug-linker using standard methods in accordance with the technology described in Doronina et al., (2003) Nat. Biotechnol. 21:778-784 and U.S. patent 2005/0238649 A1. In brief, partially restored antibodies combined with groups of drug-linker for conjugation groups of cysteine residues. The conjugation reaction was suppressed and the ADC cleared. The load of the medicinal product (the average number of groups of the drug to the antibody) for each ADC was determined by HPLC. Other suitable linkers to obtain ADC include as non-limiting examples BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, and sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate), and including bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3and BM(PEO)4.

ADC to CD22 also get conjugation to lysine residues of the antibody. Lisini antibodies in turn sulfhydryl group using, for example, reagent trout (Pierce Chemical Co.), as described herein. To obtain ADC recip is installed sulfhydryl groups capable of reacting with linkers or linker molecules-drug. Alternatively, the ADC is obtained by reaction of lysine on the antibody to CD22 with linker SPP (N-Succinimidyl-4-(2'-pyridyldithio)pentanoate, which may already be attached to the molecule drugs or may subsequently react with a molecule drugs, such as maytansinoid. For example, the antibody is modified by reaction with SPP, followed by conjugation with f, as described in Wang, L. et al., Protein Science 14:2436-2446 (2005), which included, so as a reference in full. Residues of lysine on the antibody to CD22 also can be subjected to reaction with linker SMCC (Pierce Chemical Co.), at pH 7-9, so reacting with the amine N-hydroxysuccinimide (NHS ester) SMCC forms a stable amide bond with the antibody. Reactive sulfhydryl maleimido group SMCC is subjected to reaction with the sulfhydryl group of DM1 at a pH of 6.5-7.5 (see Pierce Chemical Co., piercenet.com) with the formation of the ADC. Lysine or cysteine residues is subjected to reaction with a group of the linker-drug obtaining ADC, with an average load of drug approximately 1-8 molecules linker-drug to the antibody alternative 1-6, 1-4, 1-3 or 1-2 molecules linker-drug to the antibody.

This method has received ADC anti-CD22(RFB4)-SMCC-DM1 and anti-GP120-SMCC-DM1, where RFB4-smcc-DM1 was obtained with low (1,95), medium (3,7) and high (6.75 in) load medicinal medium spans the PTO. Was received anti-GP120-smcc-DM1 with high (6,1) load drug. Provided that these ADC are effectivein vivoas shown in example 9 and table 9 herein below.

Example 9: Effect of conjugates of the antibody to CD22-drug

Studies of the determinants of effectivenessin vitro

Identified determinants of the effectiveness of the ADC to CD22 (or TDC) in cell lines lymphoma. It is known that CD22 expressed on the surface of B-cells, internalized upon binding its ligand(s) or antibody (Sato, S. et al., Immunity 5:551-562 (1996)). For testing, does the level of expression on the surface of B-cells CD22 and/or the internalization of CD22 on efficiency and how it is conducted the following researchin vitro.

Surface expression of CD22 person on multiple cell lines lymphoma

Nineteen cell lines lymphomas expressing different amounts of CD22 on the surface, cultivated, and harvested in log-phase. Cells resuspendable in the buffer for FACS washing (PBS; 0.5% of bovine serum albumin; 0.1% of sodium azide)containing 100 μg/ml each of normal mouse IgG and normal human IgG and kept on ice. Approximately 1×106cells/100 μl were stained by APC to huCD22 (mIgG1, clone RFB4, Southern Biotech #9361-11) or isotype IgG1 APC mouse(BD Pharingen #555751) for 30 minutes on ice. Dead cells were stained by 7-AAD (BD Pharmingen #559925). Data were obtained on a flow cytometer BD FacsCalibur™ and analyzed using FlowJo software™. Determination of IC50 for hu10F4v3-SMCC-DM1 or each of the free drug (DM1, MMAF or MMAE) was performed by culturing cells lymphoma, as described above, collecting the cultured cells in log-phase and seeding 5,000 cells in 90 μl of media for culturing per well in 96-well pad. ADC and free drug were diluted serially within the range of detection (starting with 300 µg/ml for the ADC, or 90 nm for the free drugs, and breeding to almost zero amount of the analyzed target). a 10 ál aliquot of the ADC or free medicines were added in duplicate wells containing the cells and incubated for 3 days at 37°C. To each well was added 100 μl of CellTiter Glo™ and incubated for 30 minutes Spent detection of chemiluminescence and the data were analyzed using the software Prism™. The results are presented on figa that high levels of surface CD22 correlate with low IC50 (higher efficiency) hu10F4v3-SMCC-DM1. On figs shown that there is a strong correlation between the sensitivity of cells to the free drug and IC50 ADC.

u> The internalization hu10F4v3-SMCC-DM1 was determined by FACS analysis

In brief, cell lymphomas were stained by standard methods FACS using CD22-FITC (RFB4) in the presence of hu10F4v3-SMCC-DM1, and incubated on ice for 20-30 minutes. To determine levels of CD22 on the cell surface after the initial staining cells were washed in cold environments RPMI/10% FBS was added 200 μl of pre-warmed RPMI/10% FBS and incubated at 37°C for 15 minutes. Added 80 μl of buffer for staining and 20 ál V / V heat inactivated normal mouse serum (HI NMS), followed by incubation on ice for 15 minutes. Added anti-DM1-Alexa-647, incubated on ice for 20-30 minutes and the cells were washed and fixed with 200 μl of a mixture of PBS/1% paraformaldehyde before FACS analysis. For detection of surface and internal staining CD22 after the initial staining cells were washed with cold RPMI/10% FBS was added to pre-warmed RPMI/10% FBS and cells were incubated for 15 minutes at 37°C. Then cells were washed in FACS Wash and recorded Fix A Reagent (Dako™ #k2311) at room temperature for 15 minutes, and the stage was repeated with Fix Reagent B (Dako™). Added buffer for staining and HI NMS and the cell mixture was incubated on ice for 15 minutes was Added Fix Reagent B, and then anti-DM1-Alexa-647 and incubated at room temperature for 20-30 minutes. Cells were washed in FACS Wsh and were fixed with a mixture of PBS/1% paraformaldehyde. Performed FACS analysis of each cell mixture (surface, surface after internalization and internal staining) using the flow cytometer BD FacsCalibur™ and analyzed using FlowJo software™. The results are presented on FIGU, where a high number of internalized DM1 correlate with low IC50 (high efficiency); and fig.6D, which internalized DM1 visualize fluorescent microscopy.

Research on the effectiveness ofin vivo

To test the effectiveness conjugated to a toxin or unconjugated monoclonal antibodies to CD22 in terms of their ability to reduce tumor volumein vivoused the following Protocol.

Each SCID mouse was inoculable subcutaneously in the flank with 2×107cell lines of B-cell lymphoma person. Cell lines human included cell lines lymphoma Bernita human cells Daudi, Ramos and Raji (available from American Type Culture Collection, Manassas, VA, USA), and other B-cell lines, including cells U-698-M cells and Su-DHL-4 (available from DSMZ, Braunschweig, Germany; cells Su-DHL-4 was transfusional luciferase reporter gene), DoHH2 cells (Kluin-Neilemans, H.C. (1991), above), and Granta-519 (cell lymphoma, mantle cell, Jadayel, D.M. et al., Leukemia 11(1):64-72 (1997)), and cells of BJAB-luc (B-cell lymphoblastoid cell line BJAB person, which expresses reportingin luciferase). When the tumors had reached an average tumor volume between 100-200 mm3mice were divided into groups, and at 0 day and night, had intravenous injection conjugated to a toxin antibody or unconjugated antibody, as shown in table 4-16 below.

Conjugates with drug antibody to CD22-maytansine reduce B-cell tumor

Sixty-five SCID mice were subcutaneously injected with 2×107cells of BJAB-luc in the amount of 0.2 ml per mouse in the flank. Cells suspended in HBSS. When the average tumor volume reached 100-200 mm3mice were randomly divided into four groups of 9 mice in each and have a single intravenous (via tail vein) antibodies to CD22 or control antibodies listed in table 4, below.

Table 4
Reduction of tumor volumein vivo
Introduction antibodies
The injected antibodyTIPRCRDose Ab (mg/kg)The DM1 dose (µ g/m2)The ratio of the medicinal product (group drug/Ab)
anti-Her2-smcc-DM19/9004,22003,2
Mu10F4-smcc-DM19/9203,02004,6
hu10F4v2-smcc-DM19/9003,42004,0
hu10F4v19/9003,4--

"TI" is the occurrence of tumors in the latest point in time for each group; the numerator refers to the number with tumor animals, and the denominator refers to the total number of animals. "PR" refers to the number of animals with tumors subjected 50-99% regression from its original volume. "CR" refers to the number of animals that achieved complete remission.

The average tumor volume was subjected to monitoring in each group on introduction within 32 days of th the injection of antibodies. The dimension of the tumor was performed using calipers. Efficiency conjugated with toxin antibodies to CD22 was determined by comparison with control and unconjugated antibodies. The results are presented on figa. Monoclonal antibodies 10F4v1-smcc-DM1 mouse and humanized monoclonal antibodies 10F4v1-smcc-DM1 significantly slowed tumor growth relative to unconjugated antibodies to CD22 and nonspecific control antibody.

Using the same Protocol as described above, to conduct analyses comparing conjugated with toxin humanitariannet 10F4v2 with conjugated with toxin antibody mouse and simple humanized antibody, as indicated in table 5 below.

Table 5
Reduction of tumor volumein vivo
Introduction antibodies
The injected antibodyTIPRCRDose Ab (mg/kg)The DM1 dose (µ g/m2)The ratio of the medicinal product (group drug/Ab)
anti-Her2-smcc-DM1 9/9004,22003,2
Mu10F4-7/912the 4.72002,9
smcc-DM1
hu10F4v2-smcc-DM18/9114,52003,0
hu10F4v29/9004,5--

"TI"is the occurrence of tumors in the latest point in time for each group; the numerator refers to the number with tumor animals, and the denominator refers to the total number of animals. "PR" refers to the number of animals with tumors subjected 50-99% regression from its original volume. "CR" refers to the number of alive is different, achieved complete remission.

The average tumor volume was subjected to monitoring in each group on introduction within 32 days after injection of the antibody. The dimension of the tumor was performed using calipers. Efficiency conjugated with toxin antibodies to CD22 was determined by comparison with control and unconjugated antibodies. The results are presented on figv. Monoclonal antibody 10F4-smcc-DM1 mouse and humanitariannet 10F4v2-smcc-DM1 significantly slowed tumor growth relative to unconjugated antibodies to CD22 and nonspecific control antibody.

Antibody to CD22 conjugatively with DM1 through a linker spp or the linker smcc in accordance with the methods of conjugation described in this document. As a positive control used a simple antibody to CD20 and as negative controls used conjugates with a toxin, an anti-HER2-spp-DM1 and anti-HER2-smcc-DM1. Eighty SCID mice were subcutaneously injected with 2×107cells of BJAB-luc in the amount of 0.2 ml per mouse in the flank. Cells suspended in HBSS. When the average tumor size reached 100-200 mm3mice were randomly divided into six groups of 10 mice each and were performed by intravenous injection of test or control antibody. Doses were repeated once each week for three doses. Cm. table 6.

Table 6
Reduction of tumor volumein vivo
Introduction antibodies
The injected antibodyDose Ab (mg/kg)The DM1 dose (µ g/m2)
anti-Her2-spp-DM1 *4214
anti-Her2-smcc-DM16,9405
anti-CD22-spp-DM1 *5214
anti-CD22-spp-DM12,5107
anti-CD22-smcc-DM110405
simple antibody to CD2210-
* Suitable load drug.
** Suitable load drug.

The average volume of tumors were monitored twice weekly for 3 weeks and then once every week during 8 weeks. Changes in tumor volume over time (figs) shows that anti-CD22-spp-DM1, dosed in the amount of 214 and 107 g/m2 DM1 and anti-CD22-smcc-DM1, dosed in amounts of 405 mg/m2showed a strong and comparable antitumor activity in xenografts tumors BJAB-luc. All groups ADC to CD22 showed a complete response.

Antibodies to CD22, RFB4, 5E8 and 7A2 conjugatively with DM1 through a linker smcc ways conjugation described in this document. As a negative control was used conjugate with the toxin, anti-HER2-smcc-DM1 (referred to interchangeably herein as HER-smcc-DM1 or HER2-smcc-DM1).

Investigated the ability of these antibodies to reduce tumor volume in different xenografts in SCID mice. Cell lines of B-cell lymphoma person used to obtain xenografts tumors in mice, represented Ramos cells and cells of BJAB-luc. For each xenograft SCID mice were subcutaneously injected with 5×106cell B-cell lymphoma person Ramos in the amount of 0.1 ml per mouse in the flank (or 2×107cells of BJAB-luc in 0.2 ml). Cells suspended in HBSS. When the average tumor size reached 100-200 mm3mice were randomly divided into groups of 8-10 mice in each and each mouse spent a single intravenous injection of test or control antibody. Load drug DM1 resulted in 200 mg/m2for each group to obtain the dose DM1. Monitoring the average volume of the whole conducted two times each week for 4 weeks. The results are presented below in tables 7 and 8 and shown on the charts on figa and 8B, respectively.

Table 7
Reduction of tumor volumein vivoxenograft Ramos
Introduction antibodies
The injected antibodyDose Ab (mg/kg)The DM1 dose (µ g/m2)
anti-HER2-smcc-DM14,2200
anti-CD22(7A2)-smcc-DM1the 3.8200
anti-CD22(5E8)-smcc-DM1the 3.8200
anti-CD22(RFB4)-smcc-DM13,2200

Table 8
Reduction of tumor volumein vivo, xenograft BJAB-luc
Introduction antibodies
The injected antibodyDose Ab (mg/kg)The DM1 dose (µ g/m2)anti-HER2-smcc-DM14,22003,2
anti-CD22(7A2)-smcc-DM1the 3.82003,6
anti-CD22(5E8)-smcc-DM1the 3.82003,6
anti-CD22(RFB4)-smcc-DM13,22004,25

These results show that the conjugates of the antibody-drug anti-CD22-smcc-DM1 significantly reduce the amount of B-cell tumors in xenografts Ramos and BJAB-luc relative to a reference antibody or simply by antibodies to CD22.

Investigated the influence of the load of the antibody drug (the average number of conjugated molecules of the drug to the antibody in conjunction antibodies on the ability of the conjugates of the antibody-drug anti-CD22-smcc-DM1 to reduce the volume of a tumor xenografts BJAB-luc mice SCID. One hundred and forty-SCID mice were subcutaneously injected with 2×107cells of BJAB-luc in the amount of 0.2 ml per mouse in the flank. Cells suspended in HBSS. When the average resperatory reached 100-200 mm 3mice were randomly divided into groups of 8-10 mice in each and each mouse spent a single intravenous injection of test or control antibody. Test antibodies were introduced populations of the anti-CD22(RFB4)-smcc-DM1 with relatively low, medium or high loads of the medicinal product (average load drug 1,95, 3,7 or 6,75 conjugated molecules DM1 molecules per antibody, respectively). Simple antibody RFB4 and anti-GP120-smcc-DM1 (high load drug) were the controls. Doses of conjugates of the antibody-drug (test and control) led to a dose level of 5 mg/kg protein. Attaching the linker conjugate antibody was performed via lysine residues. Cm. table 9.

Table 9
Reduction of tumor volumein vivo, xenograft BJAB-luc
The introduction of anti-CD22(RFB4)-smcc-DM1
The injected antibodyDose Ab (mg/kg)The DM1 dose (µ g/m2)The ratio of the medicinal product (Group drug/Ab)
anti-CD22(RFB4) (question is th antibody) 10--
anti-CD22(RFB4)-smcc-DM1 (low)51441,95
anti-CD22(RFB4)-smcc-DM1 (average)52733,7
anti-CD22(RFB4)-smcc-DM1 (high)54976,75
anti-GP120-smcc-DM1 (high)54496,1

When dosing at selected level of protein (5 mg/kg), anti-CD22(RFB4)-smcc-DM1, loaded with high load of the medicinal product (6.75 DM1 molecules per antibody molecule), reduced tumor volume slightly larger than the conjugate antibody-drug with an average load of 3.7, while the impact of conjugate antibody-drug low load drug did not differ from control conjugate or simple antibody. The results are presented in the graph on figure 9.

Conjugates with drug antibody to CD22-auristatin reduce B-cell tumor

Studied the effects of conjugated anticalc CD22 with drug-auristatin MMAF on tumor volume in mouse xenografts. Antibody to CD22(RFB4) and control antibody to GP120 conjugatively with MMAF through a linker MC-vcPAB or linker MC in accordance with the methods described herein. The SCID mice were subcutaneously injected with 5×106of Ramos cells in a volume of 0.2 ml per mouse in the flank. Cells suspended in HBSS. When the average tumor volume reached 100-200 mm3mice were randomly divided into groups of 8-10 mice in each and each mouse spent a single intravenous injection of test or control antibody. The dose of the drug, the load of the medicinal product ratio (for drugs) and the dose of the antibody, mouse input, is presented in table 10.

Table 10
Reduction of tumor volumein vivo, Ramos xenograft
The introduction of conjugate anti-CD22(RFB4)-MMAF
The injected antibodyDose MMAF (µg/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Group drug/Ab)
anti-CD22(RFB4)-MCvcPAB-MMAF4056,64,2
anti-CD22(RFB4)-MC-MMAF4056,94,0
anti-GP120-MCvcPAB-MMAF4055,84,8
anti-GP120-MC-MMAF4055,9the 4.7

Anti-CD22-MC-MMAF showed comparable activity with anti-CD22-MC-vc-PAB-MMAF in Ramos xenografts RA1. The results are presented in the graph on figure 10.

Investigated the effect of conjugates of antibodies to CD22 with drug-auristatin MMAE and DM1 on tumor volume in mouse xenografts. Antibody to CD22(RFB4) and control antibody to GP120 conjugatively with MMAE via a linker MC-vcPAB or linker MC, or with DM1 through a linker smcc in accordance with the methods described herein. The SCID mice were subcutaneously injected with 5×106of Ramos cells in a volume of 0.1 ml per mouse in the flank. Cells suspended in HBSS. As a control, PBS was injected. When the average tumor volume reached 100-200 mm3mice were randomly divided into groups of 8-10 mice each, and each mouse spent a single intravenous injection of test or control antibody. The dose of the drug, the load of the medicinal product (ratio for medicinal medium spans the VA) and the dose of the antibody, input mouse, are presented in table 11.

Table 11
Reduction of tumor volumein vivo, Ramos xenograft
The introduction of conjugate antibodies to CD22(RFB4) MMAE and DM1
The injected antibodyDose MMAE or DM1 (µ g/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Group drug/Ab)
anti-GP120-smcc-DM14056,74,1
anti-CD22(RFB4)-smcc-DM14056,54,25
anti-GP120-MCvcPAB-MMAE4056,0the 4.7
anti-CD22(RFB4)-MCvcPAB-MMAE4056,34,5
PBS---

Anti-CD22-MCvcPAB-MMAE showed potent antitumor activity in xenografts Ramos RA1. Anti-CD22-MCvcPAB-MAE showed higher activity compared with the anti-CD22-smcc-DM1. Control ADC, anti-GP120-MCvcPAB-MMAE showed no significant activity. The results are presented on the chart of figure 11.

Investigated the effect of conjugates of antibodies to CD22 with drug-auristatin MMAF, and DM1 on tumor volume in mouse xenografts. Injected antibody to CD22 hu10F4v2-MC-MMAF, hu10F4v2-smcc-DM1 and thio-10F4v1-MC-MMAF and compared in relation to effects on tumor volume. The control antibody was an anti-Her2-MC-MMAF and anti-Her2-smcc-DM1. The SCID mice were subcutaneously injected with 2×107cells of BJAB-luc in the amount of 0.2 ml per mouse in the flank. Cells suspended in HBSS. When the average tumor volume reached 100-200 mm3mice were randomly divided into groups of 8-10 mice in each and each mouse spent a single intravenous injection of test or control antibody. "Thio" refers to Timaru, as described herein, in which the linker-molecule drugs conjugated to the antibody via a modified cysteine site on the antibody. The dose of the drug, the load of the medicinal product ratio (for drugs) and the dose of the antibody, mouse input, is presented in table 12.

Table 12
Reduction of tumor volumein vivoSinotrans Lancet BJAB-luc
The introduction of conjugate Hu10F4 with MMAF, and DM1
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Group drug/Ab)
Anti-Her2-MC-MMAF1001,16,3
Hu10F4v2-MC-MMAF1002,03,4
Hu10F4v2-MC-MMAF501,03,4
Thio-hu10F4v1-MC-MMAF1004,61,5
Thio-hu10F4v1-MC-MMAF502,31,5
Anti-Her2-smcc-DM 12004,23,2
Hu10F4v2-smcc-DM12004,53,0
Hu10F4v2-smcc-DM11002,3 3,0

ADC Hu10F4v2 showed potent antitumor activity in tumor xenograft BJAB-luc. The results are presented in the graph on Fig.

Using methods as described in the above experiments, investigated the effectiveness of ADC hu10F4v3 with-smcc-DM1 and-MC-MMAF in various xenografts in different doses. The xenografts SuDHL4-luc, DoHH2 and Granta-519 received, as described herein above. When the average tumor volume reached 100-200 mm3mice were randomly divided into groups of 8-10 mice in each and each mouse spent a single intravenous injection of test or control antibody. The dose of the drug, the load of the medicinal product ratio (for drugs) and the dose of the antibody, mouse input, are presented in tables 13A-13C and the results are presented on figa-13C.

Table 13A
Reduction of tumor volumein vivo
The introduction of conjugate Hu10F4v3 with MMAF, and DM1 in the xenografts SuDHL-4-luc
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg)ACCOR is osenia for drugs (Drug/Ab)
Anti-Her2-smcc-DM160011,93,3
Hu10F4v3-smcc-DM160013,62,9
Hu10F4v3-smcc-DM13006,82,9
Anti-Her2-MC-MMAF6009,94,0
Hu10F4v3-MC-MMAF60013,33,0
Hu10F4v3-MC-MMAF3006,63,0

Table 13B
Reduction of tumor volumein vivo
The introduction of conjugate Hu10F4v3 with MMAF, and DM1 in the DoHH2 xenografts
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Drug/Ab)
Anti-Her2-smcc-DM160011,93,3
Hu10F4v3-smcc-DM160011,83,35
Hu10F4v3-smcc-DM13005,93,35
Anti-Her2-MC-MMAF6009,94,0
Hu10F4v3-MC-MMAF60013,13.04 from
Hu10F4v3-MC-MMAF3006,63.04 from
Easy hu10F4v3-13,1-

Table 13C
Reduction of tumor volumein vivo
The introduction of conjugate Hu10F4v3 with MMAF, and DM1 in the xenografts Granta-519
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg) The ratio of the medicinal product (Drug/Ab)
Anti-Her2-smcc-DM13005,93,3
Hu10F4v3-smcc-DM13005,93,35
Hu10F4v3-smcc-DM11502,93,35
Anti-Her2-MC-MMAF300a 4.94,0
Hu10F4v3-MC-MMAF3006,63.04 from
Hu10F4v3-MC-MMAF1503,33.04 from
Easy hu10F4v3-6,6-

ADC to CD22 hu10F4v3-smcc-DM1 and-MC-MMAF showed a marked reduction of the tumor in all tested models with xenografts.

Example 10: Obtaining the modified cysteine antibodies to CD22

Receiving modified with cysteine antibodies to CD22 were performed as described in this document. DNA encoding the antibody 10F43, with the same sequences of variable and constant region that 10F4v2 (light chain, SEQ ID NO: 87; and a heavy chain, SEQ ID NO: 88, pigv)were subjected to mutagenesis methods described herein for modification of the light chain, heavy chain or the Fc region of the heavy chain. DNA encoding light chain were subjected to mutagenesis to replace the cysteine valine at Kabat position 205 in the light chain (position in the sequence 210), as shown in figa (light chain SEQ ID NO: 91 gumanitarnogo antibodies 10F4v3 timba). DNA encoding a heavy chain were subjected to mutagenesis to replace the cysteine alanine at EU position 118 in the heavy chain (position in the sequence 121), as shown in figv (heavy chain SEQ ID NO: 92 gumanitarnogo antibodies 10F4v3 timba). Fc-region was subjected to mutagenesis to replace the cysteine serine at EU position 400 in the heavy chain Fc region (the position in the sequence 403)as shown on figs (heavy chain SEQ ID NO: 93).

Receiving modified with cysteine antibodies to CD22 for conjugation through the recovery and re-oxidation

Full-length, modified cysteine monoclonal antibodies to CD22 (timeby)expressed in CHO cells, dissolved in 500 mm sodium borate and 500 mm sodium chloride at about pH 8.0 and restore approximately 50-100-fold and what BITKOM 1 mm TCEP hydrochloride (Tris(2-carboxyethyl)phosphine; Getz et al., (1999) Anal. Biochem. Vol 273: 73-80; Soltec Ventures, Beverly, MA) for approximately 1-2 hours at 37°C. the Recovered Tioman dissolved and placed on a column HiTrap S in 10 mm sodium acetate, pH 5 and elute through PBS containing 0.3 M sodium chloride. Suirvey restored Tioman 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. The oxidation of atmospheric air can also be effective. The buffer is replaced by elution through G25 resin and elute through PBS with 1 mm DTPA. The amount of thiol/Ab checked by determining the concentration of the recovered antibodies, based on the absorption at 280 nm of the solution, and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and the determination of the absorption at 412 nm.

Example 11: Obtaining the modified cysteine conjugates of the antibody to CD22-drug conjugation modified with cysteine antibodies to CD22 and intermediates drug-linker

After ways to restore and re-oxidation of example 10 modified cysteine antibody to CD22 dissolved in PBS (phosphate-buffered saline) and cooled on ice. About 1.5 molar equivalents relative to the resulting design cysteines on antibody intermediate Lek is rstone tool auristatin-linker, such as MC-MMAE (maleimidomethyl-monomethyl auristatin E), MC-MMAF, MC-val-cit-PAB-MMAE, or MC-val-cit-PAB-MMAF with reactive thiol functional group, such as maleimido, dissolved in DMSO, diluted with acetonitrile and water and add to chilled restored, re-oxidized antibody in PBS. Approximately one hour add excess maleimide for quenching the reaction and kupirovaniya any unreacted tylnej groups of antibodies. The reaction mixture is concentrated by ultrafiltration centrifugation and modified by cysteine conjugate antibody to CD22-drug cleanse and absoluut by elution through G25 resin in PBS, filtered through the 0.2-μm filters under sterile conditions, and frozen for storage.

Getting Tioman hu 10F4v3 HC(A118C)-BMPEO-DM1 was carried out as follows. The free cysteine on timebe hu 10F4v3 HC(A118C) modified bis-multimediacenter BM(PEO)4 (Pierce Chemical), leaving unreacted group maleimido on the surface of the antibody. This is carried out by dissolving BM(PEO)4 50% mixture of ethanol/water to a concentration of 10 mm and adding a tenfold molar excess of BM(PEO)4 to the solution containing ThioFab hu4D5Fabv8-(V110C) in phosphate-buffered saline at a concentration of approximately 1.6 mg/ml (10 micromolar) and allowing to react for 1 hour. Excess BM(PEO)4 was removed by gel-filtration (column HiTrap, Parmacia) in 30 mm citrate, pH 6, 150 mm NaCl buffer. To the intermediate connection hu4D5Fabv8-(VI10C) ThioFab-BMPEO was added approximately 10-fold molar excess of DM1 dissolved in dimethylacetamide (DMA). Also for the dissolution of the reagent group of medicines you can use dimethylformamide (DMF). The reaction mixture was allowed to react over night before gel filtration or dialysis in PBS to remove unreacted medicines. To remove high molecular weight aggregates and the production of purified Tioman hu 10F4v3 HC(A118C) Tioman-BMPEO-DM1 used gel filtration on columns S200 in PBS.

Through these protocols received control HC (A118C) MAb-MC-MMAF, control HC Tioman-MC-MMAF, control HC Tioman-MCvcPAB-MMAE and control HC Tioman-BMPEO-DM1.

As described above, received and tested the following modified cysteine conjugates antibody to CD22-drug:

thio hu thio-HC-10F4v3-MC-MMAF by conjugation of A118C thio hu 10F4v3 and MC-MMAF;

thio hu thio-HC-10F4v3-MC-val-cit-PAB-MMAE by conjugation of A118C thio hu 10F4v3 and MC-val-cit-PAB-MMAE;

thio hu HC-10F4v3-bmpeo-DM1 by conjugation of A118C thio hu HC-10F4v3 and bmpeo-DM1;

thio hu LC-10F4v3-MC-val-cit-PAB-MMAE by conjugation V205C thio hu LC-10F4v3 and MC-val-cit-PAB-MMAE; and

thio hu Fc-10F4v3-MC-val-cit-PAB-MMAE by conjugation S400C thio hu Fc-10F4v3 and MC-val-cit-PAB-MMAE.

Example 12: Characterization of the affinity of binding the conjugate modi is economony cysteinemia Tioman-drug to the antigen on the cell surface

The binding affinity of the conjugates thio hu 10F4v3-drug to CD22 expressed on the cells of BJAB-luc was determined by FACS analysis. In brief, approximately 1×106cells in 100 μl were subjected to contacting with varying amounts of one of the following conjugates antibodies to CD22 Tioman-drug: thio hu LC(V205C) 10F4v3-MCvcPAB-MMAE, thio hu Fc(S400C) 10F4v3-MCvcPAB-MMAE, thio hu HC(A118C) 10F4v3-MCvcPAB-MMAE, thio hu HC(A118C) 10F4v3-MC-MMAF, or thio hu HC(A118C) 10F4v3-BMPEO-DM1 (see figa-18E, respectively). Antibody to CD22, contacting the cell surface, were subjected to detection using biotinylated anti-huFc goats plus streptavidin-PE. Graphics on figa-18E indicate that the binding of antigen was approximately the same for all the tested conjugates Tioman-drug.

Example 13: the Analysis of reduction of tumor volumein vivothrough conjugates Tioman to CD22-drug

The ability of the conjugates Tioman-drug obtained in example 11, to reduce the amount of B-cell tumors in xenograft models tested in accordance with the method described in example 9, in the present document. The SCID mice with xenograft tumors from cells Granta-519, control conjugates and conjugates humanitariannet 10F4v3 Tioman to CD22-the drug was administered at 0 day in doses is provided in table 14, below. Control HC(A118C) Tioman represented antibody to HER2 4D5.

Table 14
Reduction of tumor volumein vivo
The introduction of conjugate Thio Hu10F4v3 MMAE and MMAF in xenografts Granta-519
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Drug/Ab)
Thio Control HC(A118C)-MC-MMAF1003,991,65
Thio Control HC(A118C)-MCvcPAB-MMAE1004,331,55
Thio 10F4v3-HC(A118C)-MC-MMAF1003,411,95
Thio 10F4v3-LC(V205C)-MCvcPAB-MMAE1004,231,6
Thio 10F4v3-HC(A118C)-MCvcPAB-MMAE1003,761,8
Thio 10F4v3-Fc(S400C)-MCvcPAB-MMAE1004,231,6

The results of this experiment are presented in Fig. Introduction conjugates Tioman-drug thio 10F4v3-LC(V205C)-MCvcPAB-MMAE and thio 10F4v3-HC(A118C)-MCvcPAB-MMAE in doses shown in table 14, caused a decrease in the average tumor volume throughout the study.

Additional conjugates Tioman-the drug is tested in xenografts Granta-519 mice (CB17 SCID using the specified Protocol, but with different doses of the drug. The control antibody or control Tioman represented antibody to HER2 4D5 or Tioman HC(A118C). The results are presented in table 15, below.

3,2
Table 15
Reduction of tumor volumein vivo,
The introduction of conjugate Thio Hu10F4v3 MMAE, MMAF, and DM1 in the xenografts Granta-519
The injected antibodyDose MMAF or DM1 (µg/m2)Dose Ab (mg/kg)The ratio of the medicinal product (Drug/Ab)
10F4v3-MC-MMAF1503,1
Thio Control HC(A118C)-BMPEO-DM130010,31,9
Thio 10F4v3-HC(A118C)-BMPEO-DM11505,21,9
Thio 10F4v3-HC(A118C)-BMPEO-DM130010,41,9
Thio Control HC(A118C)-MCvcPAB-MMAE1506,51,55
Thio 10F4v3-HC(A118C)-MCvcPAB-MMAE1505,31,9
Thio 10F4v3-HC(A118C)-MCvcPAB-MMAE752,71,9
Thio Control HC(A118C)-MC-MMAF1505,21,9
Thio 10F4v3-HC(A118C)-MC-MMAF1505,11,95
Thio 10F4v3-HC(A118C)-MC-MMAF752,61,95

the results of this experiment are presented in figa. The introduction of conjugate Tioman-drug thio 10F4v3-HC(A118C)-MCvcPAB-MMAE in the amount of 150 and 75 mg/m2resulted in reduction of tumor volume throughout the study. In the same study, in groups with each dosage was determined by the percentage change of body weight during the first 7 days. The results presented in the graph on FIGU, indicate that the introduction of these conjugates Tioman-the drug does not cause weight loss during this time.

In a similar study using the same study Protocol with xenografts, as described in the above examples, researched various TDC and the input dose efficiency TDC in xenografts follicular lymphoma DOHH2 mice (CB17 SCID. TDC and dose are presented in table 16, below.

On figs presents a graph which shows the change in mean tumor volume over time in xenograft follicular lymphoma DOHH2 mice (CB17 SCID, which was introduced the same TDC to CD22 with a heavy chain A118C, but in higher doses, as shown in table 16. It turned out that TDC to CD22 10F4v3-HC(A118C)-MCvcPAB-MMAE is the most effective among the tested substances in this study. However, at higher dose levels in this experiment was marked by some effectiveness controls the STI-HER2-HC(A18C)-MCvcPAB-MMAE. Probably, this activity inherent in the release of drug from the ADC in the bloodstream. Test substance to CD22 hu10F4-HC(A118C)-MC-MMAF and-BMPEO-DM1 showed intermediate performance and consistent with the high stability of these linkers, nesviazana control anti-HER2 showed little activity. On fig.20D shows a graph of the percentage change in weight of mice in the study xenograft DOHH2, showing that there is no significant weight changes during the first 14 days of the study.

Example 14: the safety of the conjugates of the antibody to CD22-the drug in rats and cynomolgus macaques

Antibody hu10F4 to CD22 cross-reacts with CD22 cynomolgus (cyno) macaques with an affinity equivalent to human CD22. Antibody hu10F4 to CD22 does not cross react with CD22 rats. As a result, evaluated independent from the target and dependent on the target safety and toxicity of the conjugates of the antibody to CD22 drug in rats and cyno, respectively.

Safety and toxicity in rats

For studies of the safety and toxicity in rats conducted two studies. In one study, rats intravenously were dosed out for 1 day conjugates hu10F4v3-SMCC-DM1-SPP-DM1, MC-vc-PAB-MMAE or-MC-MMAF, in which the drug was linked via split (-vc - or-spp-) or necessasary (MC or SMCC (also called the AK MCC)linker. As a control were injected media. Collection of blood samples was carried out on 5 days for pharmacokinetic analysis and for 12 hours (at autopsy). Clinical observation and recording of body weight was performed at least three times a week. As an indicator of toxicity monitored serum AST (aspartate aminotransferase). Serum AST levels were increased in 5 days relative to 0 days in rats that were dosed out 20 mg/kg hu10F4v3-vcMMAE and hu10F4v3-SPP-DM1 containing biodegradable linkers (figa). The levels of neutrophils was increased in 5 days relative to 0 days in rats that were dosed out 20 mg/kg hu10F4v3-MC-MMAF or hu10F4v3-MCC-DM1 (darassalam linkers, pigv). The levels of neutrophils were reduced by 5 days relative to 0 days in rats that were dosed out hu10F4v3-vc-MMAE or hu10F4v3-SPP-DM1. Increased serum AST and reduced neutrophils in rats that were dosed out ADC containing degradable linkers, indicated high toxicity of such ADC.

In the same study, rats of six animals per group were dosed out of 20, 40 or 60 mg/kg hu10F4v3-MC-MMAF or hu10F4v3-SMCC-DM1 for 1 day and were subjected to monitoring for twelve days. In animals that were dosed out hu10F4v3-MC-MMAF, was not identified the following indicators: reduced body weight, increased serum liver enzymes, reduced platelet count or a decrease in the number of neutrophils. The kr is s, which they dosaged hu10F4v3-SMCC-DM1, observed reversible reduction of body weight and reversible increase in serum liver enzymes at dose levels of 40 and 60 mg/kg, while at doses of 60 mg/kg was observed reversible reduction in the number of neutrophils and a temporary reduction in platelet count.

Safety and toxicity in cynomolgus macaques

To assess the safety and toxicity of the ADC to CD22 in the model in primates thirty cyno monkeys were divided into the following groups according to the introduction: the control in the form of a carrier (6 animals), hu10F4v3-smcc-DM1 at doses of medicines 2, 4 and 6 mg/m2(equivalent to 0, 10, 20 and 30 mg/kg dose on antibody; 4 animals per group to dosing), and hu10F4v3-MC-MMAF at doses of 2, 4 and 6 mg/m2(4 animals per group for dosing). Animals spent intravenous dosing for 1 day and 22 days. The animals were evaluated by the change in body mass, food intake and signs of pathology. Blood samples were collected and evaluated for Toxicological evaluation, pharmacodynamic effects and effects of antibodies against the drug. Half of the animals in each group were killed every 25 days and 43 days, and collected tissue samples.

There were not observed significant changes in body weight in either group at ADC. The levels of serum liver enzymes AST (aspartate aminotransferase), ALT (aminotransferase) and GGT (gamma-glutamyltransferase the basics) was estimated in accordance with standard procedures known in their respective fields. Reversible increase in serum liver enzymes was observed in animals that were dosed out of 30 mg/kg in the case of each ADC, although ALT was elevated in the DM1 group, AST and GGT were elevated in the group of MMAF. Degeneration of the sciatic nerve was minimal to mild in the DM1 group in 2 of 4 animals at a dose of 20 mg/kg and in 4 out of 4 animals at a dose of 30 mg/kg Degeneration of the sciatic nerve was minimal in the group MMAF 1 of 4 animals at a dose of 30 mg/kg of Tissue from various organs examined microscopically. Two of the four animals in group 30 mg/kg MMAF had lung injury of unknown origin, whereas it was not detected in any of the animals in the group DM1.

Depletion of peripheral B-cells by ADC hu10F4v3-MC-MMAF and-SMCC-DM1 was determined by measuring the levels of CD20 cells+blood for 43 days in cyno monkeys who have been dosing at 0 days and 22 days. Blood collected periodically during the study, was assessed by FACS using a fluorescently labeled antibody to CD20. ADC to CD22 MMAF, and DM1 leads to the depletion of the cyno peripheral B-cells, as shown in figa (group MMAF) and figv (group DM1). There were no significant effects MMAF ADC or DM1 to other populations of lymphocytes, as shown in figa and 23B, which shows that there was no significant depletion of the notches CD4 +during the same period of time.

Hu10F4v3-SMCC-DM1 resulted in depletion of B-cells its germinative center almond-shaped glands in cyno monkeys relative to the control, as shown in the microphotographs on figa and 24B. Illustrative its germinative centers are circled in figa. The complete disappearance of B-cells its germinative center was observed at the dose level of 10 mg/kg, as shown in figv. The same results were obtained after the introduction of the ADC hu10F4v3-MC-MMAF in the same conditions.

Hu10F4v3-MC-MMAF, dosed at 10 mg/kg, resulted in depletion of fissile B-cells in its germinative centers of the follicles of the spleen in cyno monkeys. Cm. the chart on figa and microphotographs of tissues on figv and 25C. The same results were obtained when tested ADC hu10F4v3-SMCC-DM1 in the same conditions. Its germinative centers are seen as dark areas on FIGU when applying staining of Ki-67 and as unpainted areas, surrounded by dark areas of the painting is labeled amenable to detection label antibody to IgD on fig.25D. The loss of its germinative centers due to the depletion of B-cells its germinative center by means of anti-10F4v3-MC-MMAF presented on figs and 25E. Thus, these protiwaritmicescie drugs affect proliferating population of B-cells.

The following hybridoma was deposited in the American Type Culture Collection PO ox 1549, Manassas, VA, 20108, USA (ATCC):

Cell lineRegistration number ATCCDate Deposit
Hybridoma 10F4.4.1PTA-7621on may 26, 2006
Hybridoma 5E8.1.8PTA-7620on may 26, 2006

These samples were deposited under the terms of the Budapest Treaty on the international recognition of the Deposit of microorganisms for purposes of patent procedure and its normative documents (Budapest Treaty). This assures maintenance of a viable deposited sample within 30 years from the date of Deposit. These cell lines will be available in the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of cell lines the company after the issue of the relevant U.S. patent or after the publication for the society of any patent application U.S. or another country, depending on what happens earlier, and guarantees the availability of cell lines to a person determined by the Commissioner of the U.S. patent and trademark office for assigned names in accordance with 5 USC §122 and the rules for commissioners according to him (including 37 CFR §1.14 with particular reference to 886 OG 638).

The patent of this application agrees that if the deposited cell line will be lost or destroyed when cultivated under suitable conditions, they will be immediately replaced when you notice a sample of the same cell line. Availability of the deposited cell lines should not be construed as permission for the practical application of the invention in violation of the rights issued under the authority of any government in accordance with its patent laws.

Although the above invention is described in some detail to illustrate examples for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. Describe all of the cited patent and scientific literature sources are incorporated herein by reference in full.

1. Antibody (Ab)binding to CD22, which contains:
(1) HVR-L1 containing the amino acid sequence selected from SEQ ID NO:9, 10, 19-23, 32 and 33, and
(2) the HVR-H1 containing the amino acid sequence of SEQ ID NO:2;
(3) the HVR-H2 containing the amino acid sequence of SEQ ID NO:4;
(4) the HVR-H3 containing the amino acid sequence of SEQ ID NO:6;
(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:12; and
(6) the HVR-L3 containing amino acid posledovatel is of SEQ ID NO:14, moreover, the antibody binds to an epitope in the region of CD22 located from amino acid 22 to amino acids 240 sequence SEQ ID NO:27.

2. The antibody according to claim 1, containing HVR-L1 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:10.

3. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:9.

4. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:19.

5. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:20.

6. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:21.

7. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:22.

8. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:23.

9. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:32.

10. The antibody according to claim 1, where the HVR-L1 contains the amino acid sequence of SEQ ID NO:33.

11. The antibody according to claim 1, additionally containing at least one frame region selected from a consensus framework region of the heavy chain (VH) subgroup III consensus framework region of the light chain (VL) subgroup I.

12. The antibody according to claim 1, containing the variable domain of the heavy chain that is at least 90% identical to the amino acid sequence with sequential matching what telectu, selected from SEQ ID NO:16.

13. The antibody according to claim 1, containing the variable domain light chain that is at least 90% identical to the sequence from amino acid sequence selected from SEQ ID NO:17.

14. The antibody according to claim 1, containing the variable domain light chain that is at least 90% identical to the sequence from amino acid sequence selected from SEQ ID NO:18.

15. The antibody according to claim 1, containing the variable domain of the heavy chain, containing one, two, three, or four amino acid sequence of framework region selected from SEQ ID NO:1, 3, 5 and 7.

16. The antibody according to claim 1, containing the variable domain of the light chain containing one, two, three, or four amino acid sequence of framework region selected from SEQ ID NO:8, 11, 13 and 15.

17. The antibody according to item 12, containing the variable domain light chain that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:17.

18. The antibody according to item 12, containing the variable domain light chain that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:18.

19. The antibody according to claim 1, containing a heavy chain that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:88.

20. The antibody according to claim 1, containing a light chain that is at least 90% of sex is in amino acid sequence, selected from SEQ ID NO:87.

21. The antibody according to claim 1, containing a heavy chain with the amino acid sequence SEQ ID NO:88 and a light chain with the amino acid sequence SEQ ID NO:87.

22. Antibody binding to CD22, containing the variable domain of the heavy chain that is at least 90% identical to the amino acid sequence of SEQ ID NO:16, and
variable domain light chain selected from the group consisting of a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:17, and a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:18,
moreover, the antibody binds to an epitope in the region of CD22,
located from amino acid 22 to amino acids 240 sequence SEQ ID NO:27.

23. The antibody according to claim 1, produced by hybridomas with the inventory number of ATSS MOUTH-7621 (10F4.4.1).

24. The antibody that binds to an epitope in the region of CD22 located from amino acid 22 to amino acid 240 sequence SEQ ID NO:27.

25. The antibody according to claim 1, which is humanized.

26. The antibody according to item 22, which is humanized.

27. The antibody according to claim 1, where CD22 CD22 is a mammal.

28. The antibody according to item 27, where CD22 selected from rodent CD22 and CD22 primates.

29. The antibody according p where CD22 CD22 is a person.

30. The antibody according to item 22, where CD22 p is ecstasy a CD22 mammal.

31. The antibody according to item 30, where CD22 selected from rodent CD22 and CD22 primacy.

32. The antibody according p where CD22 CD22 is a person.

33. Polynucleotide encoding the antibody according to claim 1.

34. The expression vector containing polynucleotide on p.

35. A host cell containing a vector according to clause 34, producing the antibody according to claim 1.

36. A host cell according p, which is eukaryotic.

37. A host cell according p, which is the cell SNO.

38. A method of obtaining antibodies against CD22, including:
a) culturing the host cell according to item 30 under conditions suitable for expression of polynucleotide encoding the antibody, and
b) isolation of antibody.

39. The antibody according to claim 1, where CD22 is expressed on the cell surface.

40. The antibody according to § 39, where the cage is a cage.

41. The antibody according to item 22, where CD22 is expressed on the cell surface.

42. The antibody according to paragraph 41, where the cage is a cage.

43. The antibody according p where In the cell is associated with impaired proliferation of b-cells.

44. The antibody according to item 43, where the violation of the proliferation of b-cells is a malignant tumor.

45. The antibody according to item 43, where the violation of the proliferation of b-cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic is th lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

46. The antibody according to § 42, where In the cell is associated with impaired proliferation of b-cells.

47. The antibody according to item 46, where the violation of the proliferation of b-cells is a malignant tumor.

48. The antibody according to item 46, where the violation of the proliferation of b-cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

49. The antibody according to any one of claims 1 to 13, 15-22 or 27-30, representing a monoclonal antibody.

50. The antibody according to claim 1, which represents the antibody fragment selected from Fab fragments, Fab'-SH, Fv, scFv, or (Fab')2.

51. The antibody according to claim 1, which is humanized.

52. The antibody according to claim 1, which represents the antibody is human.

53. The antibody according to item 22, which represents a monoclonal antibody.

54. The antibody according to item 53, representing an antibody fragment selected from Fab fragments, Fab'-SH, Fv, scFv, or (Fab')2.

55. The antibody according to item 53, which is humanized.

56. The antibody according to claim 5, representing a human antibody.

57. The antibody according to claim 1, binding to the same epitope as an antibody selected from ATS MOUTH-7621 (10F4.4.1); and the antibody containing the sequence of the heavy chain SEQ ID NO:88 and sequence of the light chain of SEQ ID NO:87.

58. The antibody according to item 22, bind to the same epitope as an antibody selected from ATS MOUTH-7621 (10F4.4.1); and the antibody containing the sequence of the heavy chain SEQ ID NO:88 and sequence of the light chain of SEQ ID NO:87.

59. Method detection CD22 in a biological sample, including:
bringing a biological sample into contact with an antibody according to claim 1 under conditions that allow the binding of an antibody to CD22, and detection is also formed complex between the antibody and CD22,
where the presence of CD22 in a biological sample is determined by the formation of a complex between the antibody and CD22.

60. The method according to p, where the biological sample taken from a patient with suspected disorders of proliferation of b-cells.

61. The method according to p where violation of proliferation In cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells manta rays who Inoi zone.

62. Method detection CD22 in a biological sample, including:
bringing a biological sample into contact with an antibody according to item 22 under conditions that allow the binding of an antibody to CD22, and detection is also formed complex between the antibody and CD22,
where the presence of CD22 in a biological sample is determined by the formation of a complex between the antibody and CD22.

63. The method according to item 62, where the biological sample taken from a patient with suspected disorders of proliferation of b-cells.

64. The method according to p where violation of proliferation In cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

65. Immunoconjugate directed against CD22, containing the antibody according to claim 1, covalently linked to a cytotoxic agent.

66. Immunoconjugate directed against CD22, containing the antibody according to item 22, covalently linked to a cytotoxic agent.

67. Immunoconjugate on p, where the cytotoxic agent is selected from a toxin, a chemotherapeutic molecule drug, an antibiotic, a radioactive isotope of the nucleus and imicheskogo enzyme.

68. Immunoconjugate on p having the formula

where: (a) Ab is the antibody of claim 1;
(b) L is a linker;
(c) D is a molecule drugs; and
(d) p is from 1 to about 20.

69. Immunoconjugate on p, where L is selected from 6-maleimidomethyl (MS), maleimidomethyl (Mr), 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-iodates)aminobenzoate (fairs are forthcoming-Siab).

70. Immunoconjugate on p, where D is selected from auristatin and dolastatin.

71. Immunoconjugate according to item 70, where D is a molecule drugs formula DEor DF:

and where each R2and R6represent methyl, each R3and R4
represent isopropyl, R7represents sec-butyl, each R8independently selected from CH3, O-CH3HE and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8-alkyl or -(CH2)2-O-(CH2)2-O-(CH2)2-O-CH3; and R18represents-C(R8)2-C( 8)2-aryl; and (d) p is in the range from approximately 1 to 8.

72. Immunoconjugate on p, possessing activity for the destruction of cells in vitro or in vivo.

73. Immunoconjugate on p, where the linker is linked to the antibody through Tilney group antibodies.

74. Immunoconjugate on p, where the linker can be broken down under the action of proteases.

75. Immunoconjugate on p, where the linker contains the dipeptide val-cit.

76. Immunoconjugate on p, where the linker contains p-aminobenzyl unit.

77. Immunoconjugate on p, where the linker contains 6-maleimidomethyl.

78. Immunoconjugate on p, where the drug is selected from MMAE and MMAF.

79. Immunoconjugate on p, where the drug is MMAE.

80. Immunoconjugate on p, where the drug is a MMAF.

81. Immunoconjugate on p, where the cytotoxic agent is selected from a toxin, a chemotherapeutic molecule drug, an antibiotic, a radioactive isotope and nucleotidase enzyme.

82. Immunoconjugate on p having the formula

where (a) Ab is the antibody according to item 22;
(b) L is a linker;
(c) D is a molecule drugs; and
(d) p is from 1 to about 20.

83. Immunoconjugate on p, where L is selected from 6-maleimido is a (MS), maleimidomethyl (Mr), 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-iodates)aminobenzoate (fairs are forthcoming-Siab).

84. Immunoconjugate on p, where the linker L may disintegrate under the action of proteases.

85. Immunoconjugate on p, where L contains the dipeptide val-cit.

86. Immunoconjugate on p, where L contains p-aminobenzyl unit.

87. Immunoconjugate on p, where p is aminoaniline unit is a p-aminobenzeneboronic (RAV).

88. Immunoconjugate on p, where L contains 6-maleimidomethyl (MS).

89. Immunoconjugate on p, where the linker contains 6-maleimidomethyl and p-aminobenzeneboronic.

90. Immunoconjugate on p having the formula

where L is a linker and R is in the range from 2 to 5.

91. Immunoconjugate on p, where L contains val-cit.

92. Immunoconjugate on p, where L contains MILLISECONDS.

93. Immunoconjugate on p, where L contains the RABBI.

94. Immunoconjugate on p, where L contains the MS-RAV.

95. Immunoconjugate on p having the formula Ab-(L-MMAE)p, where L is a linker and R is in the range from 2 to 5.

96. Immunoconjugate on p, where L contains val-cit.

97. Immunoconjugate on p, where L contains MILLISECONDS.

98. Immunoconjugate on p, where L contains the RABBI.

99. Immunoconjugate on p, where L contains the MS-RAV.

100. Immunoconjugate on p having the formula Ab-(L-MMAF)p, where L is a linker and R is in the range from 2 to 5.

101. Immunoconjugate in item 100, where L contains val-cit.

102. Immunoconjugate in item 100, where L contains MILLISECONDS.

103. Immunoconjugate in item 100, where L contains the RABBI.

104. Immunoconjugate in item 100, where L contains the MS-RAV.

105. Immunoconjugate on p having the formula Ab-(L-MMAF)p, where L is a linker and R is in the range from 2 to 5.

106. Immunoconjugate on p, where L contains val-cit.

107. Immunoconjugate on p, where L contains MILLISECONDS.

108. Immunoconjugate on p, where L contains the RABBI.

109. Immunoconjugate on p, where L contains the MS-RAV.

110. Immunoconjugate on p, where D is maytansinoid.

111. Immunoconjugate on p, where D is selected from DM1, DM3 and DM4.

112. Immunoconjugate on p, possessing activity for the destruction of cells in vitro or in vivo.

113. Immunoconjugate on p, where the linker is linked to the antibody through Tilney group antibodies.

114. Immunoconjugate on p, where the linker L is selected from N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and N-Succinimidyl-(4-iodates)aminobenzoate (fairs are forthcoming-Siab).

115. Immunoconjugate in § 111, where the drug is from the Oh DM1.

116. Immunoconjugate in § 111, where L contains SPP.

117. Immunoconjugate in § 111, where L contains SMCC.

118. Immunoconjugate in § 111, where p is from 2 to 4.

119. Immunoconjugate in § 111, where p is from 3 to 4.

120. Immunoconjugate on p, where D is maytansinoid.

121. Immunoconjugate on p, where D is selected from DM1, DM3 and DM4.

122. Immunoconjugate on p, possessing activity for the destruction of cells in vitro or in vivo.

123. Immunoconjugate on p, where the linker is linked to the antibody through Tilney group antibodies.

124. Immunoconjugate on p, where the linker L is selected from N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and N-Succinimidyl-(4-iodates)aminobenzoate (fairs are forthcoming-Siab).

125. Immunoconjugate on p, where the drug is a DM1.

126. Immunoconjugate on p, where L contains SPP.

127. Immunoconjugate on p, where L contains SMCC.

128. Immunoconjugate on p, where p is from 2 to 4.

129. Immunoconjugate on p, where p is from 3 to 4.

130. Pharmaceutical composition for the treatment of disorders of cell proliferation In cells containing an effective amount immunoconjugate on p and a pharmaceutically acceptable carrier.

131. A method of treating disorders of proliferation of b cells, comprising an introduction to the individual an effective amount of a pharmaceutical is the first song on p.

132. The method according to p where violation of proliferation In cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

133. Pharmaceutical composition for the treatment of disorders of cell proliferation In cells containing an effective amount immunoconjugate on p and a pharmaceutically acceptable carrier.

134. A method of treating disorders of proliferation of b cells, comprising an introduction to the individual an effective amount of the pharmaceutical composition according p.

135. The method according to p, where b-cell proliferative violation is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

136. Pharmaceutical composition for the treatment of disorders of cell proliferation In cells containing an effective amount immunoconjugate on PP, 66, 67 or 81, pharmace is almost acceptable carrier.

137. A method of treating disorders of proliferation of b cells, comprising an introduction to the individual an effective amount of the pharmaceutical composition according p.

138. The method according to p where violation of proliferation In cells selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

139. A method of inhibiting the proliferation of b-cells, including effects on cell immunoconjugates on p under conditions that allow immunoconjugate contact CD22.

140. The method according to p, where the proliferation of b-cells is associated with disorder is selected from lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

141. The method according to p where In the cell is a xenograft.

142. The method according to p, where the impact occurs in vitro.

143. The method according to p, where the impact of proishodit in vivo.

144. Engineered antibodies with cysteine substitutions, which binds to CD22, containing one or more free cysteine residues with the amount of thiol reactivity in the range from 0.6 to 1.0, where the engineered antibodies with cysteine substitutions get method comprising substituting one or more amino acid residues of a parent antibody cysteine residue, where the specified antibody contains:
(1) HVR-L1 containing the amino acid sequence selected from SEQ ID NO:9, 10, 19-23, 32 and 33, and
(2) the HVR-H1 containing the amino acid sequence of SEQ ID NO:2;
(3) the HVR-H2 containing the amino acid sequence of SEQ ID NO:4;
(4) the HVR-H3 containing the amino acid sequence of SEQ ID NO:6;
(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:12; and
(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO:14.

145. Engineered antibodies with cysteine substitutions on p, which is more reactive in relation to thiol reagent than the parent antibody.

146. Engineered antibodies with cysteine substitutions on p, where the method further includes determining the reactivity of thiol groups engineered antibodies with cysteine substitutions by the reaction of the engineered antibodies with cysteine substitutions with thiol re the Ghent;
where engineered antibodies with cysteine substitutions is more reactive in relation to thiol reagent than the parent antibody.

147. Engineered antibodies with cysteine substitutions on p, where one or more free cysteine residues are located in the light chain.

148. Engineered antibodies with cysteine substitutions on p, where the antibody is immunoconjugate containing engineered antibodies with cysteine substitutions, covalently linked to a cytotoxic agent.

149. Engineered antibodies with cysteine substitutions on p, where the cytotoxic agent is selected from a toxin, a chemotherapeutic molecule drug, an antibiotic, a radioactive isotope and nucleotidase enzyme.

150. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the exciting label, the label for detection or solid substrate.

151. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the exciting label is Biotin.

152. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to a label for detection - fluorescent dye.

153. Engineered antibodies with cysteine substitutions on p, the de fluorescent dye selected from dyes fluoresceine type, radominova type of dansili, lissamine, Chianina, phycoerythrin, Texas red, and their analogues.

154. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the radionuclide label for detection, selected from3H,11C,14C,18F,32P,35S64Cu68Ga86Y99Tc111In123I124I125I131I133Xe177Lu,211At and213Bi.

155. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to a label for detection - chelating ligand.

156. Engineered antibodies with cysteine substitutions on p, where the chelating ligand selected from DOTA, DOTP, DOTMA, DTPA and THETA.

157. Engineered antibodies with cysteine substitutions that communicates with CD22, containing one or more free cysteine residues with the amount of thiol reactivity in the range from 0.6 to 1.0, where the engineered antibodies with cysteine substitutions get method comprising substituting one or more amino acid residues of a parent antibody cysteine residue, where the antibody contains:
variable domain of the heavy chain that is at least 90% identical to the amino acid sequence of SEQ ID NO:16, and the variable domain of the light chain selected from the group consisting of a sequence is eljnosti, which at least 90% identical to the amino acid sequence of SEQ ID NO:17, and a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:18.

158. Engineered antibodies with cysteine substitutions on p, which is more reactive in relation to thiol reagent than the parent antibody.

159. Engineered antibodies with cysteine substitutions on p, where the method further includes determining the reactivity of thiol groups engineered antibodies with cysteine substitutions by the reaction of the engineered antibodies with cysteine substitutions with thiol reagent;
where engineered antibodies with cysteine substitutions is more reactive in relation to thiol reagent than the parent antibody.

160. Engineered antibodies with cysteine substitutions on p, where one or more free cysteine residues are located in the light chain.

161. Engineered antibodies with cysteine substitutions on p, where the antibody is immunoconjugate containing engineered antibodies with cysteine substitutions, covalently linked to a cytotoxic agent.

162. Engineered antibodies with cysteine substitutions on p, where the cytotoxic agent is selected is from the toxin, chemotherapeutic molecule drug, an antibiotic, a radioactive isotope and nucleotidase enzyme.

163. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the exciting label, the label for detection or solid substrate.

164. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the exciting label is Biotin.

165. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to a label for detection - fluorescent dye.

166. Engineered antibodies with cysteine substitutions on p, where the fluorescent dye is selected from dyes fluoresceine type radominova type of dansili, lissamine, Chianina, phycoerythrin, Texas red, and their analogues.

167. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to the radionuclide label for detection, selected from3H,11C,14C,18F,32R35S64Cu68Ga86Y99Tc111In123I124I125I131I133Xe177Lu,211At and213Bi.

168. Engineered antibodies with cysteine substitutions on p, where the antibody is covalently linked to a label for detection - chelating ligand.

169. Engineered antibodies with cysteine substitutions on p, where the chelating ligand selected from DOTA, DOTP, DOTMA, DTPA and THETA.

170. Immunoconjugate on p, where the linker is linked to the antibody through Tilney group antibodies.

171. Immunoconjugate directed against CD22, containing the antibody according to claim 1 and albuminaemia peptide.

172. Immunoconjugate on p where albuminaemia peptide selected from SEQ ID NO:42-46.

173. Immunoconjugate directed against CD22, containing the antibody according to item 22 and albuminaemia peptide.

174. Immunoconjugate on p where albuminaemia peptide selected from SEQ ID NO:42-46.

175. Engineered antibodies with cysteine substitutions (Ab)binding to CD22, which contains:
(1) HVR-L1 containing the amino acid sequence selected from SEQ ID NO:9, 10, 19-23, 32 and 33, and
(2) the HVR-H1 containing the amino acid sequence of SEQ ID NO:2;
(3) the HVR-H2 containing the amino acid sequence of SEQ ID NO:4;
(4) the HVR-H3 containing the amino acid sequence of SEQ ID NO:6;
(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:12; and
(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO:14,
moreover, the antibody binds to an epitope in the region of CD22 located from amino acid 22 to amino acids 240 sequence SEQ ID NO:27,
and additionally contains a cysteine at one or more positions selected who's 15, 43, 110, 144, 168 and 205 of the light chain according to the Kabat numbering and 41, 88, 115, 118, 120, 171, 172, 282, 375 and 400 heavy chain according to the EU numbering.

176. The antibody according p, where the cysteine is at position 205 of the light chain.

177. The antibody according p, where the cysteine is at position 118 of the heavy chain.

178. The antibody according p, where the cysteine is at position 400 of the heavy chain.

179. The antibody according p, where the antibody is selected from monoclonal antibodies, especifismo antibodies, chimeric antibodies, human antibodies and gumanitarnogo antibodies.

180. The antibody according p, which is an antibody fragment.

181. The antibody according p, where the antibody fragment is a Fab fragment.

182. The antibody according p, which is selected from a chimeric antibodies, human antibodies or gumanitarnogo antibodies.

183. The antibody according p get in bacteria.

184. The antibody according p, which is obtained in cells SNO.

185. Method detection CD22 protein in a sample suspected of containing this protein, including the impact on the specified pattern antibody on p and binding definition of the specified antibodies with the specified protein CD22 in a specified sample, where the binding of an antibody with the specified protein indicates the presence of the indicated protein in the specified pattern.

186. The method according to p, where the specified sample contains cells, presumably expr serwisie this protein CD22.

187. The method according to p where the specified cell is a cell.

188. The method according to p, where the antibody is covalently linked to a label selected from a fluorescent dye, a radioactive isotope, Biotin or ligand that forms complexes with metals.

189. Pharmaceutical composition for the treatment of disorders of cell proliferation In cells containing an effective amount of the antibody against CD22 on b and a pharmaceutically acceptable diluent, carrier or excipient.

190. Conjugate antibody-drug directed against CD22, containing antibody at p, covalently associated with auristatin or molecule maytansinoids medicines.

191. Conjugate on p containing the antibody (Ab) and auristatin or molecule maytansinoid drug (D), where the engineered antibodies with cysteine substitutions attached to D via the linker (L) through one or more free cysteine residues; and the conjugate has the formula I:

where p represents 1, 2, 3, or 4.

192. Conjugate on p, where p equals 2.

193. Conjugate on p, where L has the formula:

where a represents the extension, covalently linked to a thiol of cysteine engineered antibodies with cysteine substitutions (Ab);
and is 0 or 1;
each W follows the IMO is an amino acid unit;
w is an integer in the range from 0 to 12;
Y is a spacer elements unit covalently linked to a molecule drugs; and
y represents 0, 1 or 2.

194. Conjugate on p having the formula:

where RAV is a pair of aminomethylpropanol, a R17is a bivalent radical selected from (CH2)rWith3-C8-carbocycle, O-(CH2)rarylene, (CH2)rarylene, Allen-(CH2)r-(CH2)r-(C3-C8-carbocycle), (C3-C8-carbazolyl)-(CH2)rWith3-C8-heterocyclyl,
(CH2)r-(C3-C8-heterocyclyl)- (C3-C8-heterocyclyl)-(CH2)r-,
-(CH2)rC(O)NRb(CH2)r-, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-,
-(CH2)rC(O)NRb(CH2CH2O)r-, -(CH2)rC(O)NRb(CH2CH2O)r-CH2-,
-(CH2CH2O)rC(O)NRb(CH2CH2O)r-, -(CH2CH2O)rC(O)NRb(CH2CH2O)r-CH2and
-(CH2CH2O)rC(O)NRb(CH2)r-; where Rbrepresents H, C1-C6-alkyl, phenyl or benzyl; and r is independently researched the mo is an integer in the range from 1 to 10.

195. Conjugate on p, where Wwis a valine-citrulline.

196. Conjugate on p, where R17represents (CH2)5or (CH2)2.

197. Conjugate on p having the formula:

198. Conjugate on p, where R17represents (CH2)5or (CH2)2.

199. Conjugate on p having the formula:

200. Conjugate on p, where L represents the SMCC, SPP or UMRAO.

201. Conjugate on p, where D represents ME, with the structure:

where the wavy line indicates the binding site to the linker L.

202. Conjugate on p, where D represents the MMAF, with the structure:

where the wavy line indicates the binding site to the linker L.

203. Conjugate on p, where D represents the DM1 with structure:

where the wavy line indicates the binding site to the linker L.

204. Conjugate on p, where the parent antibody against CD22 selected from monoclonal antibodies, especifismo antibodies, chimeric antibodies, human antibodies, gumanitarnogo antibodies and fragments of antibodies.

205. Conjugate on p, where the antibody fragment is a Fab fragment.

206. Conjugate antibody-drug, nab is allendy against CD22, selected from the structures:

where Val is a valine; Cit is citrulline; p represents 1, 2, 3 or 4; and Ab is an antibody against CD22 on p.

207. Conjugate on p where auristatin represents MMAE or MMAF.

208. Conjugate on p, where L is a MC-val-cit-PAB or MS.

209. The method of detection of b-cells by binding conjugate antibody-drug on p with CD22 antibody, including
(a) effects on cells conjugate antibody-drug; and
(b) determining the degree of binding of the conjugate antibody-drug cells.

210. Method of inhibiting cell proliferation, comprising the treatment of malignant b-cells of mammals in the environment for culturing cells conjugate antibody-drug on p, resulting in the proliferation of malignant b-cells inhibited.

211. Pharmaceutical composition for the treatment of disorders of cell proliferation In cells containing an effective amount of a conjugate of the antibody-drug on p and a pharmaceutically acceptable diluent, carrier or excipient.

212. A method of treating a malignant tumor, comprising the administration to a patient the pharmaceutical composition according to p.

213. The method according to p, where slocate is to develop a tumor selected from the group consisting of lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

214. The method according to p, where the patient is administered a cytotoxic agent in combination with a conjugate of the antibody-drug.

215. Product for the treatment of disorders of cell proliferation In cells containing
pharmaceutical composition according to p;
container; and
- insert or label indicating that the compound can be used for the treatment of a malignant tumor characterized by overexpression of the polypeptide CD22.

216. Product by p, where a malignant tumor selected from the group consisting of lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed slow-growing NHL, refractory NHL, refractory slow-growing NHL, chronic lymphocytic leukemia (CLL), small cell lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and lymphoma cells of the mantle zone.

217. A method of obtaining a conjugate of the antibody-drug containing antibody against CD22 (Ab) p, and auristatin Il the molecule maytansinoid drug (D), where the antibody is attached to D via the linker (L) through one or more free cysteine residues engineered antibodies with cysteine substitutions; and the conjugate has the formula I:

where p is 1, 2, 3 or 4;
where the method involves the following stages:
(a) interaction resulting from the design group of the cysteine antibody with a linker reagent with the formation of an intermediate product of the antibody-linker (Ab-L); and
(b) interaction Ab-L with activated molecule drugs D; resulting in a conjugate of the antibody-drug;
or where the method comprises the stages:
(c) interaction of nucleophilic groups of the molecule drugs with the linker reagent with the formation of the intermediate product, the drug-linker (D-L); and
(d) interaction of D-L with the resulting design group of cysteine antibodies, resulting in a conjugate of the antibody-drug.

218. The method according to p additionally includes the stage of expression of antibodies in the cells of the Chinese hamster ovary (Cho).

219. The method according to p additionally includes the stage of processing of the expressed antibodies reducing agent.

220. The method according to p, where the reducing agent is selected from TSER and DTT.

221. The method according to p, additionally including the surrounding stage of processing expressed antibodies oxidant after treatment with reducing agent.

222. The method according to p, where the oxidant is selected from copper sulfate, dehydroascorbic acid and air.

223. The antibody according p, where the antibody contains a sequence of the heavy chain selected from any of the sequences SEQ ID NO:88, 92 or 93.

224. The antibody according p, where the antibody contains a sequence of light chain selected from SEQ ID NO:87 or 91.

225. The antibody according p, where the antibody contains a sequence of light chain SEQ ID NO:87 and sequence of the heavy chain SEQ ID NO:92.

226. The antibody according p, where the antibody contains a sequence of light chain SEQ ID NO:87 and sequence of the heavy chain SEQ ID NO:93.

227. The antibody according p, where the antibody contains a sequence of light chain SEQ ID NO:91 and the sequence of the heavy chain SEQ ID NO:88.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method involves cultivation of an obligate methanol-assimilating bacterium Methylophilus methylotrophus or Methylobacillus glycogens in a fluid medium with the bacterium secreting an end protein from a bacterial cell where said bacterium has a DNA structure containing a promoter sequence functioning in the methanol-assimilating bacterium, a nucleotide sequence coding a polypeptide containing a signal sequence which functions in the methanol-assimilating bacterium, and a sequence of the end protein functionally connected with the promoter sequence.

EFFECT: method allows producing the protein effectively by means of extracellular secretion, difficult-to-produce by means of secretory production with application of Escherichia coli bacteria.

5 cl, 7 ex

FIELD: medicine.

SUBSTANCE: there are presented version forms of uricase containing sequences referred in the description. Also, there is presented a recovered nucleic acid containing a nucleotide sequence which codes said uricase, and said nucleic acid is operatively bound with a heterological promoter. There is described an expression vector containing said nucleic acid and a host cell containing this vector and being an uricase producer. There are described a method for producing uricase involving stages of cultivation of said host cells under conditions when the nucleic acid sequence is expressed by the host cells, and of uricase recovery.

EFFECT: invention allows relieving hyperuricemia and hyperuricosuria.

13 cl, 8 dwg, 7 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacology, namely a method for producing cytochrome C. The method for producing cytochrome C involving degreasing of pig, cattle and horse hearts, removal of ligaments and vessels, grinding and extraction in a trichloracetic acid solution under certain conditions, separation of the extract from stuffing, sorption of the filtered extract at extract pH in the UNOsphere™ S cation exchanger balanced with a starter buffer solution at pH 3.9-4.2, after completion of the sorption process, the cation exchanger is washed with the starter buffer solution; ballast proteins are removed from the UNOsphere™ S cation exchanger by a tris solution, and it is followed with elution of cytochrome C from the UNOsphere™ S cation exchanger with an ammonium sulphate solution, concentration and dialysis purification to remove ammonium sulphate ions completely.

EFFECT: method allows increasing product yield, providing higher purity and reducing production process.

4 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method involves: (a) culturing E.coli cells containing polypeptide-coding nucleic acid in a culture medium into which transportable organophosphate is fed, said organophosphate being selected from a group comprising alpha-glycerophosphate, beta-glycerophosphate, glycerol-3-phosphate, and a mixture of glycerol-2-phosphate and glycerol-3-phosphate, such that nucleic acid is expressed, and (b) extracting polypeptide from cells, wherein during the culturing step, inorganic phosphate is added to the culture medium.

EFFECT: improved heterologous protein expression and high output of the end product.

29 cl, 10 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is composition for causing immune reaction against Neisseria meningitidis basing on vesicles, separated from bacteria N. meningitidis, in which endogenous gen GNA 1870 is inactivated and which is transformed by gene construction, coding polypeptide GNA 1870. Such bacterium insures super-expression of GNA 1870 polypeptide in vesicles of N. meningitidis. Composition can additionally contain antigen vesicle from second, third bacteria of N. meningitidis, on condition that both bacteria are genetically different from each other and from said first bacteria. GNA 1870 polypeptides, expressed by first, second and third bacteria are also genetically different from each other. Invention describes method of exciting immune reaction against bacteria of species Neisseria by introduction to mammal of composition based on vesicles, containing polypeptide GNA 1870, as well as method of obtaining composition based on vesicles, obtained as a result of culturing in proper way prepared bacteria, by their mixing with pharmaceutically acceptable carrier.

EFFECT: invention provides protective immunity against broad spectrum of N meningitidis strains, including strains N meningitidis of serogroup B.

45 cl, 20 dwg, 5 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: what is offered is a new method for producing human methionine-free interferon-alpha2b. The method starts with recombinant plasmid DNA containing a human interferon-alpha2b gene which is foregone by an enteropeptidase proteolysis site, and used to transform Escherichia coli cells. The cells are cultured, and inclusion bodies of the synthesised predecessor are isolated. It is followed by partial renaturation of the isolated predecessor in the presence of dithioerythrole preventing closure of disulphide bonds. The predecessor is hydrolysed by the enteropeptidase enzyme with producing human methionine-free interferon-alpha2b. After completion of the predecessor hydrolysis reaction, complete renaturation of human interferon-alpha2b is carried out in the presence of a pair of cystine and cysteine compounds promoting closure of disulphide bonds. The produced protein is purified by a chromatography in a KM-sepharose.

EFFECT: higher yield.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method for producing an antigene preparation from Micobacterium tuberculosis involves mycobacteria cultivation on a Lowenstein-Jensen medium, three-fold washing of bacterial cells from the residual nutrient medium. The washed bacterial mass is treated by aqueous acetone solutions of the concentrations 25 % and 50 %, and each acetone treatment the cells are precipitated by centrifugation. Further, the extracts containing an antigen are produced by three-fold sequential treatment of the bacterial mass deposited by centrifugation, by aqueous dimethylsulphoxide solutions with the increasing concentrations 5 % to 20 % with an insoluble fraction every time separated by centrifugation. The produced three portions of dimethylsulphoxide extracts are mixed and exposed to in-depth dialysis against distilled water. The prepared solution is used as an antigen-containing material for preparing a nitrocellulose membrane immunosorbent.

EFFECT: antigen preparation according to the invention exhibits extended-spectrum serum-positive fractions in a western blotting reaction.

4 dwg, 2 ex

Fused partner cell // 2431667

FIELD: medicine.

SUBSTANCE: invention describes fused partner cells making it possible to produce heterohybridomas of cells of biological species different from a mouse. Heterohybridomas are produced by fused myeloma cells produced from an animal of a first biological species with leukemia cells produced from an animal of a second biological species which have an additional S-phase in a cell cycle and exhibit a diploidisation property. The fusion partner cell SPYMEG is deposited, No. FERM BP-10761 and can be used for hybridoma production. What is described is hybridoma producing antibodies produced by fusion of fusion partner cell and a third cell with antibody-producing ability. The invention describes methods for producing a fusion partner cell, a hybridoma and an antibody-producing cell, and also methods for producing antibodies. Use of the invention provides stable and simple production of the substances with the use of hybridomas in a wide range of species of animals.

EFFECT: hybridomas stably keep a phenotype throughout the whole process of cloning.

20 cl, 10 dwg, 2 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: described is a novel cyclodecapeptide antibiotic laterocin, produced by the Brevibacillus laterosporus VKPM V-10531 strain. Laterocin is a white amorphous solid substance of formula [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Met-)] and molecular weight 1240.6 Da. Laterocin has algicidal (antagonistic) effect on various types of microalgae, particularly blue-green algae Nostoc, Anabaena, Microcystis aeruginosa.

EFFECT: biologically active compound laterocin can be used as an agent for inhibiting growth of blue-green microalgae.

3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: method involves cultivation of an obligate methanol-assimilating bacterium Methylophilus methylotrophus or Methylobacillus glycogens in a fluid medium with the bacterium secreting an end protein from a bacterial cell where said bacterium has a DNA structure containing a promoter sequence functioning in the methanol-assimilating bacterium, a nucleotide sequence coding a polypeptide containing a signal sequence which functions in the methanol-assimilating bacterium, and a sequence of the end protein functionally connected with the promoter sequence.

EFFECT: method allows producing the protein effectively by means of extracellular secretion, difficult-to-produce by means of secretory production with application of Escherichia coli bacteria.

5 cl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.

EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.

3 cl, 1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: vector carries an additional transcription unit containing a foreign gene functionally coupled with an overlying starting sequence of a virus gene (GS) of Mononegavirales order and an underlaying end sequence of a virus gene (GE) of Mononegavirales order; between the GS sequence and an initiator codon of the foreign gene and between a stop sign of the foreign gene and the GE sequence there are respectively 3'-noncoding area and 5'-noncoding area (a genome sense chain) of the virus gene of Mononegavirales order.

EFFECT: higher expression level of the protein coded by the foreign gene.

22 cl, 9 dwg, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is offered is a DNA molecule recovered from Bifidobacterium bifidum, with a sequence presented in the description, and its degenerate derivative coding β-galactosidase. Also, what is presented is a p-galactosidase enzyme having a sequence presented in the description. What is offered is an expression vector containing said DNA, and a bacterial host cell containing said vector. What is offered is application of the enzyme or the host cell for preparing a mixture of oligosaccharides which can be used for preparation of foodstuff, forages for animals and medicines. What is described is a method for producing the enzyme involving cultivation of said host cell in an appropriate culture medium under conditions supposing expression of said enzyme and recovery of the prepared enzyme from the culture.

EFFECT: invention allows transforming lactose into a mixture of oligosaccharides.

12 cl, 5 dwg, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: claimed invention can be used in microbiligical industry for obtaining recombinant strains of fungi Acremonium chrysogenum - superproducents of cephalosporin C. Plasmid contains expression cassette, which consists of promoter of Aspergillus nidulans glyceraldehide phosphate dehydrogenase gene, terminator of Saccharomyces cerevisiae phosphoglycerate kinase gene, separated by polylinker site. As dominant selective marker it contains gene of Escherichia coli hygromycin-B phosphotransferase under control of promoter trpC Aspergillus nidulans, which determines stability to hygromycin of recombinant strains of mycelial fungi, transformed by plasmid pZEN16 and its derivatives.

EFFECT: claimed invention makes it possible to receive unique vector for agrobacterial transformation of Acremonium chrysogenum, which makes it possible to realise constitutive expression of target genes, responsible for biosynthesis and transport of cephalosporin C.

1 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to isolated nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, expression vector, containing said nucleic acid, cell of E.coli, producing protein, encoded by said nucleic acid, and isolated protein, with activity of fluorescent biosensor for hydrogen peroxide detection. Isolated protein, with activity of fluorescent biosensor for hydrogen peroxide detection, has amino acid sequence, presented in form SEQ ID NO: 2. Expression vector contains nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, under control of regulatory elements, necessary for expression of nucleic acid in host cell. Cell of E.coli is modifies due to introduction into it of nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, functionally connected with regulatory sequence.

EFFECT: claimed invention is applied for detection of hydrogen peroxide inside live cells.

4 cl, 3 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: polypeptide, which is used in composition of pharmaceutical composition and in sets for screening of adhesion inhibitors of platelet adhesion or aggregation, is obtained in recombinant way applying matrix of cDNA of Anopheles stephensi salivary gland.

EFFECT: invention makes it possible to obtain polypeptide which possesses inhibiting activity with respect to platelet aggregation or inhibiting activity with respect to platelet adhesion.

10 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: claimed is version of neutral metaloproteinase of wild type Bacillus, which has, at least, 80% identity with sequence, presented in description and having mutations in amino acid sequence in positions, also described in description. Described is method of ferment obtaining, which includes transformation of host-cell by vector, which contains polynucleotide, which codes one of metaloproteinase versions, and culturing of transformed cell in suitable conditions. Described are purifying composition, which contains claimed ferment, and method of fabric and textile purification, which includes stage of bringing surface and/or product from fabric or textile into contact with purifying composition.

EFFECT: claimed invention makes it possible to expand spectrum of means applied for surfaces purification, products of fabric or textile.

28 cl, 12 tbl, 35 dwg, 29 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

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