Monomethylvaline compounds capable of forming conjugates with ligands

FIELD: chemistry.

SUBSTANCE: invention relates to auristatin peptides, including MeVal-Val-Dil-Dap-norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), and these peptides were attached to ligands through various linkers, including maleimidocaproyl-val-cit-PAB. The resulting "ligand-drug" conjugates are active in vitro and in vivo.

EFFECT: high activity of the compounds.

118 cl, 19 dwg, 12 tbl, 33 ex

 

Links to previous applications

The present application claims the priority of provisional application for U.S. patent, No. 60/518534, filed November 6, 2003; and provisional application for U.S. patent, No. 60/5557116 filed March 26, 2004; provisional application for U.S. patent, No. 60/598899, filed August 4, 2004; and provisional application for U.S. patent, No. 60/622455, filed October 27, 2004, the description of which is incorporated into this description by reference.

1. Prior art

The present invention relates to a medicinal compound, and in particular conjugates “drug-linker-ligand”, to compounds drug-linker and conjugates “drug-ligand”, to compositions comprising the above compounds and conjugates, and to methods of their use for treating cancer, autoimmune disease or infectious disease. The present invention also relates to conjugates of the antibody-drug”, to compositions comprising these conjugates, and to methods of their use for the treatment of cancer, autoimmune diseases and infectious diseases. The present invention also relates to methods of using compounds, representing the conjugates of the antibody-drug” forin vitro,in situandin vivodiagnosis or treatment of glue is OK mammals or for the treatment of pathological conditions.

2. Prior art

For many years, intensive research is being conducted to improve the delivery of drugs and other agents in cells, tissues and tumor target in order to achieve maximum efficiency and minimal toxicity. Although it has taken many attempts to develop effective methods for delivering biologically active molecules into cells, asin vivoandin vitrobut none of them gave satisfactory results. Optimization of the conjugation of drugs with its intracellular target, and minimize the intracellular redistribution of medicines, for example, in neighboring cells often presents certain difficulties or is ineffective.

Most modern means, parenterally injected to the patient, not aimed at the target, which leads to their systemic delivery into cells and tissues of the body, in which the presence of these tools is not required and is often undesirable. As a result, these drugs cause unwanted side effects, which often leads to limitation of doses of a medicinal product (for example, chemotherapy (anti-cancer) funds cytotoxic funds inhibitor of enzymes and antiviral or antimicrobial agents), is the output for the introduction. Although oral administration of drugs, compared with parenteral introduction, is considered as a convenient and economical way of introduction, however, in this case, the problem of non-specific toxic effects of drugs, after falling in the circulatory system, normal cells remains relevant. Other problems that can arise when administered orally, are associated with the bioavailability of injected drugs and delay in the intestine, which leads to additional effects of the drug to the intestine, and consequently increase the risk of toxic effects on the intestine. In accordance with this, the main aim of the present invention is to develop methods specific delivery of agents to cells and tissues. The advantages of this treatment include the possibility of preventing the General physiological effects associated with unwanted delivery of these agents to other cells and tissues, such as uninfected cells. Intracellular delivery can be achieved by methods that ensure the accumulation or the presence of active agents, that is, the active metabolites within cells.

Was developed therapy using monoclonal antibodies for targeted treatment of patients suffering from cancer, immune responsive ness going down the practical and angiogenic disorders.

The use of conjugates of the antibody-drug” for local delivery of cytotoxic or cytostatic agents, such as drugs for destruction or suppression of tumor cells in the treatment of cancer (Syrigos & Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz & Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. patent No. 4975278)theoretically allows targeted delivery of the drug in the tumor and to ensure their accumulation inside the cells, whereas systemic administration of these unconjugated drug can lead to the production of toxic levels that are unacceptable for normal cells, as well as insufficient for 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 Antobodies 84:Biological And Clinical Applications, A. Pinchera et al. (eds.), p. 475-506). Thus, it is desirable that the maximum efficiency combined with minimal toxicity. It was reported that this strategy can be used as polyclonal and monoclonal antibodies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods are daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, see above). Toxins used in the conjugates of the antibody-toxi is”, are bacterial toxins such as diphtheria toxin, plant toxins such as ricin, and small molecule toxins such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784; 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 may exert their zitotoksicescoe and cytotoxic effects in accordance with the mechanisms, including binding to tubulin, binding to DNA or inhibition of topoisomerase (D.L. Meyer & P.D. Senter “Recent Advances in Antibody Drug Conjugates for Cancer Therapy”, in Annual Report in Medicinal Chemistry, Vol. 38(2003) Chapter 23, 229-237). Some cytotoxic drugs in their conjugation with major antibodies or ligands of proteins-receptors, have a tendency to loss of activity or reduced activity.

Of zevalin (Zevalin®) (ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioisotope consisting of murine monoclonal antibodies IgG1-Kappa directed against the CD20 antigen present on the surface of normal and malignant b-lymphocytes, and radioactive isotopes111In or90Y associated with chelat forming a complex of thiourea-linker” (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):76-77; 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-jackinsky lymphoma (NHL), but its introduction leads to a severe and chronic cytopenias in most patients. In 2000, he was given permission to use the drug mylotarg (Mylotarg®) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), i.e. conjugate the antibody-drug”consisting of antibodies against human CD33-related calicheamicin, for the treatment of acute myeloid leukemia by injection of the indicated drug (Drugs of the Future(2000) 25(7):686; U.S. patents№ 4970198; 5079233; 5585989; 5606040; 5693762, 5739116, 5767285, 5773001). Cantuzumab mertansine (Immunogen, Inc.), conjugate the antibody-drug”consisting of antibodies against human C242, linked via a disulfide linker SPP to maytansinoid drug DM1, was used in phase II trials for the treatment of cancers that Express CanAg, such as cancers of the colon, pancreas, stomach, etc. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), conjugate the antibody-drug”consisting of monoclonal antibodies against membrane-bound antigen of the prostate (PSMA), attached to maytansinoid drug, DM1, is at the stage of research is that its possible application for the treatment of prostate tumors. This maytansinoid drug, DM1, was conjugated with mouse monoclonal antibody TO through non-disulfide linker SMCC (Chari et al. (1992) Cancer Research 52:127-131). It was reported that the conjugate was 200 times less effective than the corresponding conjugate linked via a disulfide linker. In this work, the linker SMCC is considered “non-split.”

From the marine molluskDolabella auriculariathere are several short-chain peptide compounds, and found that they have a biological activity (Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al. (1995) Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour. Org. Chem. 60:4474). Were also obtained analogues of these compounds, and it was found that some of them have biological activity (for an overview, see Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277). For example, auristatin E (U.S. patent No. 5635483) is a synthetic analogue of a natural marine product dolastatin 10, i.e. an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as anticancer drug vincristine (G.R. Pettit (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10, auristatin RE and auristatin E are linear peptides having four amino acids, three of which are unique to compounds of the class of dolastatin, and-to Navoi amide.

Auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), i.e., synthetic analogues of dolastatin, were conjugated to: (i) with chimeric monoclonal antibodies cBR96 (specific to the antigen Lewis Y on carcinomas); (ii) with SAS, which is specific to CD30, present on hematological malignancies (Klussman et al. (2004) Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation to Ligands”; Francisco et al. (2003) Blood 102(4):1458-1465; publication of patent application U.S. 2004/0018194); (iii) with antibodies against CD20, such as Rituxan® (WO 04/032828)used for the treatment of CD20-expressing cancers and immune disorders; (iv) with anti-EphB2 antibody N and with anti-IL-8 antibody used for the treatment of colon cancer (Mao et al. (2004) Cancer Research 64(3):781-788)); (v) with the antibody against E-selectin (Bhaskar et al. (2003) Cancer Res. 63:6387-6394) and (vi) with other anti-CD30 antibodies (WO 03/043583).

Auristatin E, conjugated with monoclonal antibodies described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published 28 March 2004.

In spite ofin vitrodata obtained in clinical studies of compounds of the class of dolastatins and their analogues, the dose needed to achieve therapeutic effect, produce significant General toxicity, which reduces their effectiveness. In accordance with this heart and soul is for the development of derivative dolastatin/auristatin, with significantly less toxicity, but valuable therapeutic activity, remains relevant. Thus, the present invention is directed at overcoming these and other limitations and problems that still remain unsolved.

Tyrosinekinase receptors of the ErbB family are important mediators of growth, differentiation and survival of cells. This family of receptors consists of four different members, including the receptor for epidermal growth factor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185neu), HER3, (ErbB3) and HER4 (ErbB4 or tyro2). Panel anti-ErbB2 antibody was characterized using cell lines of human breast tumors SCBR3 (Hudziak et al. (1989) Mol. Cell. Biol. 9(3):1165-1172. Maximum inhibition was achieved with the use of antibodies, called 4D5, which is 56% inhibited cell proliferation. In this analysis other antibodies in this panel of antibodies reduced cell proliferation to a lesser extent. In addition, it was found that the antibody 4D5 sencibilisiruet ErbB2-sverkhekspressiya cell lines of breast cancer by producing cytotoxic effects of TNF-α (U.S. patent No. 5677171). Anti-ErbB2 antibodies, discussed in Hudziak et al., were further characterized Fendly et al. (1990), Cancer Research 50:1550-1558; Kotts et al. (1990) In vitro 26(3):59A; Sarup et al. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin. Immunol. 11(3):117-127; Kumar et al. (1991) Mol. ell. Biol. 11(2):979-986; Lewis et al. (1993) Cancer Immunol. Immunother. 37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al. (1994) Cancer Research 54:5301-5309; Silwkowski et al. (1994) J. Biol. Chem. 269(20):14661-14665; Scott et al. (1991) J. Biol. Chem. 266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465 and Schaefer et al. (1997) Oncogene 15:1385-1394.

Other anti-ErbB2 antibody with different properties, described Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. Proc. Natl. Acad. Sci., USA, 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); in WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Handcock et al. (1991) Cancer Res. 51:4575-4580; Shawver et al. (1994) Cancer Res. 54:1367-1373; Arteaga et al. (1994) Cancer Res. 54:3758-3765; Harwerth et al. (1992) J. Biol. Chem. 267:15160-15167; in U.S. patent No. 5783186 and Klapper et al. (1997) Oncogene 14:2099-2109.

Screening for homology allowed us to identify the other two members of the family of ErbB receptors, namely ErbB3 (U.S. patent No. 5183884; U.S. patent No. 5480968; KraU.S. et al. (1989) Proc. Natl. Acad. Sci., USA, 86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc. Natl. Acad. Sci., USA, 90:1746-1750; and Plowman et al. (1993) Nature 366:473-475). Both of these receptor was found increased expression of at least some cell lines breast cancer.

Herceptin® (trastuzumab) is a recombinant DNA gumanitarnoe” monoclonal antibody, which is in cell analysis selectively and with high affinity (Kd=5 nm) was associated with the extracellular domain of the protein of prescriptions the ora of the human epidermal growth factor -2, HER2 (ErbB2) (U.S. patent No. 5821337; U.S. patent No. 6054297; U.S. patent No. 6407213; U.S. patent No. 6639055; Coussens L. et al. (1985) Science 230:1132-9; D.J. Slamon et al. (1989) Science 244:707-12). Trastuzumab is an antibody IgG1-Kappa that contains a frame region of a human antibody hypervariable region (complementarity-determining region) of a murine antibody (4D5)that binds to HER2. Trastuzumab binds to HER2 antigen and, thus, inhibits the growth of cancer cells. Because trastuzumab is gumanitarnym antibody, it minimizes any NAMA-response in patients. Gumanitarnoe antibody against HER2 was produced in suspension culture of mammalian cells (cells of Chinese hamster ovary, Cho). Protooncogen HER2 (or C-erbB2) encodes a transmembrane receptor protein with a size of 185 kDa, which is related to the structure of the receptor for epidermal growth factor. Overexpression of HER2 protein is observed in 25%-30% of primary breast tumors and can be determined by immunohistochemical evaluation of fixed tumor sections (Press M.F. et al. (1993) Cancer Res. 53:4960-70). Analyses conducted byin vitroand on animals have shown that trastuzumab inhibits proliferation of human tumor cells that sverkhekspressiya HER2 (Hudziak R. et al. (1989) Mol. Cell. Biol. 9:1165-72; G.D. Lewis et al. (1993) Cancer Immunol. Immunother; 37:255-63; Baselga J. e al. (1998) Cancer Res. 58:2825-2831). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling T.E. et al. (1996) [abstract]. Proc. Annual. Meeting Am. Assoc. Cancer Res; 37:471; Pegram M.D. et al. (1997) [abstract] Proc. Am. Assoc. Cancer Res; 38:602).In vitroit was shown that mediated by trastuzumab ADCC occurs predominantly in the HER2-sverkhekspressiya cancer cells compared with cancer cells, which do not detect the overexpression of HER2. Herceptin® is the only remedy that is indicated for the treatment of patients suffering from cancers of the breast with metastases, which sverkhekspressiya protein HER2, and for those who received one or more courses of chemotherapy for cancer with metastasis. Herceptin® in combination with paclitaxel is indicated for the treatment of patients suffering from cancers of the breast with metastases, which sverkhekspressiya protein HER2, and for patients who are not held chemotherapy for cancer with metastasis. Herceptin® is clinically active in patients with ErbB2-sverkhekspressiya cancer metastatic tumors of the breast, where these patients before anti-cancer therapy, underwent an intensive course of treatment (Baselga et al. (1996) J. Clin. Oncol. 14:737-744).

Mouse monoclonal anti-HER2 antibody inhibits the growth of cell lines of breast cancer, PR is the observed overexpression of HER2 at level 2+ and 3+ (1-2×10 6of HER2 receptors per cell), but this antibody had no activity against the cells that expressed low levels of HER2 (Lewis et al. (1993) Cancer Immunol. Immunother. 37:255-263). Taking into account these observations, the antibody 4D5 was humanitarno (huMab4D5-8, rhuMab HER2, U.S. patent No. 5821337; Carter et al. (1992) Proc. Natl. Acad. Sci., USA, 89:4285-4289) and has been tested in patients with cancers of the breast, which was sverkhekspressiya HER2, but progressed after standard chemotherapy (Cobleigh et al. (1999) J. Clin. Oncol. 17:2639-2648).

Although getting Herceptin is outstanding scientific achievement in the treatment of patients with ErbB2-sverkhekspressiya cancers of the breast, which were previously subjected to intense anti-cancer therapy, but some patients in this group did not show any response or found only a slight response to treatment with Herceptin.

Therefore remains an urgent need to develop other, with important clinical methods HER2-targeted anticancer therapy for patients with HER2-sverkhekspressiya tumors or other diseases associated with expression of HER2 that don't respond or not respond to treatment with Herceptin.

Any works cited in the present application should not be considered as prototypes for the present invention.

3. Description of the invention

In one of its aspects the present invention relates to compounds “drug-linker-ligand, having the formula Ia:

L--(Aa--Ww--Yy-D)P(Ia)

or their pharmaceutically acceptable salts or solvate,

where: L represents a ligand component;

-Aand-Ww-Yy- is a linker component (LU), where specified in the linker component:

-A - is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

-Y - spacer elements means the component

y is 0, 1 or 2;

R is in the range from 1 to about 20; and

-D is a component of the “drug”that has formula DEand DF;

where in each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH, where n is an integer from 0 to 6; and

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).

In another aspect the present invention relates to pharmaceutical compounds of the formula Ib:

or their pharmaceutically acceptable salts or solvate,

where: R2selected from hydrogen and C1-C8of alkyl;

R3selected from hydrogen, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from hydrogen is, With1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle), where R5selected from H and methyl, or R4and R5taken together with the carbon atom to which they are linked, form a ring of formula -(CRaRb)n-where Raand Rbindependently selected from H, C1-C8the alkyl and C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl group or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH, and

n is an integer from 0 to 6.

The compounds of formula (Ib) can be used to treat cancer, autoimmune disease or infectious disease in a patient, or they can be used as intermediate compounds for the synthesis of conjugates “drug-linker”, “drug-linker-ligand” and “drug-ligand”, in which this drug is otdalennym component.

In another aspect the present invention relates to compositions comprising an effective amount of the conjugate “drug-linker-ligand” and a pharmaceutically acceptable carrier or excipient.

In another aspect the present invention relates to pharmaceutical compositions comprising an effective amount is in connection “drug-linker” and a pharmaceutically acceptable carrier or excipient.

In yet another aspect the present invention relates to compositions comprising an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”, and a pharmaceutically acceptable carrier or excipient.

In another aspect the present invention relates to methods of lysis or inhibiting the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods of lysis or inhibiting the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods of lysis or inhibiting the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In still another aspect of us is Aasee the invention relates to methods for treating cancer includes introduction to the patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods for treating cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods for treating cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In another aspect the present invention relates to methods of lysis or inhibition of cell replication, which expresses an autoimmune antibody, where the method includes the administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods of lysis or inhibition of cell replication, which expresses an autoimmune antibody, where the method includes the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect n the present invention relates to methods of lysis or inhibition of cell replication, which expresses an autoimmune antibody, where the method includes the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to methods for treating autoimmune diseases comprising administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods for treating autoimmune diseases comprising administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods for treating autoimmune diseases comprising administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to methods for treating an infectious disease comprising the administration to a patient in need, an effective number is the number of connection “drug-linker”.

In another aspect the present invention relates to methods for treating an infectious disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods for treating an infectious disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to methods of lysis multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods for preventing the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods for preventing the multiplication of tumor cells or cancer cells comprising the administration to a patient in need this, effetive the number of conjugate “drug-ligand”, contains the component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to a method of preventing cancer, comprising the administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to a method of preventing cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to a method of preventing cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to methods for preventing the multiplication of cells expressing autoimmune antibody, where these methods include administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to methods for preventing the multiplication of cells, expressyou the autoimmune antibody where these methods include administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to methods for preventing the multiplication of cells expressing autoimmune antibody, where these methods include administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to a method of preventing an autoimmune disease comprising the administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to a method of preventing an autoimmune disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to a method of preventing an autoimmune disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component drugs is the authorized agent”, ottsepleny from the specified conjugate “drug-ligand”.

In yet another aspect the present invention relates to a method of preventing an infectious disease, comprising administration to a patient in need, an effective amount of compounds of “a drug-linker”.

In another aspect the present invention relates to a method of preventing an infectious disease, comprising administration to a patient in need, an effective amount of the conjugate “drug-linker-ligand”.

In another aspect the present invention relates to a method of preventing an infectious disease, comprising administration to a patient in need, an effective amount of the conjugate “drug-ligand containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In another aspect the present invention relates to a medicinal compound that can be used as intermediate compounds for the synthesis of compounds of “a drug-linker containing component “drug”, ottsepleny from the specified conjugate “drug-ligand”.

In another aspect the present invention relates what I connection “drug-linker”, which can be used as intermediate compounds for the synthesis of conjugate “drug-linker-ligand”.

In another aspect the present invention relates to compounds of the formula Ia':

Ab--(Aa--Ww--Yy--D)P(Ia')

or their pharmaceutically acceptable salts or solvate,

where: Ab is an antibody, including an antibody that binds to CD30, CD40, CD70 and antigen Lewis Y;

But is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer elements the component

y is 0, 1 or 2,

R is in the range from 1 to about 20; and

D is a component of “drug”, selected from compounds of formulas DEand DF;

where in each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8g is teracycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH, and

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 from 0 to 6.

In one embodiment, the invention is not Ab is an antibody which binds to an ErbB receptor, or which binds to one or more of receptors (1)-(35), such as:

(1) BMPR1B (the receptor protein of bone morphogenesis type IB, Genbank reg. No. NM_001203);

(2) e (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate, Genbank reg. No. NM_012449);

(4) R (SA, MUC16, Genbank reg. No. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank reg. No. NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, member 2 family R is storymy media 34 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II, Genbank reg. No. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5B, Genbank reg. No. AV);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, cDNA gene RIKEN 2700050C12, Genbank reg. No. AY358628);

(9) ETBR (endothelin receptor type b, Genbank reg. No. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of prostate cancer, Genbank reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M, Genbank reg. No. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma, Genbank reg. No. NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2)or C3DR (C3d/receptor of the virus of Epstein-Barr), or Hs.73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), V, Genbank reg. No. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C, Genbank reg. No. NM_030764);

(1) HER2 (Genbank reg. No. M11730);

(18) NCA (Genbank reg. No. m);

(19) MDP (Genbank reg. No. BC017023);

(20) IL20Rα (Genbank reg. No. AF184971);

(21) Brevican (Genbank reg. No. AF229053);

(22) Ephb2R (Genbank reg. No. NM_004442);

(23) ASLG659 (Genbank reg. No. AX092328);

(24) PSCA (Genbank reg. No. AJ297436);

(25) GEDA (Genbank reg. No. AY260763);

(26) BAFF-R (Genbank reg. No. NP_443177.1);

(27) CD22 (Genbank reg. No. NP-001762.1);

(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha-protein-klecko-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with molecules of IgM on the cell surface, passing the signal that is involved in the differentiation of b-cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (receptor 1 lymphoma Burkitt, the receptor-associated G-protein and activated by the chemokine CXCL13, provides migration of lymphocytes and humoral defense, plays a role in infection by HIV-2 and probably in the development SPEED and, lymphoma, myeloma and leukemia, Genbank reg. No. NP_001707.1);

(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes, Genbank reg. No. NP_002111.1);

(31) RG (ion channel -5 opened ligand purinergic receptor RH; ion channel opening extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may play a role in the pathophysiology of idiopathic dysfunction of the bladder, Genank reg. No. NP_002552.2);

(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2, Genbank reg. No. NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I, which regulates the activation and apoptosis of b-cells, and loss of function of this protein is associated with progression in patients of systemic lupus erythematosus, Genbank reg. No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, the presumed receptor for the Fc domain of immunoglobulin, which contains Ig-like domains of type C2 and ITAM domains and may play a role in the differentiation of b-lymphocytes, Genbank reg. No. NP_443170.1);

(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily 2, the estimated immunoreceptor, which might play a role in the development of b cells and lymphomagenesis; and in some malignant b cells observed dysregulation of the gene by translocation, Genbank reg. No. NP_112571.1).

In yet another aspect the present invention relates to pharmaceutical compositions comprising an effective amount of the conjugate “drug-linker-antibody” and a pharmaceutically acceptable carrier or excipient.

In yet another aspect the present invention relates to compositions comprising effective amounts of the conjugate “drug-antibody”, where the specified drug (part) cleaved from the specified conjugate “drug-antibody”, and a pharmaceutically acceptable carrier or excipient.

In another aspect the present invention relates to methods of lysis or inhibiting the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods of lysis or inhibiting the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to methods for treating cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods for treating cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component, in addition to the public means”, ottsepleny from the specified conjugate “drug-antibody”.

In another aspect the present invention relates to methods of lysis or inhibition of cell replication, which expresses an autoimmune antibody, where the method includes the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods of lysis or inhibition of cell replication, which expresses an autoimmune antibody, where the method includes the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to methods for treating autoimmune diseases comprising administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In yet another aspect the present invention relates to methods for treating autoimmune diseases comprising administration to a patient in need, an effective amount of the conjugate “drug-antibody containing comp the element “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to methods for treating an infectious disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods for treating an infectious disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to methods for preventing the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods for preventing the multiplication of tumor cells or cancer cells comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate drug is sredstvo-antibody”.

In yet another aspect the present invention relates to a method of preventing cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In yet another aspect the present invention relates to a method of preventing cancer, comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In another aspect the present invention relates to methods for preventing the multiplication of cells expressing autoimmune antibody, where these methods include administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to methods for preventing the multiplication of cells expressing autoimmune antibody, where these methods include administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to methods for prevention of autoimmune disease, includes introduction to the patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to a method of preventing an autoimmune disease comprising the administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In yet another aspect the present invention relates to a method of preventing an infectious disease, comprising administration to a patient in need, an effective amount of the conjugate “drug-linker-antibody”.

In another aspect the present invention relates to a method of preventing an infectious disease, comprising administration to a patient in need, an effective amount of the conjugate “drug-antibody containing component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In another aspect the present invention relates to a medicinal compound that can be used as intermediate compounds for the synthesis of compounds of medicines is-linker”, contains the component “drug”, ottsepleny from the specified conjugate “drug-antibody”.

In another aspect the present invention relates to a connection “drug-linker”, which can be used as intermediate compounds for the synthesis of conjugate “drug-linker-antibody”.

In one of its aspects the present invention relates to conjugates of “drug-linker-antibody” (also called the conjugates of the antibody-drug”), having the formula Ic:

Ab--(Aa--Ww--Yy--D)P(Ic)

or their pharmaceutically acceptable salts or solvate,

where: Ab is an antibody that binds to one or more antigens (1)-(35), such as:

(1) BMPR1B (the receptor protein of bone morphogenesis type IB, Genbank reg. No. NM_001203);

(2) e (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate, Genbank reg. No. NM_012449);

(4) R (SA, MUC16, Genbank reg. No. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank reg. No. NM_005823);

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 family soluble carriers 34 (f the SFAT sodium), sodium-dependent phosphate-transferring protein 3b type II, Genbank reg. No. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5B, Genbank reg. No. AV);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, cDNA gene RIKEN 2700050C12, Genbank reg. No. AY358628);

(9) ETBR (endothelin receptor type b, Genbank reg. No. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of prostate cancer, Genbank reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M, Genbank reg. No. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma, Genbank reg. No. NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2)or C3DR (C3d/receptor of the virus of Epstein-Barr), or Hs.73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), V, Genbank reg. No. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C, Genbank reg. No. NM_030764);

(1) HER2 (Genbank reg. No. M11730);

(18) NCA (Genbank reg. No. m);

(19) MDP (Genbank reg. No. BC017023);

(20) IL20Rα (Genbank reg. No. AF184971);

(21) Brevican (Genbank reg. No. AF229053);

(22) Ephb2R (Genbank reg. No. NM_004442);

(23) ASLG659 (Genbank reg. No. AX092328);

(24) PSCA (Genbank reg. No. AJ297436);

(25) GEDA (Genbank reg. No. AY260763);

(26) BAFF-R (Genbank reg. No. NP_443177.1);

(27) CD22 (Genbank reg. No. NP-001762.1);

(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha-protein-klecko-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with molecules of IgM on the cell surface, passing the signal that is involved in the differentiation of b-cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (receptor 1 lymphoma Burkitt, the receptor-associated G-protein and activated by the chemokine CXCL13, provides migration of lymphocytes and humoral defense, plays a role in infection by HIV-2 and probably in the development SPEED and, lymphoma, myeloma and leukemia, Genbank reg. No. NP_001707.1);

(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes, Genbank reg. No. NP_002111.1);

(31) RG (ion channel -5 opened ligand purinergic receptor RH; ion channel opening extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may play a role in the pathophysiology of idiopathic dysfunction of the bladder, Genank reg. No. NP_002552.2);

(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2, Genbank reg. No. NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I, which regulates the activation and apoptosis of b-cells, and loss of function of this protein is associated with progression in patients of systemic lupus erythematosus, Genbank reg. No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, the presumed receptor for the Fc domain of immunoglobulin, which contains Ig-like domains of type C2 and ITAM domains and may play a role in the differentiation of b-lymphocytes, Genbank reg. No. NP_443170.1);

(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily 2, the estimated immunoreceptor, which might play a role in the development of b cells and lymphomagenesis; and in some malignant b cells observed dysregulation of the gene by translocation, Genbank reg. No. NP_112571.1).

But is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer elements the component

y is 0, 1 or 2;

R is in the range from 1 to about 20; and

D is a drug selected from the compounds is of oral D Eand DF;

where: the wavy line in formula DEand DFmeans the website covalent binding to A, W or Y, and where at each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

R7selected from H and C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1With 8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH, and

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 from 0 to 6.

In another aspect of the invention, the antibody in the conjugate is the antibody-drug” (ADC) according to the invention specifically binds to the receptor, encoded by the gene ErbB2.

In another aspect of the invention, the antibody in the conjugate antibody-drug” represents gumanitarnoe antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

In another aspect the present invention relates to a product containing the compound in the form of conjugate “antibody-drug” according to the invention; the container and the liner, is attached to the packaging, or label, indicating that this compound may be used to treat cancer characterized by overexpression of ErbB2 receptor.

In another aspect the present invention relates to a method of treating cancer in a mammal, where the specified cancer is characterized by overexpression of ErbB2 receptor and do not respond or responds poorly to treatment with anti-ErbB2 antibody and where the method includes the introduction of the specified mammal a therapeutically effective amount of the conjugate the antibody-drug” according to the invention.

In another aspect of the present invention a significant number of the specified molecule drugs are not cleaved from the antibody up until the specified connection-conjugate antibody-drug” will not penetrate a cell having on its surface of prescriptions is PR, non-specific antibodies present in the specified conjugate antibody-drug”, and this molecule drug is cleaved from the specified antibodies only when the conjugate antibody-drug” into the specified cell.

In another aspect of the present invention, the bioavailability of the compound-conjugate “antibody-drug or an intracellular metabolite of the compounds in the body of a mammal is better than the bioavailability of pharmaceutical compounds containing molecule drugs present in the compound-conjugate antibody-drug”, or better, than the bioavailability of similar compounds that do not contain the molecule drugs.

In another aspect of the present invention the specified molecule drug is a molecule that the body of the mammal undergoes intracellular cleavage from antibodies, which is a component of this compound or an intracellular metabolite of this compound.

In another aspect the present invention relates to pharmaceutical compositions containing an effective amount of the compound-conjugate “antibody-drug” according to the invention, the sludge is its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent, the carrier or filler. This composition may further comprise a therapeutically effective amount of a chemotherapeutic agent, such as a tubulin-forming inhibitor, a topoisomerase inhibitor and a DNA-binding agent.

In another aspect the present invention relates to a method of lysis or inhibition of proliferation of tumor or cancer cells, including the treatment of tumor or cancer cells in a certain number of connections-conjugate “antibody-drug” according to the invention or its pharmaceutically acceptable salt or MES, which is effective for lysis or inhibition of proliferation of these tumor or cancer cells.

In another aspect the present invention relates to a method of inhibiting cell proliferation, comprising the treatment of mammalian cells present in the medium for culturing cells, compound-conjugate antibody-drug” according to the invention, where the specified connection-conjugate antibody-drug” enters the cell, and then the drug is cleaved from the specified connection-conjugate “antibody-drug”, resulting in inhibition of cell proliferation.

In another aspect of nastoyascheevremya relates to a method of treatment of cancer, includes introduction to the patient a composition containing the compound-conjugate antibody-drug” and a pharmaceutically acceptable diluent, carrier or filler.

In another aspect the present invention relates to the analysis for the detection of cancer cells, including:

(a) treatment of cells with compound-conjugate antibody-drug” according to the invention and

(b) determining the degree of binding of the compound-conjugate “antibody-drug” with these cells.

For a better understanding of the present invention below is a detailed description of examples of implementation, illustrated in the accompanying graphic material and schemes. The following discussion is only descriptive, illustrative and representative in nature and should not be construed as limiting the scope of the below claims.

Brief description of the graphical material

Figure 1 illustrates the effective analysis ofin vivoconducted using a single dose SAS-mcMMAF in subcutaneous xenografts ALCL Karpas-299.

Figure 2 illustrates the effective analysis ofin vivoconducted by a single dose SAS-mcMMAF in subcutaneous L540cy. This study was carried out on 4 mice group that has not undergone processing, and 10 bat is in each group processing.

On figa and 3b illustratedin vivothe effectiveness of cBR96-mcMMAF in subcutaneous L2987. Shaded triangles on figa and arrows on fig.3b indicate the days of therapy.

On figa and 4b illustratedin vitrothe activity of conjugates of the antibody SAS-drug”directed against the CD30+-cell lines.

On figa and 5b illustratedin vitrothe activity of conjugates of the antibody cBR96-drug”against Ley+-cell lines.

On figa and 6b illustratedin vitrothe activity of conjugates of the antibody c1F6-drug”against CD70+cell lines of renal carcinoma.

Figure 7 illustrates thein vitroanalysis of cell proliferation using cells SK-BR-3, treated with conjugates of the antibody-drug” (ADC): -●- trastuzumab-MC-vc-RAV-MMAF, 3,8 MMAF/Ab, -o-trastuzumab-MC-MMAF, A 4.1 MMAF/Ab, and-Δ-trastuzumab-MC-MMAF, 4,8 MMAF/Ab, measured in relative fluorescence units (RLU) relative to the ADC concentration (µg/ml). H=trastuzumab, where H is attached via a cysteine [cys].

On Fig illustratedin vitroanalysis of cell proliferation using cell BT-474, processed ADC: -●- trastuzumab-MC-vc-RAV-MMAF, 3,8 MMAF/Ab, -o-trastuzumab-MC-MMAF, A 4.1 MMAF/Ab, and-Δ - trastuzumab-MC-MMAF, 4,8 MMAF/Ab.

Figure 9 illustrates thein vtro analysis of cell proliferation using MCF-7 cells treated ADC: -●- trastuzumab-MC-vc-RAV-MMAF, 3,8 MMAF/Ab, -o-trastuzumab-MC-(N-Me)-vc-PAB-MMAF, 3,9 MMAF/Ab, and-Δ - trastuzumab-MC-MMAF, A 4.1 MMAF/Ab.

Figure 10 illustrates thein vitroanalysis of cell proliferation using cell MDA-MB-468, processed ADC: -●- trastuzumab-MC-vc-RAV-MMAE, 4,1 MMAE/Ab, -o - trastuzumab-MC-vc-RAV-MMAE, 3,3 MMAE/Ab, and-Δ - trastuzumab-MC-vc-RAV-MMAF, 3,7 MMAF/Ab.

Figure 11 illustrates the study on the removal of concentrations of plasma after the introduction of the H-MC-vc-RAV-MMAF-TEG and H-MC-vc-RAV-MMAF rats Sprague-Dawley: injected dose was 2 mg ADC per kg of body weight of the rat. Concentrations of total antibody and ADC was measured in dependence on time (N=trastuzumab).

On Fig illustrated study on the removal of concentrations of plasma after the introduction of the H-MC-vc-MMAE abacadabra monkeys in different doses: 0,5; 1,5; 2,5; 3,0 mg/kg on days 1 and 21. Concentrations of total antibody and ADC was measured in dependence on time (N=trastuzumab).

On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5 that on day 0 was introduced: the media, trastuzumab-MC-vc-RAV-MMAE (1250 mg/m2) and trastuzumab-MC-vc-RAV-MMAF (555 mg/m2) (H=trastuzumab).

On Fig illustrates the change in average volume is the tumor depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5, on day 0 were injected: 10 mg/kg (660 g/m2) trastuzumab-MC-MMAE and 1250 mg/m2trastuzumab-MC-vc-RAV-MAE.

On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5 that on day 0 was introduced: the media and 650 mg/m2trastuzumab-MC-MMAF.

On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5 that on day 0 was introduced: the media and 350 mg/m2four conjugates trastuzumab-MC-MMAF, where the ratio of MMAF/trastuzumab (N) was 2, 4, 5.9 and 6.

On Fig illustrates the change in mean body mass, with the magnitude of the error for groups of animals (rats) (mean ± cf. square from.) after the introduction of media, trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-RAV-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-RAV-MMAF.

On Fig illustrates the change in the average body weight for groups of animals (rats) (mean ± cf. square from.) after the introduction 9,94 mg/kg H-MC-vc-MMAF, of 24.90 mg/kg H-MC-vc-MMAF, 10,69 mg/kg N-IU(IU)-vc-RAV-MMAF, 26,78 mg/kg N-IU(IU)-vc-RAV-MMAF, 10,17 mg/kg N-MC-MMAF, 25.50 mg/kg N-MC-MMAF and 21.85 mg/kg H-MC-vc-RAV-MMAF. H=trastuzumab. Each conjugate linker MS is linked via a cysteine of trastuzumab.

On Fig polluter the Vano change in average body weight, with the magnitude of the error for groups of rats Sprague-Dawley (mean ± cf. square from.) after the introduction of trastuzumab(H)-MC-MMAF in doses 2105, 3158 and 4210 mg/m2. Each conjugate linker MS is linked via a cysteine of trastuzumab.

4. Detailed description of representative embodiments of the invention

4.1. Definitions and abbreviations

If it is not specifically mentioned, then used herein, the terms and expressions shall have the following meanings.

Used by the applicants, the term “trademarks” means independently protected trademark of the finished product, generic drug and active(e) ingredient(s) product-protected(s) trademark.

Used herein, the term “antibody” is used in its broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bespecifically antibodies)formed by at least two intact antibodies, and antibody fragments, provided that they possess the desired biological activity. Antibody is a protein that is generated by the immune system and which is able to recognize specific antigen and binds to this antigen. As to the structure of antibodies, it is usually a Y-shaped protein consisting of a couple, the ex amino acid chains, two heavy and two light chains. Each antibody has mainly two areas: variable region and a constant region. Variable region located at the ends of the branches of the Y, communicates and interacts with the target antigen. This variable region comprises a hypervariable region (complementarity-determining region CDR), which recognizes a specific binding site on a particular antigen and binds to it. Constant region located at the “tail” of Y, is recognized by the immune system and interacts with this immune system (Janeway C. Travers, P., Walport M. Shlomchik (2001) Measurement Biology, 5th Ed., Garland Publishing, New York). Antigen-target usually has multiple binding sites, the so-called epitopes recognized by CDR on multiple antibodies. Each antibody that specifically binds to a different epitope has a structure different from the structure of another antibody. Thus, a single antigen can have more than one corresponding antibody.

Used herein, the term “antibody” also means a full-size immunoglobulin molecule or immunologically active portion of the full-length immunoglobulin molecules, i.e. molecules containing antigennegative site that immunospecificity binds to the antigen of interest target or its part where indicated and targets are, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. Described here immunoglobulins can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Immunoglobulins can occur from molecules of any species. However, in one aspect of the invention, the immunoglobulin is of human, mouse or rabbit immunoglobulin. In another aspect of the invention, these antibodies are polyclonal, monoclonal, bespecifically, human, gumanitarnye or chimeric antibodies, single-chain antibody, an Fv, Fab, F(ab')-, F(ab')2fragments, fragments produced library of expressed Fab fragments, antiidiotypic (anti-Id) antibodies, CDR, and epitope-binding fragments of any of the above antibodies that immunospecificity contact with antigens on cancer cells, viral antigens or microbial antigens.

Used herein, the term “monoclonal antibody” means an antibody obtained from a population of mostly homogeneous antibodies, the antibodies included in this population and are identical except for possible natural mutations that may be present in small to the likesto. Monoclonal antibodies are highly specific and are directed against the same antigenic determinants. In addition, unlike drugs polyclonal antibodies, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized so that they do not contain impurities of other antibodies. The term “monoclonal” indicates the type of antibodies obtained essentially homogeneous population of antibodies, but this term does not imply that this antibody must be produced to any particular method. For example, the monoclonal antibodies according to the invention can be produced using hybridoma technology, first described by Kohler et al. (1975) Nature 256:495, or methods of recombinant DNA (see U.S. patent No. 4816567). “Monoclonal antibodies” may also be isolated from phage libraries of antibodies by methods described, for example, Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597.

Used here monoclonal antibodies include, in particular, “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding consequently the values of antibodies, derived from a particular species or belonging to a particular class or subclass of antibody, and the other(s) chain(s) is identical to(s) or homologous(s) corresponding sequences of antibodies derived from another species or belonging to another class or subclass of antibody, and include fragments of such antibodies, provided that they possess the desired biological activity (U.S. patent No. 4816567 and Morrison et al. (1984) Proc. Natl. Acad. Sci., USA, 81:6851-6855).

For production of monoclonal antibodies (MAb) were used several methods. In hybridoma technology applied to the cloned cell lines that produce the antibody of the same type, are cells of various species, including mice, hamsters, rats, and humans. Another method to obtain MAb involves the application of techniques of genetic engineering, including methods of recombinant DNA. Monoclonal antibodies obtained by such methods are, among others, chimeric antibodies and gumanitarnye antibodies. Chimeric antibody combines region encoded by DNA derived from more than one species. For example, the chimeric antibody can be derived from variable regions of the mouse antibody and the constant region of a human antibody. Gumanitarnoe antibody occurs mainly from human antibodies, even if it contains about the region of non-human antibodies. Like chimeric antibody, gumanitarnoe antibody can contain a fully human constant region. However, in contrast to the chimeric antibody, its variable region may partially come from human antibodies. Non-human synthetic part gumanitarnogo antibodies often come from a CDR of a mouse antibody. In any case, these areas play a crucial role in the detection antibody specific antigen and its binding to this antigen.

As already noted, can be used murine antibodies. Although murine antibodies can be used in the diagnosis and short courses of therapy, however, their introduction to a person for a long period of time increases the risk of producing a person undesirable immunogenic response. This response is the production of human antimelanoma antibodies (HAMA), which is produced in the case, when the human immune system recognizes the murine antibody as foreign and attacks it. HAMA response can cause toxic shock or even lead to death.

Chimeric and gumanitarnye antibodies reduce NAMA-answer by minimizing the non-human parts of the injected antibodies. In addition, chimeric and gumanitarnye antibodies have the advantage that they and teleroute secondary immune responses in humans, such as antibody-dependent cellular cytotoxicity.

“Antibody fragments” contain some of the intact antibody, and preferably antigennegative or variable region. Examples of fragments of antibodies are Fab-, Fab'and F(ab')2and Fv fragments; dyatel; linear antibodies; single-chain molecule antibodies and multispecific antibodies, derived from the fragment(s) of the antibody.

“Intact” antibody is an antibody containing antigennegative variable region and the constant domain of the light chain (CL) and constant domains of the heavy chain, CN, CH2 and CH3. The constant domains may represent a constant domains of the native sequence (e.g., the constant domains of human native sequence) or their variants amino acid sequence.

The intact antibody may have one or more “effector” functions”, which means the biological activity attributed to the Fc region (Fc-region of a native sequence or a Fc region of the variant amino acid sequence) antibodies. Examples of effector functions of antibodies are binding to C1q; complement dependent cytotoxicity; binding to an Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; inhibition of receptor glue the internal surface (for example, B-cell receptor; BCR), etc

Intact antibodies, depending on the amino acid sequence of the constant domain of their heavy chains, can be attributed to different “classes”. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain, which correspond to the different classes of antibodies are called α, δ, ε, γ and µ, respectively. Structures of subunits and three-dimensional configuration of immunoglobulins of different classes are well known.

Used herein, the term “ErbB2” and “HER2” are interchangeable and mean the human HER2 protein described, for example, in Semba et al. Proc. Natl. Acad. Sci., USA, 82:6497-6501 (1985) and Yamamoto et al. (1986) Nature, 319:230-234 (Genbank reg. No. h). The term “erbB2” means the gene encoding human ErbB2 and “neu” refers to the gene encoding rat p185neu. Preferred is human ErbB2 ErbB2 with native sequence.

Antibodies against ErbB receptors are commercially available and can be obtained from various sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.

“The ErbB ligand” means a polypeptide that binds to an ErbB receptor and/or activates this receptor. ErbB ligand may be human ErbB ligand having the provide sequence, such as epidermal growth factor (EGF) (Savage et al. (1972) J. Biol. Chem. 247:7612-7621); transforming growth factor-alpha (TGF-α) (Marquardt et al. (1984) Science 223:1079-1082); amphiregulin, also known as Sanoma or autocrine keratinocyte growth factor (Shoyab et al. (1989) Science 243:1074-1076; Kimura et al. Nature, 348:257-260 (1990) and Cook et al. Mol. Cell. Biol. 11:2547-2557 (1991)); betacellulin (Shing et al. Science, 259:1604-1607 (1993) & Sasada et al., Biochem. Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem. 270:7495-7500 (1995) & Komurasaki et al. Oncogene, 15:2841-2848 (1997)); heregulin (see below); neuregulin-2 (NRG-2) (Caraway et al. Nature 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al., Oncogene 18:2681-89 (1999)) or crypto (CR-1) (Kannan et al., J. Biol. Chem. 272(6):3330-3335 (1997)). ErbB ligands that bind to EGFR, are EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands that bind to ErbB3, are heregulin. ErbB ligands, the ability to communicate with ErbB4, are betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulin. ErbB ligand may also be a synthetic ligand of ErbB. This synthetic ligand may be specific to a specific ErbB receptor, or it can recognize the specific complexes of ErbB receptors. An example of a synthetic ligand is a synthetic heregulin/Chimera “EGF-biregular” (see, e.g., Jones et l. (1999) FEBS Letters 447:227-231, which is introduced in the present description by reference).

“Heregulin” (HRG) refers to the polypeptide encoded by the gene product heregulin described in U.S. patent No. 5641869 or Marchionni et al., Nature, 362:312-318 (1993). Examples of heregulin are heregulin-α, heregulin-β1, heregulin-β2 and heregulin-β3 (Holmes et al. Science, 256:1205-1210 (1992) and U.S. patent No. 5641869); the factor of neu differentiation (NDF) (Peles et al. Cell 69:205-216 (1992)); the factor inducing acetylcholine receptor (ARIA) (Falls et al. (1993) Cell 72:801-815); growth factors glial cells (GGF) (Marchionni et al., Nature 362:312-318 (1993)); the factor derived from afferent and motoneurons (SMDF) (Ho et al., J. Biol. Chem. 270:14523-14532 (1995)); γ-heregulin (Shaefer et al., Oncogene, 15:1385-1394 (1997)). The term includes biologically active fragments and/or variants of the amino acid sequence of native HRG polypeptide, such as a fragment of the EGF-like domain (for example, HRGβ1177-244).

“Heterooligomeric ErbB represents ecovalence linked oligomer comprising at least two different ErbB receptor. “ErbB dimer” is ecovalence linked oligomer comprising at least two different ErbB receptor. Such complexes can be formed in the case, if a cell expressing two or more ErbB receptors, is exposed to an ErbB ligand. Oligomers ErbB, such as ErbB dimers, can be selected PU is eat and thus analyzed by electrophoresis in SDS page with LTOs, as described, for example, Sliwkowski et al. J. Biol. Chem., 269(20):14661-14665 (1994). Examples of such heterooligomeric ErbB are complexes of EGFR-ErbB2 (also known as HER1/HER2), ErbB2-ErbB3 (HER2/HER3,) and ErbB3-ErbB4 (HER3,/HER4). In addition, heterooligomeric ErbB may contain two or more receptors ErbB2, combined with other ErbB receptors, such as ErbB3, ErbB4, or EGFR (ErbB1). The composition of heterooligomeric may include other proteins, such as receptor subunit cytokines (e.g., gp130).

Polypeptide with a “native sequence” is a polypeptide that has the same amino acid sequence as the polypeptide, for example, the natural receptor for tumor-associated antigen. Such polypeptides with a native sequence can be isolated from natural sources or they may be obtained by recombinant methods or methods of synthesis. For example, the polypeptide with a native sequence can have the amino acid sequence of natural human polypeptide, mouse polypeptide or polypeptide derived from mammals of any other kind.

The term “variant amino acid sequences” means polypeptides having amino acid sequences that are to some extent different from the native polypeptide sequence. Usually options amino is slotnik sequences at least about 70% homologous, at least one binding to the receptor domain of the natural ligand or at least one binding ligand domain of the native receptor, such as tumor-associated antigen, and preferably they can be at least about 80%, and more preferably at least about 90%homologous domains, bind to this receptor or ligand. Variants of amino acid sequences have substitutions, deletions and/or insertions at certain positions of the native amino acid sequence.

“Sequence identity” is defined as the percentage of residues in the variant amino acid sequence that are identical after aligning the sequences and include gaps, if necessary to achieve the maximum percent identity of the sequences. Methods and computer programs for such justification. One such computer program is “Align 2”, developed by Genentech, Inc. and submitted along with the documentation for a user in the Office for the protection of copyright, Washington, DC 20559, December 10, 1991.

“Antibody-dependent cellular cytotoxicity” and “ADCC” means a cell-mediated reaction, to the th nonspecific cytotoxic cells, that Express Fc receptors (FcRs) (e.g., natural killer cells (NK), neutrophils and macrophages), recognize bound antibody on the target cell, resulting in lysis of the target cells. Primary cells mediating ADCC, that is, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. Data FcR expression on hematopoietic cells systematized in table 3 on page 464 of Ravetch & Kinet (1991) Annu. Rev. Immunol. 9:457-92. To assess ADCC activity of interest molecules can be evaluated ADCCin vitrosuch as analysis, described in U.S. patent No. 5500362 or 5821337. Suitable effector cells for such assays are mononuclear cells of peripheral blood (MCPC) and natural killer cells (NK). Alternative or additionally, ADCC activity of interest of a molecule can be evaluated byin vivofor example, in murine models, such as the model described Clynes et al., Proc. Natl. Acad. Sci., USA, 95:652-656 (1998).

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

“Complement-dependent cytotoxicity” or “CDC” means the ability of a molecule to undergo lysis of the target in the presence of complement. Path activation of complement is initiated by binding of the first component of the complement system (C1q) with a molecule (e.g. antibody), forming a complex with cognatum antigen. To assess activation of complement can be evaluated CDC, e.g. the measures as described in Gazzano-three-bet et al., J. Immunol. Methods, 202:163 (1996).

The term “variable” refers to certain portions of the variable domains, which have significant differences in the sequences of different antibodies and are involved in the binding of each antibody with its specific antigen, as well as determine the specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across all variable domains of antibodies. It is concentrated in three segments called hypervariable regions that are present in the variable domains, both light and heavy chains. The most highly conserved part of the variable domains are called the frame regions (FR). Each variable domain of native heavy and light chains contain four FR with mainly β-folded configuration and connected by three hypervariable regions, which form loops connecting, and in some cases forming part of β-folded structure. Hypervariable region in each chain are held in close proximity to each other through FR and, with the hypervariable regions of the other chain, involved in the formation antigennegative site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Healt, Bethesda, MD). The constant domains are not directly involved in the binding of an antibody to an antigen, but have different effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

Used herein, the term “hypervariable region” means amino acid residues of an antibody which are responsible for binding to the antigen. This hypervariable region generally contains amino acid residues, derived from the “hypervariable region” or “CDR (complementarity-determining region) (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., see above), and/or residues originating from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia &Lesk (1987) J. Mol. Biol., 196:901-917). The remains of the “framework region” or “FR” represents the remains of the variable domain of the non-specific residues here hypervariable region.

As a result of hydrolysis of the antibody with papain formed two identical antigenspecific fragment, called “Fab”fragments, each of which has one antigennegative website, and one remaining the Fc-fragment, the name of which refers to his ability to easily crystallize. Treatment with pepsin leads to the formation of F(ab')2fragment that has two antigenspecific site and capable of cross-linking antigen.

“Fv” is the minimum antibody fragment that contains a full-sized antigen-recognizing site and antigennegative website. This region consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected with each other by non-covalent bond. This area has such a configuration in which the three hypervariable region of each variable domain interact with each other so that they form antigennegative site on the surface of the dimer VH-VL. In General, six hypervariable regions are reported to the antibody binding specificity to the antigen. However, even a single variable domain (or half of an Fv, containing only three hypervariable region that is specific for an antigen) has the ability to recognize the antigen and to contact him, although with lower affinity than the entire binding site.

Fab-fragment also contains the constant domain of the light chain and the first constant domain (SN) the heavy chain. Fab'fragments differ from Fab fragments by the fact that they have a few extra OST the Cove at the carboxy-end SN-domain heavy chain, including one or more cysteines, derived from the hinge region of the antibody. Used herein, the designation of Fab'-SH means Fab'in which the cysteine(C) residue(s) constant domain has(have)at least one free thiol group. F(ab')2-fragments of antibodies, by their nature, are produced as pairs of Fab'-fragments, among which are cysteine hinge region. Also known and other chemical bond linking the fragments of antibodies.

The “light chains” of antibodies derived from any vertebrate species can be assigned to one of two different types of circuits, called Kappa (k) and lambda (λ), based on amino acid sequences of their constant domains.

“Single-chain Fv fragment or scFv-fragment” of an antibody include the domains VH and VL of the antibody, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further contains a polypeptide linker between domains, VH and VL, which provides education scFv with the structure required for binding to the antigen. Description scFv can be found at Plückthun the Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg & Moore eds., Springer-Verlag, New York, p.269-315 (1994).

The term “diately” means a small antibody fragments with two antihistamine sites, where these fragments comprise variable heavy domain t is PI (VH), coupled with the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). If the linker is too short for pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific site. Diately described in more detail, for example, in EP 404097; WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci., USA, 90:6444-6448.

“Gumanitarnye forms of non-human antibodies (e.g., antibodies rodents) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part gumanitarnye antibodies are human immunoglobulins (recipient antibody)in which residues, derived from the hypervariable region of this antibody-recipient, replaced by residues derived from the hypervariable region of a non-human antibody (donor antibody)such as mouse antibody, rat antibody, rabbit antibody or antibody primates, non-human, where these antibodies possess the desired specificity, affinity and binding capacity. In some cases, remnants of the framework region (FR) of a human immunoglobulin are replaced by corresponding non-human residues. In addition to t the th, gumanitarnye antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These changes were introduced to improve properties of antibodies. In General gumanitarnoe antibody may contain substantially all or at least one, and typically two, variable domain, in which all or nearly all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin and all or nearly all FR are FR from a human immunoglobulin sequence. Gumanitarnoe antibody also includes, but not necessarily, at least a portion of the constant region of immunoglobulin (Fc), typically human immunoglobulin. A more detailed description, see Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1998) Nature 332:323-329 and Presta (1992) Curr. Op. Struct. Biol., 2:593-596.

Gumanitarnye anti-ErbB2 antibody is huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®)as described in Table 3 of U.S. patent No. 5821337, which is fully introduced into the present description by reference; and gumanitarnye antibodies S (WO 93/21319) and IS described below.

“Isolated” antibody is an antibody that was identified and isolated and/or purified from components of its natural environment. Contaminant components of its natural environment are material is, adversely affect diagnostic or therapeutic efficacy of antibodies, and such components may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments of the invention, the specified antibody can be purified (1) to more than 95% by weight of antibody as may be determined by the method of Lowry, and more preferably more than 99% by weight antibodies, (2) to the extent sufficient for the introduction of at least 15 residues of N-terminal or internal part of the amino acid sequence, using a sequencing machine, equipped with a centrifuge vessel, or (3) to homogeneity by electrophoresis in LTO-page in reducing or non conditions with staining of Kumasi preferably blue or silver. The term “isolated antibody” includes antibodyin situin recombinant cells, if there is at least one natural component of this antibody. Typically, however, the selected antibody can be obtained, at least in one stage of cleaning.

An antibody that “binds” with interest antigen, the ability to communicate with the specified antigen with an affinity that is sufficient for this antibody could be used for delivery to a cell expressing this antig the N.

An antibody that “induces apoptosis”is an antibody that induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, the shrinkage of the cells, the expansion of the endoplasmic reticulum, fragmentation of cells and/or formation of membrane vesicles (called apoptotic bodies). This cell is a tumor cell, for example cell breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder. To assess the cellular events associated with apoptosis, there are different methods. So, for example, translocation of phosphatidylserine (PS) can be determined by binding of annexin; DNA fragmentation can be evaluated by the formation of DNA ladder; and condensation cores/chromatin with DNA fragmentation can be evaluated by any increase hypodiploidy cells.

The term “disturbance or disorder” means any condition, the symptoms of which may be impaired as a result of treatment according to the invention. Such disorders or disorders are chronic and acute disorders or diseases including pathological condition that can lead to the development of the mammal in question is ustroystva or violations. Non-limiting examples of disorders, being treated in accordance with the present invention, are benign and malignant tumors; leukemias and lymphoid malignancies, particularly breast cancer, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder; disorders associated with damage to the nerve cells, glial cells, astrocytes, hypothalamic cells and other glandular cells, macrophages, epithelial cells, stromal cells and blastcleaning cells, and inflammatory, angiogenic and immunologic disorders or disorders.

The term “therapeutically effective amount” means a quantity of a drug effective for treating diseases or disorders in a mammal. In the case of such therapeutically effective quantity of a drug may reduce the number of cancer cells and tumor cells; inhibiting (that is, slow to some extent and preferably stop) infiltration of cancer cells into peripheral organs; inhibit (i.e., to slow to some extent and preferably stop) the development of tumor metastasis; inhibition, to some extent, increased the tumor; and/or alleviate to some extent one or more symptoms associated with cancer. Drug, to some extent, can prevent the growth of cancer cells and/or to destroy cancer cells, and it may be cytostatic and/or cytotoxic. The effectiveness of anticancer therapy can be assessed, for example, by determining the time before disease progression (TTP) and/or determine the speed of response (RR).

The term “significant amount” means the greater part, that is >50% of the entire population, the entire collection or just a sample.

The term “intracellular metabolite” means a compound formed as a result of intracellular metabolic processes or reactions, which is subjected to conjugate the antibody-drug” (ADC). Such metabolic processes or reactions can be enzymatic reaction, such as proteolytic cleavage of the peptide linker ADC or hydrolysis of functional groups, such as hidradenoma group, ester group or amide group. Intracellular metabolites include, but are not limited to, antibodies and available medicines, which are intracellular cleavage after delivery into the cell, diffusion in the cell, the absorption cell or transport into the cell.

The terminology is s “split inside the cell and intracellular cleavage” refers to intracellular metabolic process or reaction, faced conjugate “drug-ligand”conjugate “drug-linker-ligand, the conjugate antibody-drug” (ADC) or the like, resulting in a covalent bond, such as a linker between the component “drug” (D) and antibody (Ab) is cleaved, resulting in the removal of free drug from the antibody inside the cell. Thus, molecules, derived from conjugate “drug-ligand”conjugate “drug-linker-ligand” or ADC are intracellular metabolites.

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

The term “cytotoxic activity” means cytolytic, cytostatic or antiproliferative effect of connection type conjugate the antibody-drug” or intracellular metabolite. Cytotoxic activity can be expressed as the value of the IC50that made the focus of a concentration (molar or mass) per unit volume, at which survives half cells.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by dysregulation of cell growth. The term “tumor” includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers are squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer (“nmcl”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the abdominal cavity, hepatocellular cancer, gastric cancer, including cancer of the gastrointestinal tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, cancer of the colon, carcinoma of the endometrium or uterine carcinoma, salivary gland, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, carcinoma of the anus, penis carcinoma, and head and neck cancer.

“A cancerous tumor that expresses ErbB2 is a tumor on the surface of cells which are produced is definitely the levels of ErbB2, sufficient for the anti-ErbB2 antibody could bind to ErbB2 and had a therapeutic anti-cancer effect.

Cancer, “characterized by excessive activation of the ErbB2 receptor, is a cancer, wherein the degree of activation of the ErbB2 receptor in cancer cells significantly exceeds the degree of activation of this receptor in non-cancerous tissue cells of the same type. Such excessive activation may be a result of overexpression of ErbB2 receptor and/or the presence of levels higher than the normal level of ErbB2 ligand, available for activation of the ErbB2 receptor in cancer cells. This over-activation can lead to malignant cancer cells and/or may be caused by cancer cells. In some embodiments of the invention can be carried out diagnostic or prognostic analysis of cancer in order to determine whether the amplification and/or overexpression of ErbB2 receptor, leading to such excessive activation of the ErbB2 receptor. Alternative or in addition may be carried out diagnostic or prognostic analysis of cancer in order to determine whether the amplification and/or overexpression of ErbB2 ligand in cancer, which causes excessive activation of this receptor. In subpopulations such cancers over-activation on the frame of the receptor may be due to autocrine mechanism of stimulation.

Cancer, “sverkhekspressiya” ErbB2-receptor, is a cancerous tumor, which, on the surface of their cells have significantly higher levels of ErbB2-receptor compared to non-cancerous tissue cells of the same type. Such overexpression may be due to amplification or increased transcription or translation of the gene. Overexpression of ErbB2-receptor can be determined in a diagnostic or prognostic analysis by detection of elevated levels of ErbB2 protein present on the cell surface (for example, using immunohistochemical analysis; tissues. Alternative or additionally can be measured levels of ErbB2-coding nucleic acid in the cell, for example, by fluorescent hybridizationin situ(FISH; see WO 98/45479), southern blotting, or polymerase chain reaction (PCR), such as quantitative real-time PCR (RT-PCR). Overexpression of ErbB2 ligand can be defined in the diagnostic analysis by measuring levels of the ligand (or nucleic acid that encodes the ligand) in a patient, for example, by biopsy of the tumor or by using various diagnostic tests, such as tissues, FISH, southern blotting, PCR orin vivothe analyses described above. Can be also carried out studies on the overexpression of ErbB2 receptor by measuring the level of“exfoliating” antigen (for example, the extracellular domain of ErbB2) in a biological fluid such as serum (see, for example, U.S. patent No. 4933294; WO 91/05264; U.S. patent No. 5401638 and Sias et al. (1990) J. Immunol. Methods, 132:73-80). In addition to the above analyses, practitioners known, and various other testsin vivo. For example, cells in the body of the patient can be treated with an antibody that marked optional detectable label such as a radioactive isotope, and the binding of an antibody with cells of a patient can be assessed, for example, by external scanning for radioactivity or by analysis of the biopsy specimen taken from a patient previously treated with antibody.

Tumor sverkhekspressiya HER2, assessed by immunohistochemical scale corresponding to the number of copies expressed HER2 molecules on the cell, and such assessment may be carried out using biochemical analysis, where the scores of this scale mean: 0=0-10000 copies/cell; 1+=at least about 200,000 copies/cell; 2+=at least about 500000 copies/cell; 3+=1-2 x 106copies/cell. Overexpression of HER2 at level 3+, which leads to ligand-independent activation of the tyrosine kinase (Hudziak et al. (1987) Proc. Natl. Acad. Sci., USA, 84:7159-7163), is observed in approximately 30% of patients with breast cancer, and these patients there is a decrease in the duration of relapse-free period is Yes and life expectancy in General (Slamon et al. (1989) Science, 244:707-712; Slamon et al. (1987) Science, 235:177-182).

Conversely, cancer, which is not characterized by overexpression of ErbB2 receptor”is defined as cancer, in which according to the diagnostic analysis is not expressed levels of ErbB2-receptor, higher than normal levels of this receptor, compared to non-cancerous tissue cells of the same type.

Used herein, the term “cytotoxic agent” means a substance that inhibits or prevents the function of cells and/or causes destruction of cells. This term shall include radioactive isotopes (e.g.,211At,131I125I90Y186Re,188Re,153Sm212Bi32R60C and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, including their synthetic analogs and derivatives. In one aspect of the invention, the term does not include radioactive isotopes.

“Chemotherapeutic agent” is a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents are alkylating agents, such as thiotepa and cyclophosphamide CYTOXAN®; acesulphame, such as busulfan, improsulfan and piposulfan; aziridines, is such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; TLK 286 (TELCYTATM); acetogenin (in particular, bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulin acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMTOSAR®), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podofillina acid; teniposide; cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and SW-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard gas analogues, such as chlorambucil, chlornaphazine, chloroformed, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy similar mustard gas; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; bisphosphonates, such as clodronate; antibiotics such as enediyne antibiotics (for example, calicheamicin, while cha is in the surrounding area calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)) and the anthracyclines, such as annamycin, AD 32, algarobilla, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubitsin, KRN5500, menogaril, dynemicin, including dynemicin And, spiramycin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotics-chromophores, aclacinomycin, cactinomycin, autralian, azaserine, bleomycin, aktinomitinov, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morphosyntactical, cyanomethaemoglobin, 2-pyrroline doxorubicin, liposomal doxorubicin and desoxidation), zorubicin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, clamycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; analogs of folic acid, such as deeperin, peripherin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre and tioguanin; pyrimidine analogs, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane and testolactone; antiadrenergics means, such as aminoglutethimide, mitotane, trilostane; means supplying the deficiency of folic acid, such as folinovaya acid (leucovorin); Eagleton; antitumor folate antagonists, such as ALIMTA®, pemetrexed LY231514, inhibitors of dihydrofolate-reductase, such as methotrexate, an antimetabolite such as 5-fluorouracil (5-FU) and its prodrugs, such as UFT, S-1 and capecitabine, inhibitors of thymidylate synthase, inhibitors glycinamide-ribonucleotide-formyltransferase, such as raltitrexed (TOMUDEXRM, TDX); inhibitors dihydropyrimidine-dehydrogenase, such as eniluracil; glycoside aldophosphamide; aminolevulinic acid; amsacrine; astroball; bisantrene; edatrexate; definin; demecolcine; diazinon; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; 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; tenotomy acid; creation; 2,2',2”-trihlortrietilamin; trichothecenes (in particular, the toxin T-2, verrucarin And, roridin and unguided); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman Galitsin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid and taxanes, such as paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETMthat does not contain cremophor drug paclitaxel from the nanoparticles obtained on the basis of albumin (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-tioguanin; mercaptopurine; platinum; platinum analogues or analogues obtained on the basis of platinum, such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinylchloride; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, oxides or derivatives of all the above compounds, and combinations of two or more of the above compounds, such as CHOP, where this abbreviation refers to the combination of therapeutic agents, such as cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, where this abbreviation refers to the combination of therapeutic agents, such as oxaliplatin (ELOXATINTM), 5-FU and leucovorin.

In the scope of this term are also antihormone means of regulating or inhibiting the action of hormones on SDA is Oli, such as antiestrogens and selective modulators of estrogen receptor (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; aromatase inhibitors that inhibit the enzyme aromatase and regulate the production of estrogen in the adrenal cortex, such as 4(5)-imidazoles, aminoglutetimid, acetate megestrol MEGASE®, exemestane AROMASIN®, formestane, fadrozole, vorozole RIVISOR®, letrozole, FEMARA® and anastrozole ARIMIDEX®; and anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide and goserelin; as well as troxacitabine (nucleoside casinoby similar 1,3-dioxolane); antisense oligonucleotides, particularly oligonucleotides that inhibit expression of genes in signal transduction pathways involved in proliferation of undesirable cells, such as, for example, PKC-alpha, Raf, H-Ras and the receptor for epidermal growth factor (EGF-R); vaccines such as vaccines for gene therapy, for example a vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®; rIL-2 PROLEUKIN®; topoisomerase inhibitor 1 LURTOTECAN®; rmRH ABARELIX® and pharmaceutically acceptable salts, acids or derivatives of all the above compounds.

Used herein, the term “EGFR-drug” means a therapeutic agent that binds to EGFR and, optionally, inhib which induces the activation of EGFR. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR, are MAb 579 (ATSS CRL HB 8506), MAb 455 (ATSS CRL HB 8507), MAb 225 (ATSS CRL 8508), MAb 528 (ATSS CRL 8509) (see U.S. patent No. 4943533, Mendelsohn et al.) and their variants, such as chimeric antibody 225 (S or cetuximab; ERBITUX®) and reconstructed human antibody 225 (N) (see WO 96/40210, Imclone Systems Inc.); antibodies that bind to mutant EGFR type II (U.S. patent No. 5212290); gumanitarnye and chimeric antibodies that bind to EGFR, as described in U.S. patent No. 5891996; and human antibodies that bind to EGFR, such as ABX-EGF (see WO 98/50433, Abgenix). Antibody against EGFR may be conjugated with a cytotoxic agent, forming, thereby, immunoconjugate (see, for example, EP A, Merck Patent GmbH). Examples of small molecules that bind to EGFR, are ZD1839 or Gefitinib (IRESSATM, Astra Zeneca), erlotinib-HCl (CP-358774, TARCEVATM; Genentech/OSI) and AG1478 effect, AG1571 (SU 5271; Sugen).

“Tyrosine kinase inhibitor” is a molecule, which to some extent inhibits tyrosinekinase tyrosine kinase activity, such as the ErbB2 receptor. Examples of such inhibitors are EGFR-drugs”described in the previous paragraph, as well as hintline, such as PD 153035, 4-(3-chloroanilino)hinzelin, pyridopyrimidines, pyrimidopyrimidine pyrrolopyrimidine, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloylmethane, 4,5-bis(4-foronline)phthalamide), tyrphostin containing nitrothiophene group; PD-0183805 (Warner-Lambert); antisense molecules (e.g. molecules that bind to ErbB-encoding nucleic acid); cinoxacin (U.S. patent No. 5804396); triforine (U.S. patent No. 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); inhibitors pan-ErbB, such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); mesilate of imatinib (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone), or compounds described in the following publications patents and patent applications: U.S. patent No. 5804396; WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc.); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca) and WO 96/33980 (Zeneca).

The term “angiogenic agent” means a compound that inhibits the development of blood vessels or to some extent hamper such development. This angiogenic factor may be, for example, a small molecule or antibody that bind to a growth factor or growth factor receptor involved in the stimulation of angiogenesis. In one embodiment of the invention specified angiogenic factor is an antibody that binds to VA the molecular endothelial growth factor (VEGF).

The term “cytokine” is a generic term that refers to proteins that are released by one cell population and which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and standard polypeptide hormones. In addition to the above definitions, the term “cytokine” includes growth hormones such as human growth hormone, N-nationally human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); a growth factor for hepatocytes, fibroblast growth factor; prolactin; placental lactogenic; tumor necrosis factor-α and-β; Millerovskiy inhibitory factor; peptide associated with mouse gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors tissues, such as NGF-β; platelet-growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); the factors that induce osteogenesis; interferons such as interferon-α, -β and-γ; colony stimulating factors (CSFs)such as macrophage CSF (M-CSF); granulocyte-Christ.tion-macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (IL)such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and ligand it (KL). Used herein, the term “cytokine” includes proteins derived from natural sources or from recombinant cell culture and biologically active equivalents of the cytokines with the native sequence.

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

“Liposome” is a small vesicles composed of lipids of different types of phospholipids and/or surfactant that can be used to deliver drugs, such as anti-CD30, anti-CD40, anti-CD70 antibody or antibodies against Lewis-Y and, optionally, a chemotherapeutic agent) to a mammal. Components of liposomes are usually located so that they form a bilayer, similar to the lipid bilayer in biological membranes. Used herein, the term “liner in packaging” means instructions, which are usually invested in available-for-sale packaging of therapeutic products and in which there is information concerning the indications, route of administration, dose, method of administration, contraindications and/or warnings concerning the use of such therapeutically the products.

“Isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one impurity molecules of nucleic acid with which it is normally associated in the natural source of the nucleic acid that encodes the antibody. The selected nucleic acid molecule, in its form or structure, different from the molecules found in nature. Therefore, the selected nucleic acid molecules differ from the nucleic acid molecules contained in natural cells. However, the selected nucleic acid molecule includes a nucleic acid molecule contained in cells that normally Express the antibody, where, for example, the nucleic acid molecule is located in the chromosome at the position different from its position in natural cells.

The term “regulatory sequence” means a DNA sequence necessary for the expression of the operatively linked coding sequence in a particular organism, the host. Regulatory sequences that are suitable for prokaryotes, for example, are the promoter, optional operator sequence and the site of binding to the ribosome. It is known that eukaryotic cells are used promoters, polyadenylation signals shall enhancers.

Nucleic acid is operatively linked”when it is in functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader sequence is functionally attached to DNA that encodes a polypeptide if it is expressed in the form of a protein precursor which participates in the secretion of the polypeptide; or a promoter or enhancer functionally attached to a coding sequence if it affects the transcription of the sequence; or a binding site with the ribosome is functionally attached to the coding sequence, if the location of this site makes the broadcast. In General, the term “functionally linked” means that the DNA sequences are contiguous and, in the case of a secretory leader sequence they are adjacent and are in the same reading frame. However, the enhancers should not be adjacent to the join sequence. Connection can be made by ligating the appropriate restriction sites. If such sites are present, in accordance with standard practice can be used synthetic oligonucleotide adaptors or linkers.

Used herein, the terms “cell”, “cell is ine” and “cell culture” are used interchangeably, and they are all the progeny of these cells. For example, the words “transformants” and “transformed cells” include the primary cells and stem from them culture without specifying the number of subcultures. It should be noted that all progeny may not be precisely identical in composition DNA, due to the artificially introduced or spontaneous mutations. These terms also include mutant progeny that have the same function or biological activity, which was established during the initial screening of the transformed cells. A more precise definition of these terms will be clear from the context of the following description.

Used herein, the term “autoimmune disease” refers to diseases or disorders that occur in native tissues of the individual and against these tissues, or accompanying diseases or their symptoms or condition developing on the basis of these disorders. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis and ankylosing spondylitis, psoriasis, dermatitis, including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria; polymyositis/dermatomyositis, toxi is a mini-epidermal necrosis, systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis and inflammatory bowel diseases with accompanying pyoderma gangrenosum, nodular erythema, primary sclerosing cholangitis and/or episcleritis; respiratory distress syndrome, including respiratory distress syndrome, adult (rdsw); meningitis, IgE-mediated diseases such as anafilaktichesky and allergic rhinitis, encephalitis such as Rasmussen encephalitis; uveitis; colitis, such as histologically diagnosed with colitis and collagenosis colitis; glomerulonephritis (GN), such as membranous GN, idiopathic membranous GN, membranous proliferative GN (MPGN), including glomerulonephritis type I and type II, and rapidly progressive GN, allergic conditions, eczema, asthma, conditions associated with infiltration of T cells and chronic inflammatory responses; atherosclerosis; autoimmune myocarditis; condition associated with impaired leukocyte adhesion; systemic lupus erythematosus (SLE)such as cutaneous SLE, lupus (including nephritis, cerebra, children lupus, non-renal lupus, discoid lupus, alopecia); juvenile diabetes; multiple sclerosis (PC), such as spinal-ophthalmic PC; allergic encephalomyelitis; immune reactions, associated the e with acute and delayed hypersensitivity, mediated by cytokines and T-lymphocytes; tuberculosis; sarcoidosis; Wegener, including Wegener's granulomatosis, agranulocytosis, vasculitis (including vasculitis of large vessels (including rheumatic rheumatica and giant cell arteritis diagnostics (arthritis, Takayasu's)), vasculitis of medium vessels (including disease Kawasaki and nodular polyarteritis), CNS vasculitis, and ANCA-associated vasculitis, such as vasculitis or syndrome charge-Strauss (CSS)); aplastic anemia; positive anemia of Coombs; anemia diamond-Blackfan; immune hemolytic anemia including autoimmune hemolytic anemia (AIG); pernicious anemia; true red blood cell aplasia (IEA); diseases associated with deficiency of factor VIII; hemophilia A, autoimmune neutropenia; pancytopenia; leukopenia; diseases associated with leukocyte diapedesis; inflammatory disorders of the CNS; syndrome lesions multiple organs; male heavy; diseases, mediated by complex antigen-antibody”; disease glomerular basement membranes catalyzed reaction of the antibody-antigen”; syndrome associated with the production of antibodies against phospholipids; allergic neuritis; Behcet's disease; syndrome Kalman; syndrome?; myasthenic syndrome Lambert-Eaton; Raynaud's syndrome; syndrome of Segren; syndrome Stevens-On the Onsong; rejection of solid organ transplant (including reactions to the preliminary introduction of high titers test angegeben reactive antibodies to IgA deposition in tissues and graft rejection kidney transplant liver transplant small bowel transplant hearts, etc.); graft-versus-host (GVHD); bullous pemphigoid; bladderwort (including vulgar disease, the leaf bladderwort and pemphigoid mucous membranes, resembling a disease); autoimmune polyendocrinopathy; disease Reiter; syndrome stiff man”; immune complex nephritis; IgM polyneuropathy or IgM-mediated neuropathy; idiopathic thrombocytopenic purpura (ETC); thrombotic thrombocytopenic purpura (TTP); thrombocytopenia (developing, for example, in patients with myocardial infarction), including autoimmune thrombocytopenia; autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary gipoterioz; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis; idiopathic hypoparathyroidism; Addison disease; graves ' disease; autoimmune polyglandular syndromes or syndromes polyglandular endocrinopathies); diabetes type I, also called insulin-dependent diabetes di is beta (IDDM), including IDDM in children and Sheehan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), obliterating brodholt (not transmitted), in contrast to the NSIP; Guillain-Barre syndrome; disease Berger (IgA nephropathy), primary biliary cirrhosis; intestinal sprue (gluten enteropathy); refractory sprue with extensive lesions dermatitis herpetiformis; cryoglobulinemia; amyotrophies lateral sclerosis (ABS; disease Louis Gehrig); ischemic heart disease; autoimmune disease of the inner ear (CBA); autoimmune alopecia; syndrome “dancing eyes” (LNG); polyhedric such as intractable polyhedra; pulmonary alveolar proteins; amyloidosis; giant cell hepatitis; scleritis; monoclonal gammopathy unclear/unknown etiology (MGUS); peripheral neuropathy, paraneoplastic syndrome; “kalapati”, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, “kalapati” of the CNS, autism, inflammatory myopathy, and focal segmental glomerulosclerosis (OSGS).

“Alkyl” represents a C1-C18the hydrocarbon containing normal, secondary, tertiary carbon atom or a carbon atom in the ring. Examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH ), 2-propyl (i-Pr, isopropyl, CH2(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, C(CH3)3), 1 pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2(CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)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).

“Alkene which l represents a C 2-C18the hydrocarbon containing normal, secondary, tertiary carbon atom or a carbon atom in the ring, at least one unsaturated bond, i.e. a carbon-carbon bond andsp2-double bond. Examples include, but are not limited to, ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (C5H7) and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

“Quinil” is a2-C18the hydrocarbon containing normal, secondary, tertiary carbon atom or the carbon atom on the ring, at least one unsaturated bond, i.e. a carbon-carbon bond andspa triple bond. Examples include, but are not limited to, acetylene (-C≡CH) and propargyl (-CH2WITH≡CH).

“Alkylene” represents a saturated, branched-chain or cyclic hydrocarbon moiety consisting of 1 to 18 carbon atoms and having two monovalent radical center formed by removing two hydrogen atoms from two identical or different carbon atoms of the original alkane. Typical alkionovymi radicals include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2With the 2and so on

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

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

“Aryl” represents a monovalent aromatic hydrocarbon radical consisting of 6-20 carbon atoms and formed by removing one hydrogen atom from a single carbon atom of the original aromatic system. Some of the aryl group in the representative structures designated as “Ar”. Typical aryl groups include, but are not limited to, Radik the crystals, derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc.

“Arylalkyl” is an acyclic alkyl radical in which one of the hydrogen atoms associated with carbon atom, typically a terminal carbon atom, orsp3-carbon atom, substituted aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-Penilaian-1-yl, 2-Penilaian-1-yl, naphthylmethyl, 2-Nettleton-1-yl, 2-naphthalate-1-yl, naphthalenyl, 2-naphthenate-1-yl, etc. Arylalkyl group contains from 6 to 20 carbon atoms; for example, the alkyl part, including albanello, alkenylphenol or alkenylphenol part arylalkyl group has 1-6 carbon atoms and the aryl part has from 5 to 14 carbon atoms.

“Heteroaromatic” is an acyclic alkyl radical in which one of the hydrogen atoms associated with carbon atom, typically a terminal carbon atom, orsp3-carbon atom, substituted heteroaryl radical. Typical heteroarylboronic groups include, but are not limited to, 2-benzimidazolylthio, 2-purolater etc. Heteroallyl group has from 6 to 20 carbon atoms; for example, the alkyl part, including albanello, alkenylphenol or alkenylphenol part heteroallyl group has 1-6 carbon atoms, and heteroaryl the portion has 5 to 14 carbon atoms and 1-3 heteroatoms, selected from N, O, P and S. the Heteroaryl part heteroallyl group may be a monocycle having 3 to 7 members in the ring (2-6 carbon atoms), or Bicycle having from 7 to 10 members on the ring (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P and S), for example bicyclo[4,5]-, [5,5]-, [5,6]- or [6,6]system.

The terms “substituted alkyl”, “substituted aryl” and “substituted arylalkyl” mean alkyl, aryl and arylalkyl respectively, in which one or more hydrogen atoms are independently replaced with a substituent. Typical substituents include, but are not limited to, -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, RHO3-, RHO3H2-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-C18alkyl, C6-C20aryl, C3-C14a heterocycle, a protective group or a group of prodrugs. Allenova, Alcanena and akinlana group described above may also be substituted analogues is mainly.

“Heteroaryl” and “heterocycle” represents a cyclic system in which one or more atoms on the ring are heteroatoms, such as nitrogen atoms, oxygen and sulfur. Heterocyclic radical contains 1-20 carbon atoms and 1-3 heteroatoms selected from N, O, P and S. the Heterocycle may be a monocycle having 3 to 7 members in the ring (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P and S), or Bicycle having from 7 to 10 members on the ring (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P and S), for example bicyclo[4,5]-, [5,5]-, [5,6]- or [6,6]system.

The heterocycles described in 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 is, for example, but not limited to, pyridyl, dihydropyridin, tetrahydropyranyl (piperidyl), thiazolyl, tetrahydrothiophene, oxidized grey tetrahydrothiophene, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianeptine, indolyl, indolinyl, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, b is s-tetrahydropyranyl, tetrahydroquinoline, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6N-1,2,5-thiadiazine, 2N,6N-1,5,2-detainer, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine 2N-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazole, purinol, 4H-hemolysins, phthalazine, apterygial, honokalani, hintline, cinnoline, pteridine, an-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, oxindoles, benzoxazolyl and satanail.

Non-limiting examples linked through carbon heterocycles heterocycles are linked in position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5 or 6 pyridazine, in position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5 or 6 pyrazine, in position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, in position 2, 4 or 5 oxazole, imidazole or thiazole, position 3, 4 or 5 isoxazol, pyrazole or isothiazole, in position 2 or 3 of aziridine, put in the 2, 3 or 4 azetidine, in position 2, 3, 4, 5, 6, 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 of isoquinoline. More typical connected through carbon heterocycles are 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

Non-limiting examples linked through nitrogen heterocycles 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; in position 2 of a isoindole, or isoindoline in position 4 of the research and in position 9 carbazole or β-carboline. The most typical linked through nitrogen heterocycles are 1-ezyrider, 1-azetidin, 2-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

“Carbocycle” represents a saturated or unsaturated monocyclic ring having 3-7 carbon atoms, or a bicyclic ring having 7-12 carbon atoms. Monocyclic carbocycle have 3-6 carbon atoms in the ring, and usually 5 or 6 carbon atoms in the ring. Bicyclic carbocycle have 7-12 atmosukarto on the ring, for example, in the form of bicyclo-[4,5]-, -[5,5]-, -[5,6]- or [6,6]system, or 9 or 10 carbon atoms in the ring, in the form of bicyclo-[5,6]or [6,6]system. Examples of monocyclic carbocycles are 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.

The terms “linker”, “linker component” or “link” means a chemical group that contains covalent bond or a chain of atoms that covalently bind the antibody to the molecule drugs. In various embodiments of the invention the linker is denoted by LU. The linker is dwuhvalentny radical, such as alkerdeel, areldil, heteroaryl, groups such as -(CR2)nO(CR2)n-, repeating units of alkyloxy (for example, polietilene, PEG, polymethylenes), alkylamino (for example, polyethylenimine, JeffamineTM), and dibasic ester and amides including succinate, succinamide, diglycolate, malonate and caproamide.

The term “chiral” refers to molecules that do not align the overlay with its mirrored counterpart, while the term “achiral” refers to molecules that are superimposed with its mirrored counterpart.

The term “stereoisomers” refers to compounds that have the t identical chemical structure, but different spatial arrangement of atoms or groups.

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

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

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

Examples of “patient” include, but are not limited to, human, rat, mouse, Guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird, and poultry. In a representative embodiment of the invention the patient is a man.

The term “aryl” means a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracene. Carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or may be substituted by one or more groups of the mi, including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl), aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2-C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, -NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

Used herein, the term “C1-C8alkyl” means straight or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “C1-C8alkyl” groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl, and branched C1-C8alkilani are, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl; unsaturated With1-C8alkilani are, but are not limited to, 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-pentini, 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-dime alpental, 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. With1-C8the alkyl group may be unsubstituted or may be substituted by one or more groups including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2-C(O)other', -C(O)N(R')2, -NHC(O)R', -SO3R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, -NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

The term “C3-C8carbocycle” means 3-, 4-, 5-, 6-, 7 - or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative3-C8carbocycle are, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, cycloheptyl, -1,3-cycloheptadiene, -1,3,5-cycloheptatriene, cyclooctyl and cyclooctadiene. With3-C8the carbocyclic group may be unsubstituted or may be substituted by one or more groups including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl)-, aryl-C(O)R', -OC(O)', -C(O)OR', -C(O)NH2-C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

The term “C3-C8carbocycle” means3-C8carbocyclic group defined above, where one of the hydrogen atoms of the carbocyclic group is replaced by a link.

The term “C1-C10alkylene” means a straight saturated hydrocarbon group of the formula -(CH2)1-10-. Examples1-C10alkylene are methylene, ethylene, propylene, butylene, pentile, hexylen, reptile, octiles, Nonlin and decalin.

The term “Allen” means an aryl group which has two covalent bonds and may be present in ortho-, meta - or para-configurations, as shown in the following structures:

where the phenyl group can be unsubstituted or may be substituted by one to four groups including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl)-, aryl-C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2-C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, -NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

The term “C3-C8a heterocycle” oznake is aromatic or nonaromatic 3-C8carbocycle, in which one to four carbon atoms in the ring is independently replaced by a heteroatom selected from the group consisting of O, S and N. Representative examples3-C8heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzimidazolyl, coumarinyl, ethenolysis, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, chinoline, pyrimidinyl, pyridinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolin, isoxazolyl and tetrazolyl. With3-C8the heterocycle may be unsubstituted or may be substituted by 1 to 7 groups, including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl)-, -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2-C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, -NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

The term “C3-C8heterocycle” means3-C8heterocyclic group as defined above where one of the hydrogen atoms of the heterocyclic group is replaced by a link. With3-C8heterocycle may be unsubstituted or may be substituted by 1-6 groups, including, but not limited to, -C1-C8alkyl, -O-(C1-C8alkyl)-, -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2-C(O)other', -C(O)NR') 2, -NHC(O)R', -S(O)2R', -S(O)R', -HE, halogen, N3, -NH2-, -NH(R'), -N(R')2and-CN, where each R' is independently selected from H, -C1-C8of alkyl and aryl.

The term “representative compound” means a drug connection or “drug-linker”.

The term “representative conjugate” means the conjugate drug-ligand, in which the drug is cleaved from the specified conjugate “drug-ligand or conjugate “drug-linker-ligand”.

In some embodiments of the invention the above representative compounds and conjugates are present in the selection or in purified form. Used herein, the term “isolated” means a component that is separated from other components, (a) a natural source, such as a cell or cell culture of plant or animal, or (b) a synthetic organic chemical reaction mixture. Used herein, the term “purified” means that after selecting the isolate contains at least 95%, and in another aspect of the invention, at least 98% of a representative compound or a representative of the conjugate by weight of the isolate.

Examples of the “hydroxyl protective group” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl the th ether, tetrahydropyranyloxy ether, benzyl ether, p-methoxybenzyloxy ether, trimethylsilyloxy ether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyloxy ether, triphenylmethylchloride ether, ester acetic acid, substituted esters of acetic acid, pivaloate, benzoate, methanesulfonate and p-toluensulfonate.

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

Used herein, the term “pharmaceutically acceptable salt” means a pharmaceutically acceptable organic or inorganic salt of a representative compound or a representative conjugate. These representative compounds and representative conjugates contain at least one amino group, this amino group can form the corresponding acid salt additive. Examples of such salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, sa is illat, acid citrate, tartrate, oleate, tannat, Pantothenate, bitartrate, ascorbate, succinate, maleate, getitemat, fumarate, gluconate, glucuronate, saharat, formate, benzoate, glutamate, methanesulfonate, aconsultant, bansilalpet, p-toluensulfonate, pamoate (i.e. 1,1'-methylene-bis-(2-hydroxy-3-aftout)). The term “pharmaceutically acceptable salt” may include another molecule, such as acetate ion, succinate ion, or other counterion. This counterion can be any organic or inorganic molecule that stabilizes the charge on the source connection. In addition, pharmaceutically acceptable salt can have in its structure more than one charged atom. If many of charged atoms are part of a pharmaceutically acceptable salt, this salt, there can be many counterions. Consequently, the pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

The terms “pharmaceutically acceptable MES” or “MES” means the complex of one or more solvent molecules and compounds of the present invention, for example, a representative compound or a representative conjugate. Examples of solvents which form pharmaceutically acceptable solvate, include, but are not limited to, the ode, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.

Abbreviations used in this description have the following values: AE means auristatin E.; Vos means N-(tert-butoxycarbonyl), cit is citrulline, dap means dalapon, DCC means 1,3-dicyclohexylcarbodiimide, DCM (DHM) means dichloromethane, DEA means diethylamin, DEAD means diethylazodicarboxylate, DEPC means diethylphosphoramidite, DIAD means diisopropylsalicylic, DIEA means N,N-diisopropylethylamine, dil means daisosasen, DMAP means 4-dimethylaminopyridine, DME means dimethyl ether of ethylene glycol (or 1,2-dimethoxyethane), DMF (DMF means N,N-dimethylformamide, DMSO (DMSO means dimethyl sulfoxide, doe means draftin, dov means N,N-dimethylamine, DNTB means 5,5'-dithiobis(2-nitrobenzoic acid), DTPA means diethylenetriaminopentaacetic acid, DTT means dithiothreitol, EDCI means of the hydrochloride of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EEDQ means 2 ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline, ES-MS stands for mass spectrometry by elektrorazpredelenie, EtOAc means ethyl acetate, Fmoc means N-(9-fluorenylmethoxycarbonyl), gly glycine means, HATU means hexaphosphate O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea, HOBt is 1-hydroxybenzotriazole, HPLC (HPLC) means liquid chromatog is AFEW high pressure, ile means isoleucine, lys means lysine, MeCN (CH3CN) means acetonitrile, MeOH means methanol, Mtr means 4-insidefamily (or 4-methoxytrityl), nor (nor) means (1S,2R)-(+)-norephedrine, RAV means p-aminobenzyl, PBS means phosphate buffered saline (pH 7.4), PEG (PEG) means polyethylene glycol, Ph means phenyl, Pnp means p-nitrophenyl, MS means 6-maleimidomethyl, phe means of L-phenylalanine, PyBrop means hexaphosphate bromo-Tris-pyrrolidinone, SEC means exclusion chromatography, Su means succinimide, TBTU means tetrafluoroborate O-benzotriazol-1-yl-N,N,N,N-tetramethylurea, TFA means triperoxonane acid, TLC (TLC means thin layer chromatography, UV means ultraviolet radiation, and val denotes valine.

Abbreviations used in this description for the linkers have the following values: Val-Cit means valine-citrulline, dipeptide site cleaved by the protease linker; EQUAL means t-aminobenzimidazole; (IU)vc means N-methyl-valine-citrulline, where the linker peptide bond has been modified to prevent cleavage of the linker by cathepsin B; MS(PEG)6HE means maleimidomethyl-glycol; SPP means N-Succinimidyl-4-(2-pyridylthio)pentanoate and SMCC means N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-to rboxylic.

The terms “treat” or “treatment”, if in the context of the description they do not have any other sense, means therapeutic treatment and prophylactic or preventative measures, which are aimed at preventing or slowing down (weakening) of the individual undesired physiological change or disorder, such as the development or progression of cancer. In accordance with this invention, beneficial or desired clinical effects include, but are not limited to, relieving symptoms, reducing the severity of the disease, stable (i.e. not worsening) state, delay or slowing of disease progression, reduction or temporary relief of symptoms and remission (partial or complete), regardless of whether these symptoms detectivesyme or neglectible. The term “treatment” can also mean a longer life expectancy compared with life expectancy in the absence of treatment. The term “individuals in need of treatment” refers to individuals who already suffer from this condition or disorder, and individuals with a predisposition to the condition or disorder, or to individuals who need the warning of this condition or disorder.

What concerns the cancer I, the term “treatment” includes any or all of the following effects, such as preventing the growth of tumor cells, cancer cells or a tumor, preventing replication of tumor cells or cancer cells, the decrease in total tumor mass or a decrease in the number of cancer cells and the weakening of one or more symptoms associated with the illness.

With regard to autoimmune diseases, the term “treatment” includes any or all of the following effects, such as preventing replication of cells associated with autoimmune disease, including, but not limited to, cells producing autoimmune antibody; lower titers of autoimmune antibodies and the weakening of one or more symptoms of an autoimmune disease.

With regard to infectious diseases, the term “treatment” includes any or all of the following effects, such as preventing the growth, reproduction or replication of pathogens causing infectious disease and weakening of one or more symptoms of an infectious disease.

Abbreviations used in the present description for cytotoxic drugs, have the following values: MMAE means monomethyl-auristatin E (mol. weight (MW 718); MMAF means N-methylvaline-valine-daisosasen Dol is informed-phenylalanine (MW 731,5); MMAF-DMEAEA means MMAF with DMAEA (diethylaminoethylamine)having an amide bond with the C-terminal phenylalanine (MW 801,5); MMAF-TEG means MMAF with tetraethylene glycol, etherified with phenylalanine; MMAF-NtBu means N-tert-butyl, attached in the form of amide to-end MMAF; AEVB means valerianaceae of auristatin E associated acid-labile linker with-end AE (732 MW), and AFP means monoamide p-phenylenediamine with C-terminal phenylalanine of auristatin F (732 MW).

4.2. Compounds according to the invention

4.2.1. The compound of formula (Ia)

In one of its aspects the present invention relates to conjugates of “drug-linker-ligand, having the formula Ia:

L--(Aa--Ww--Yy-D)P(Ia)

or their pharmaceutically acceptable salts or solvate,

where: L represents a ligand component;

-Aand-Ww-Yy- is a linker component (LU), where specified in the linker component:

-A - is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

-Y - spacer elements means the component

y is 0, 1 or 2;

R is within the t 1 to about 20; and

-D is a component of the “drug”that has formula DEand DF;

where in each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(the 3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH;

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

where n is an integer from 0 to 6.

In another embodiment, the present invention relates to Lech is the only compounds of the formula Ib:

or their pharmaceutically acceptable salts or solvate,

where: R2selected from hydrogen and C1-C8of alkyl;

R3selected from hydrogen, C1-C8of alkyl, C3-C8carbocycle, aryl, -C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle, and-C1-C8alkyl-(C3-C8heterocycle);

R4selected from hydrogen, -C1-C8of alkyl, -C3-C8carbocycle, -aryl, -C1-C8alkylaryl, -C1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle), where R5selected from H and methyl, or R4and R5taken together with the carbon atom to which they are linked, form a ring of formula -(CRaRb)n-where Raand Rbindependently selected from-H, -C1-C8the alkyl and C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R6selected from H and-C1-C8of alkyl;

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

8independently selected from H, HE, -C1-C8of alkyl, -C3-C8carbocycle and-(C1-C8the alkyl);

R9selected from H and-C1-C8of alkyl;

R10selected from aryl group or-C3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents-C2-C8-alkyl;

R14represents H or-C1-C8alkyl;

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

each of R16independently represents H, -C1-C8alkyl or -(CH2)n-COOH, and

n is an integer from 0 to 6.

In yet another embodiment, the present invention relates to conjugates of “drug-linker-ligand of formula Ia':

Ab--(Aa--Ww--Yy--D)P(Ia')

or to their farmaci is almost acceptable salt or solvate,

where: Ab is an antibody;

But is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer elements the component

y is 0, 1 or 2,

R is in the range from 1 to about 20; and

D is a drug selected from the compounds of formulas DEand DF:

where in each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

or R4and R5taken together form a carbocyclic ring and have the formula -(CRaRb)n-where Raand Rbindependently selected from H, C1-C8the alkyl and C3-C8it is recicla, and n is selected from 2, 3, 4, 5 and 6;

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH;

R18 selected 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 from 0 to 6.

Ab is an antibody covalently associated with one or more molecules of the drug. Antibody Ab is the antibody that binds to CD30, CD40, CD70 and Lewis antigen-Y. In another embodiment of the invention Ab does not include an antibody that binds to ErbB receptor or one or more of receptors (1)-(35), such as:

(1) BMPR1B (the receptor protein of bone morphogenesis type IB, Genbank reg. No. NM_001203);

(2) e (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate, Genbank reg. No. NM_012449);

(4) R (SA, MUC16, Genbank reg. No. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank reg. No. NM_005823);

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 family soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II, Genbank reg. No. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TC) and short cytoplasmic domain (semaphorin) 5B, Genbank reg. No. AV);

(8) PSCA hlg (2700050C12Rik, C530008O1Rik, cDNA RIKEN 2700050C12, cDNA gene RIKEN 2700050C12, Genbank reg. No. AY358628);

(9) ETBR (endothelin receptor type b, Genbank reg. No. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of prostate cancer, Genbank reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M, Genbank reg. No. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma, Genbank reg. No. NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2)or C3DR (C3d/receptor of the virus of Epstein-Barr), or Hs.73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), V, Genbank reg. No. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C, Genbank reg. No. NM_030764);

(17) HER2 (Genbank reg. No. M11730);

(18) NCA (Genbank reg. No. m);

(19) MDP (Genbank reg. No. BC017023);

(20) IL20Rα (Genbank reg. No. AF184971);

(21) Brevican (Genbank reg. No. AF229053);

(22) Ephb2R (Genbank reg. No. NM_004442);

(23) ASLG659 (Genbank reg. No. AX092328);

(24) PSCA (Genbank reg. No. AJ297436);

(25) GEDA (Genbank reg. No. AY260763);

(26) BAFF-R (Genbank reg. No. NP_443177.1);

(27) CD22 (Genbank reg. No. NP-001762.1);

(28) CD79a (CD79A, C79α, associated with immunoglobulin alpha-protein-cladoselache protein that covalently interacts with Ig beta (CD79B) and forms a complex with molecules of IgM on the cell surface, passing the signal that is involved in the differentiation of b-cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (receptor 1 Burkitt lymphoma associated with G-protein receptor that is activated by the chemokine CXCL13, provides migration of lymphocytes and humoral defense, plays a role in infection by HIV-2 and probably in the development SPEED and, lymphoma, myeloma and leukemia, Genbank reg. No. NP_001707.1);

(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes, Genbank reg. No. NP_002111.1);

(31) RG (ion channel opened by the ligand purinergic receptor RH 5; ion channel opening extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may play a role in the pathophysiology of idiopathic dysfunction of the bladder, Genbank reg. No. NP_002552.2);

(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2, Genbank reg. No. NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein of the family of leucine-rich repeats (LRR) type I, which regulates the activation and apoptosis of b-cells, and loss of its function is associated with progressioni the m disease in patients with systemic lupus erythematosus, Genbank reg. No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, the presumed receptor for the Fc domain of immunoglobulin, which contains Ig-like domains of type C2 and ITAM domains, may play a role in the differentiation of b-lymphocytes, Genbank reg. No. NP_443170.1); and/or

(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily 2, the estimated immunoreceptor, which possibly plays a role in the development of b-cells and lymphomagenesis; in some malignant b cells observed dysregulation of the gene by translocation, Genbank reg. No. NP_112571.1).

In one embodiment of the invention-Ww - is a Val-Cit.

In another embodiment of the invention R3, R4and R7independently represent an isopropyl or sec-butyl, and R5represents a-N. In a representative embodiment of the invention, each of R3and R4represents isopropyl, R5represents-H, R7represents a sec-butyl. In yet another variant of the invention, each of R2and R6represents methyl, and R9represents-N.

In another embodiment of the invention each R8represents-OCH3.

In a representative embodiment of the invention, each of R3and R4represents isopropyl, each of R2and R6the stand is made by a methyl, R5represents H, R7represents sec-butyl, each of R8represents-OCH3and R9represents-N.

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

In one embodiment of the invention R10represents aryl.

In a representative embodiment of the invention R10represents phenyl.

In a representative embodiment of the invention when Z represents-O-, R11represents H, methyl or tert-butyl.

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

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

In one aspect of the invention Ab represents SAS, cBR96, cS2C6, c1F6, c2F2, hAc10, hBR96, hS2C6, h1F6 and h2F2.

Representative variants of compounds of formula Ia have the following structure:

or

where L is an antibody, Val represents valine, and Cit is citrulline.

Load the medicinal product is determined by the number R, i.e. the average number of drug molecules per antibody in a molecule (for example, the compounds of formula Ia, Ia' and Ic). Load medicinal product may vary from 1 to 20 molecules of the drug (D) to the ligand (e.g., Ab or mAb). Compositions of the compounds of formulas Ia and Ia' include sets of antibodies conjugated 1-20 molecules drugs. The average number of molecules of drugs to the antibody in the product obtained by the reaction of conjugation can be determined by standard methods such as mass spectroscopy, ELISA analysis and HPLC. Quantitative distribution of conjugates of the ligand-drug” can also be expressed by the symbolp. In some cases, isolation, purification and identification of homogeneous conjugates “ligand-drug”, where p is a certain value, from homogeneous conjugates “ligand-drug” with another load of drugs can be achieved by methods such as reverse-phase HPLC or electrophoresis.

4.2.2. COMPOUND MEDICINES of the FORMULA (Ib)

In another aspect the present invention relates to pharmaceutical compounds of the formula (Ib):

or their pharmaceutically acceptable the salt or the solvate,

where: R2selected from hydrogen and-C1-C8of alkyl;

R3selected from hydrogen, -C1-C8of alkyl, -C3-C8carbocycle, aryl, -C1-C8alkylaryl, -C1-C8alkyl-(C3-C8carbocycle)- C3-C8heterocycle, and-C1-C8alkyl-(C3-C8heterocycle);

R4selected from hydrogen, -C1-C8of alkyl, -C3-C8carbocycle, aryl, -C1-C8alkylaryl, -C1-C8alkyl-(C3-C8carbocycle)- C3-C8heterocycle, and-C1-C8alkyl-(C3-C8heterocycle), where R5selected from H and methyl, or R4and R5taken together with the carbon atom to which they are linked, form a ring of formula -(CRaRb)n-where Raand Rbindependently selected from H, -C1-C8the alkyl and-C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R6selected from-H and-C1-C8of alkyl;

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

each of R8independently selected from-H, -OH, -C1-C8of alkyl, -C3-C8 carbocycle and-O-(C1-C8the alkyl);

R9selected from H and C1-C8of alkyl;

R10selected from aryl group or-C3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents-C2-C8-alkyl;

R14represents H or-C1-C8alkyl;

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

each of R16independently represents-H, -C1-C8alkyl or -(CH2)n-COOH, and

n is an integer from 0 to 6.

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

In another embodiment of the invention, each of R2The R 6represents methyl, and R9represents-N.

In yet another variant of the invention, each R8represents-OCH3.

In a representative embodiment of the invention, each of R3and R4represents isopropyl, each of R2and R6represents methyl, R5represents-H, R7represents sec-butyl, each of R8represents-OCH3and R9represents-N.

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

In one embodiment of the invention R10represents aryl.

In a representative embodiment of the invention R10represents phenyl.

In a representative embodiment of the invention, if Z represents-O-, R11represents H, methyl or tert-butyl.

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

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

Representative compounds of formulas is (Ib), each of which can be used as a molecule drugs (D) in the ADC, are compounds having the following structures:

and their pharmaceutically acceptable salts or solvate.

The compounds of FORMULA (Ic)

In another aspect the present invention relates to compounds of the conjugates of the antibody-drug” (ADC) of formula Ic:

Ab--(Aa--Ww--Yy--D)P(Ic)

containing antibody covalently associated with one or more components (molecules) of the medicinal product. These connections in the form of conjugate “antibody-drug” include their pharmaceutically acceptable salt or solvate.

In the compounds of formula Ic:

Ab is an antibody that binds to one or more receptors of the tumor-associated antigen, such as receptors(1)-(35):

(1) BMPR1B (the receptor protein of bone morphogenesis type IB, Genbank reg. No. NM_001203);

(2) e (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate, Genbank reg. No. NM_012449);

<> (4) R (SA, MUC16, Genbank reg. No. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank reg. No. NM_005823);

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 family soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II, Genbank reg. No. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5B, Genbank reg. No. AV);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, cDNA gene RIKEN 2700050C12, Genbank reg. No. AY358628);

(9) ETBR (endothelin receptor type b, Genbank reg. No. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of prostate cancer, Genbank reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M, Genbank reg. No. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma, Genbank reg. No. NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2)or C3DR (C3d/receptor of the virus of Epstein-Barr), or Hs73792, Genbank reg. No. M26004);

(15) CD79b (CD79B, CD79β, IGb (beta protein associated with immunoglobulin), V, Genbank reg. No. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C, Genbank reg. No. NM_030764);

(17) HER2 (Genbank reg. No. M11730);

(18) NCA (Genbank reg. No. m);

(19) MDP (Genbank reg. No. BC017023);

(20) IL20Rα (Genbank reg. No. AF184971);

(21) Brevican (Genbank reg. No. AF229053);

(22) Ephb2R (Genbank reg. No. NM_004442);

(23) ASLG659 (Genbank reg. No. AX092328);

(24) PSCA (Genbank reg. No. AJ297436);

(25) GEDA (Genbank reg. No. AY260763);

(26) BAFF-R (factor receptor activation In cells, BLys receptor 3, BR3, NP_443177.1);

(27) CD22 (b-cell receptor, isoform CD22 -, NP-001762.1);

(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha-protein-klecko-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with molecules of IgM on the cell surface, passing the signal that is involved in the differentiation of b-cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (receptor 1 lymphoma Burkitt, the receptor-associated G-protein and activated by the chemokine CXCL13, provides migration of lymphocytes and humoral defense, plays a role in infection by HIV-2 and probably in the development SPEED and, lymphoma, myeloma and leukemia, Genbank reg. No. NP_001707.1);

(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes, Genbank reg. No. NP_002111.1);

(31) the 2X5 (ion channel -5, open ligand purinergic receptor RH; ion channel opening extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may play a role in the pathophysiology of idiopathic dysfunction of the bladder, Genbank reg. No. NP_002552.2);

(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2, Genbank reg. No. NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I, which regulates the activation and apoptosis of b-cells, and loss of function of this protein is associated with progression in patients of systemic lupus erythematosus, Genbank reg. No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, the presumed receptor for the Fc domain of immunoglobulin, which contains Ig-like domains of type C2 and ITAM domains and may play a role in the differentiation of b-lymphocytes, Genbank reg. No. NP_443170.1); and

(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily 2, the estimated immunoreceptor, which might play a role in the development of b cells and lymphomagenesis; and in some malignant b cells observed dysregulation of the gene by translocation, Genbank reg. No. NP_112571.1).

But is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer elements the component

y is 0, 1 or 2,

R is in the range from 1 to about 8; and

D is a drug selected from the compounds of formulas DEand DF:

where the wavy line in the DEand DFmeans the website covalent binding to A, W or Y, and where at each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

<> R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH,

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

n is an integer from 0 to 6.

In one embodiment of the invention-Ww - is-Val-Cit.

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

In yet another variant of the invention, each of R2and R6represents methyl, and R9represents-N.

In another embodiment of the invention each R8represents-OCH3.

In a representative embodiment of the invention, each of R3and R4represents isopropyl, each of R2and R6represents methyl, R5represents-H, R7represents sec-butyl, each of R8represents-OCH3and R9represents-N.

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

In one embodiment of the invention R10represents aryl.

In a representative embodiment of the invention R10represents phenyl.

In representation the m variant of the invention, if Z represents-O-, R11represents H, methyl or tert-butyl.

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

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

Representative variants of the ADC of formula Ic have the following structure:

where Ab is an antibody that binds to one or more receptors of the tumor-associated antigen (1)-(35); Val represents valine, and Cit is citrulline.

Load the medicinal product is determined by the number R, i.e. the average number of drug molecules per antibody molecule of formula I. Load medicinal product may vary from 1 to 20 molecules of the drug (D) to the antibody (Ab or mAb). The composition of the ADC of formula I include sets of antibodies conjugated with 1-20 molecules drugs. The average number of molecules of drug antibody in preparations of the ADC obtained by Rea is the conjugation, can be determined by standard methods, such as spectroscopy in the UV/visible range of the spectrum, mass spectroscopy, ELISA analysis and HPLC. Quantitative distribution of the ADC can also be expressed by the symbolp. In some cases, isolation, purification and identification of homogeneous conjugates ADC, where p is a certain value, from conjugates ADC with another load of drugs can be achieved by methods such as reverse-phase HPLC or electrophoresis.

For some conjugates of the antibody-drug”pmay be limited by the number of binding sites on the antibody. For example, if a website's accession is a thiol of cysteine, as defined above in the representative embodiments of the invention, the antibody may have one or more thiol groups of cysteine, or it may have only one or few enough reactive thiol groups, through which can be attached to the linker.

Usually, the reaction of conjugation, the antibody may be conjugated to less than theoretical maximum number of molecules of the drug. The antibody may contain, for example, many lysine residues, which do not react with the intermediate connection “drug-linker or the linker reagent is. With a linker reagent reacting with an amine, can communicate only the most reactive group of lysine. Generally speaking, antibodies do not contain many, if present at all, free and reactive thiol groups of cysteine, which may be associated with a molecule drugs. Most of thiol groups of cysteine residues in the antibody compounds according to the invention is present in the form disulfide bridges and has to be restored under the action of a reducing agent, such as dithiothreitol (DTT). In addition, for the detection of reactive nucleophilic groups, such as lysine or cysteine groups, this antibody should be placed under denaturing conditions. Load ratio (drug/antibody) of the ADC can be adjusted in several different ways, including: (i) limiting molar excess of intermediate connection “drug-linker or the linker reagent with respect to the antibody, (ii) restrictions on time or reaction temperature of conjugation, and (iii) the creation of a partial or limiting recovery conditions for modification of the thiol group of cysteine.

It should be noted that in the case with intermediate connection “drug-linker or linker reagent, followed by races who alogena molecule drugs, reacts more than one nucleophilic group, the resulting product is a mixture of compounds of the ADC with the appropriate distribution of one or more drug molecules attached to the antibody. The average number of molecules of the drug to the antibody can be calculated using ELISA analysis of this mixture using two antibodies, one of which (“second” antibody) that is specific for the antibody, and the other is specific to the drug. Individual molecules of the ADC can be identified in a mixture using mass spectroscopy and separated by HPLC, for example, hydrophobic chromatography (“Effect of drug loading on the pharmacology, pharmacokinetics and toxicity of an anti-CD30 antibody-drug conjugate”, Hamblett K.J. et al., Abstract No. 624, American Association for Cancer Research; 2004 Annual Meeting, March 27-31, 2004, Proccedings of the AACR, Volume 45, March 2004; “Controlling the Location of Drug Attachement in Antibody-Drug Conjugates”, Alley, S.C. et al. Abstract No. 627, American Association for Cancer Research; 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). Thus, homogeneous ADC with a single load value may be selected from a mixture of conjugates by electrophoresis or chromatography.

4.3. The linker component

“Linker component (LU) is a bifunctional compound which can be used to bind molecules of a drug and a ligand with formation of conjuga the s “drug-linker-ligand or which can be used to obtain immunoconjugates, directed against tumor-associated antigens. Such immunoconjugate can provide selective delivery of toxic drugs in tumor cells. In one embodiment of the invention the linker component connection “drug-linker” and conjugate “drug-linker-ligand has the formula:

-Aa--Ww--Yy-

where: -A - is an extension component;

and is 0 or 1,

each W independently represents an amino acid component,

w is independently an integer from 0 to 12,

-Y - spacer elements means the component

y is 0, 1 or 2.

To conjugate the drug-linker-ligand” linker capable of binding a molecule of a drug and a ligand.

4.3.1. The extension component

Extension component (-A-), if present, are capable of binding ligand component with the amino acid component (-W-). In this case, the ligand (L) has a functional group that can form a bond with the functional group of the extension component. Suitable functional groups that may be present on the ligand and which may be either natural or chemically synthesized, are, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, anomeric hydroxyl GRU is PA carbohydrate and carboxyl. In one aspect of the invention the functional groups of the ligand are sulfhydryl and amino. Sulfhydryl groups can be generated by recovery of the intramolecular disulfide bonds of the ligand. Alternative sulfhydryl groups can be formed through the interaction of the amino groups of the lysine molecule ligand in the presence of 2-aminothieno (reagent trout) or other sulfhydryl-forming reagent.

In one embodiment of the invention the specified extension component forms a bond with a sulfur atom ligand component. The sulfur atom can occur from the sulfhydryl group of the ligand. A representative of the extension component of this variant of the present invention is indicated by brackets in formulas IIIa and IIIb, where L-, -W-, -Y-, -D, w and y are defined above, and R17selected from-C1-C10alkylene, -C3-C8carbocycle-, -O-(C1-C8the alkyl)-, arylene, -C1-C10alkylenediamine, Allen-C1-C10alkylene, -C1-C10alkylene-(C3-C8carbocycle)-, -(C3-C8carbocycle)-C1-C10alkylene, -C3-C8heterocycle, -C1-C10alkylene-(C3-C8heterocycle)-, -(C3-C8heterocycle)-C1-C10alkylene, -(CH2CH2O)rand -(CH2CH2O)r -CH2-; and r is an integer from 1 to 10. It should be noted that all representative variants of compounds of formula Ia, such as III-VI, which, even if it is not specifically contain from 1 to 20 molecules of the drug are associated with ligand (R=1-20).

A representative example of the extension is the component of the formula IIIa, where R17represents -(CH2)5is:

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

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

In another embodiment of the invention the extension component is associated with the ligand component via disulfide bond between the sulfur atom of ligand component and a sulfur atom extension component. A representative of the extension component of this variant of the present invention is indicated by brackets in the formula IV, where R17L-, -W-, -Y-, -D, w and y are defined above.

L-[-S-R17-C(O)-]-Ww-Yy-D(IV)

In yet another variant of the invention, the reactive group of the specified extension component contains a reactive site that can form a bond with a primary or secondary amino group of the ligand. Examples of such reactive sites include, but are not limited to, activated esters, such as succinimide, 4-nitrophenolate esters, pentafluorophenyl esters, tetraterpene esters, anhydrides, acid chlorides, sulphonylchloride, isocyanates and isothiocyanates. A representative of the extension component of this variant of the present invention is indicated by brackets in formulas Va and Vb, where R17L-, -W-, -Y-, -D, w and y are defined above;

In yet another variant of the invention, the reactive group of the specified extension component contains a reactive site capable of reacting with the modified carbohydrate group (-Cho), which may be present on the ligand. For example, the carbohydrate may be weakly oxidized in such a reagent as periodate sodium, and the resulting group (-Cho) oxidized carbohydrate may be condensed with an extension component containing a functional group, such as a hydrazide, an oxime, a primary or secondary amine, hydrazine, tiosamteroba is he, the hydrazine carboxylate and originated described in T. Kaneko et al. (1991) Bioconjugate Chem. 2:133-41. A representative of the extension component of this variant of the present invention is indicated by brackets in formulas VIa, VIb, and VIc, where-R17L-, -W-, -Y-, -D, w and y are defined above.

4.3.2. Amino acid component

Amino acid component (-W-), if present, binds extension component with spacer elements element, if such spacer elements component is present, binds the specified extension component molecule drugs, if the specified component is missing spacer elements, and binds the ligand component molecule drugs, if the specified extension component and component spacer elements are missing.

Ww- is a dipeptide, Tripeptide, tetrapeptide, pentapeptidnogo, Hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptides, undecapeptide or dodecapeptide component. Each of the components-W - is independently has the formula shown below in square brackets, and w denotes an integer from 0 to 12:

where R19represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2HE, -CH(OH)CH 3, -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)4NHCONH2, -CH2CH2CH(OH)CH2NH22-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, phenyl, cyclohexyl.

Amino acid component can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, with the release of a molecule drugs (-D), which, In one embodiment of the invention after release undergoes protonation ofin vivowith the formation of a drug (D). Representative components of Wwrepresented by formulas (VII)to(IX):

where R20and R21have the following meanings:

R20R21
benzil(CH2)4NH2
met the l (CH2)4NH2
isopropyl(CH2)4NH2
isopropyl(CH2)3NHCONH2
benzil(CH2)3NHCONH2
isobutyl(CH2)3NHCONH2
second-butyl(CH2)3NHCONH2
(CH2)3NHCONH2
benzilmethyl
benzil(CH2)3NHC(=NH)NH2

where R20, R21and R22have the following meanings:

R20R21R22
benzilbenzil(CH2)4NH2
isopropylbenzil (CH2)4NH2
Nbenzil(CH2)4NH2

where R20, R21, R22and R23have the following meanings:

R20R21R22R23
NbenzilisobutylN
methylisobutylmethylisobutyl

Representative amino acid components include, but are not limited to, components of the formula (VII), where: R20represents benzyl, and R21represents -(CH2)4NH2; R20represents isopropyl, and R21represents -(CH2)4NH2; R20represents isopropyl, and R21represents -(CH2)3NHCONH2. Other representative amino acid component is a component of the formula (VIII), where R20represents benzyl, R21isone benzyl, and R22represents -(CH2)4NH2.

Suitable components-Ww- can be obtained and optimized in their selectivity against enzymatic degradation by specific enzymes, such as tumor-associated protease. In one embodiment of the invention component-Wwis a component, the separation of which is catalyzed by cathepsin b, C and D, or a plasmin is a protease.

In one embodiment of the invention component-Ww- is a dipeptide, Tripeptide, tetrapeptide or Pentapeptide.

If R19, R20, R21, R22or R23are not hydrogen, the carbon atom through which the bound R19, R20, R21, R22or R23is chiral.

Each carbon atom through which the bound R19, R20, R21, R22or R23independently is (S)- or (R)-configuration.

In one embodiment, the amino acid component of the specified amino acid is valine-citrulline. In another embodiment, the amino acid component is a phenylalanine-lysine (that is, fk). In another embodiment, the amino acid component of the specified amino acid component is N-methylvaline-citrulline. In yet another aspect, the amino acid component is a 5-aminovaleric acid, g is materialand-lysine, tetraethylammonium-lysine, cyclohexylamin-lysine, isonipecotic acid-lysine, beta-alanine-lysine, glycine-serine-valine-glutamine and isonicotinoyl acid.

In some embodiments of the invention the amino acid component may contain natural amino acids. In other embodiments of the invention the amino acid component may contain non-natural amino acids.

4.3.3. Spacer elements component

Spacer elements component (-Y-), if present, binds to the amino acid component with a molecule drugs, when such amino acid component is present. Alternative spacer elements specified component connects the extension component molecule drugs, when such amino acid component is missing. The specified component spacer elements also binds the molecule drugs with the ligand component, if there are no amino acid component and extension component.

Spacer elements components are components of two General types: smolinerwien and non-smolinerwien. Not-carolinensis spacer elements component is a component, where the entire spacer elements component or part thereof remains bound to the molecule drugs after removal, and in particular enzymatic cleavage aminocyclohexanone from conjugate “drug-linker-ligand” or connection “drug-linker”. Examples of non-somalimerirosvot spacer elements component include, but are not limited to, glycine-glycine spacer elements component and glycine spacer elements component (both are depicted in figure 1) (see below). If a representative compound containing a glycine-glycine spacer elements component or a glycine spacer elements component undergoes enzymatic cleavage via a tumor-associated protease associated with cancer cells protease or associated with lymphocyte protease, the molecule glycine-glycine-drug or molecule glycine-drug is cleaved from L-Aa-Ww. In one embodiment of the invention independent hydrolysis reaction occurs in the target cells and leads to the breakdown of communication in the molecule glycine-drug with the release of the drug.

In another embodiment of the invention-Yy- represents the p-aminobenzoyl alcohol (RAV) (see schemes 2 and 3), fenelonov part of which is substituted Qmwhere Q represents-C1-C8alkyl, -O-(C1-C8alkyl), halogen, nitro or cyano, and m is an integer from 0 to 4.

Scheme 1

In one embodiment, the invention is not-carolinensis spacer elements component (Y) is a-Gly-Gly-. In others the d variant of the invention not-carolinensis spacer elements component (Y) is a-Gly-.

In one of its variants the present invention relates to a connection “drug-linker” or conjugate the drug-linker-ligand, where spacer elements component is absent (y=0), or their pharmaceutically acceptable salts or MES.

Alternatively, a representative compound containing carolinensis spacer elements component can liberate-D, and thus no need for a separate hydrolysis step. In this embodiment,- Y - is a group of RAV, which is associated with Wwthrough the nitrogen atom of the amino group RAV and which is directly linked to-D via a carbonate, urethane or ester group. Not limited to any particular theory or mechanism, the authors present the following scheme 2, which illustrates a possible mechanism of removal of drug from the group of the AM, which is directly attached to D via urethane or carbonate group, and this mechanism is described by Toki et al., (2002) J.Org.Chem.67:1866-1872.

Scheme 2

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

Not limited to any particular theory or mechanism, the authors present nor the e scheme 3, which illustrates a possible mechanism of removal of drug from the group of the AM, which is connected to D by ether or amine linkages.

Scheme 3

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

Other examples carolinensis spacers include, but are not limited to, aromatic compounds that, because of their electronic properties, similar to the RAV group, such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para-aminobenzoate. Can also be used the spacers that are subjected to cyclization after hydrolysis of the amide bond, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1]- bicyclo[2.2.2]-cyclic system (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). Similarmusical amine-containing drugs, which are substituted in the α-position of glycine (Kingsbury et al., J. Med. Chem. 1984, 27, 1447), are also examples carolinensis spacers used in the representative compounds.

In one and the variants of the invention spacer elements component is a branched bis(hydroxymethyl)styrene (BHMS), shown in figure 4, and can be used to activate and release of many drugs.

Scheme 4

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

In one embodiment of the invention the molecule-D are the same. In another embodiment of the invention molecules D are different.

In one aspect of the invention, the spacer elements components (-Yy-) is represented by formulas (X)-(XII):

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

and

Variants of compounds of conjugates of the antibody-drug” formula 1A' and Ic are:

where each of w and y is equal to 0.

and

4.4. Component “drug” (molecule)

The molecule drugs (D) of conjugates of the antibody-drug” (ADC) refers to the type of dolastatin/auristatin (U.S. patent No. 5635483; 5780588), which, as shown, is egative affect the dynamics of the formation of microvessels, the hydrolysis of GTP and division of nuclei and cells (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anti-cancer (U.S. patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents. Chemother. 42:2961-2965).

D is a component of “drug” (molecule)having a nitrogen atom that can form a bond with the spacer elements component, if y=1 or 2, with the C-terminal carboxyl group of the amino acid component, if y=0, with a carboxyl group of the extension component, if w and y =0, and with a carboxyl group of the component “drug”if a, w and y=0. It should be noted that the component terms “drug” and “molecule drugs” are synonymous and interchangeable terms.

In one embodiment of the invention, D is a compound of formula DEor DF:

where in each position independently:

R2selected from H and C1-C8of alkyl;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R4selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1 8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

R5selected from H and methyl;

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

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl,1-C8alkyl-(C3-C8carbocycle)3-C8heterocycle and C1-C8alkyl-(C3-C8heterocycle);

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

R9selected from H and C1-C8of alkyl;

R10selected from aryl or3-C8heterocycle;

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

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

m is an integer from 1 to 1000;

R13 represents a C2-C8-alkyl;

R14represents N or C1-C8alkyl;

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

each of R16independently represents H, C1-C8alkyl or -(CH2)n-COOH,

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

n is an integer from 0 to 6.

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

In another embodiment of the invention, each of R2and R6represents methyl, and R9represents-N.

In yet another variant of the invention, each of R8represents-OCH3.

In a representative embodiment of the invention, each of R3and R4represents isopropyl, each of R2 6represents methyl, R5represents H, R7represents sec-butyl, each of R8represents-OCH3and R9represents-N.

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

In one embodiment of the invention R10represents aryl.

In a representative embodiment of the invention R10represents phenyl.

In a representative embodiment of the invention, if Z represents-O-, R11represents H, methyl or tert-butyl.

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

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

Representative components of the “drug” (-D) are molecules of medicinal product having the following structure:

and their pharmaceutically acceptable salts or with whom livity.

In one aspect of the invention, the hydrophilic group, such as, without limitation, esters of triethylene glycol (TEG), as shown above, can be attached to a component of a “drug”, presents R11. Without pretending to any particular theory, the authors believe that the hydrophilic group contribute to the internalization and prevent agglomeration of the component “drug”.

4.5. Ligand component

In the amount of ligand component (L) is any component of a ligand (L), which binds, or through reactions associated with, or forms complexes with a receptor, antigen or other sensitive molecule associated with this population of target cells. A ligand is a molecule that is bound, forms complexes or react with part of the cell population analyzed for therapeutic or any other biological modification. In one aspect of the invention, the ligand component component delivers medicine to specific populations of target cells, which reacts this ligand component. Such ligands include, but are not limited to, high molecular weight proteins, such as, for example, full-length antibodies, antibody fragments, proteins with lower molecular weight, polypep the IDA or peptides, lectins, glycoproteins, substances which are not peptides, vitamins, molecules that carry trace elements (such as, but not limited to, transferrin), or any other molecule or any other substance that communicates with the cell.

Ligand component can form a connection with the extension component, the amino acid component, spacer elements component or component “drug”. Ligand component can form a bond with the linker component via a heteroatom of this ligand. Heteroatoms, which may be present on the ligand component, are sulfur (In one embodiment of the invention derived from the sulfhydryl group of the ligand), oxygen (In one embodiment of the invention derived from carbonyl, carboxyl or hydroxyl group of the ligand) and nitrogen (In one embodiment of the invention derived from primary or secondary amino group of the ligand). These heteroatoms may be present on the ligand in its natural state, for example in natural antibody, or they can be introduced into the ligand by chemical modification.

In one embodiment of the invention, the ligand has a sulfhydryl group, and the ligand attached to a linker via the sulfur atom of sulfhydryl groups.

In another aspect of the invention, the ligand has one or more Lisi is new balances which can be chemically modified by introducing one or more sulfhydryl groups. Specified ligand component is connected to the linker component via the sulfur atom of sulfhydryl groups. Reagents that can be used for modification of lysine are, but are not limited to, N-Succinimidyl-S-acetylthiourea (SATA) hydrochloride and 2-aminosilane (reagent trout).

In another embodiment of the invention, the ligand may have one or more carbohydrate groups that can be chemically modified by introducing one or more sulfhydryl groups. Specified ligand component is connected to the linker component, such as lengthening the component via the sulfur atom of sulfhydryl groups.

In yet another variant of the invention, the ligand may have one or more carbohydrate groups that can be oxidized with the formation of aldehyde groups (-Cho) (see, for example, Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). The corresponding aldehyde can form a bond with a reactive site of the extension component. The reactive sites of the extension component that can interact with the carbonyl group on the ligand include, but are not limited to, hydrazine and hydroxylamine. Other protocols for conducting modification of proteins to attach to components of the drug is the first tool or to associate with these components are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), which is introduced in the present description by reference.

Suitable for use by non-immunoreactive protein, polypeptide, or peptide ligands include, but are not limited to, transferrin, epidermal growth factors (“EGF”), bombezin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF”), such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, lectins and apoprotein, derived from low density lipoprotein.

Suitable for use polyclonal antibodies are heterogeneous populations of antibody molecules isolated from serum immunogenic animals. For production of polyclonal antibodies against the interest of the antigen can be conducted by various procedures well known in the art. For example, for the production of polyclonal antibodies, various animal hosts can be immunitary by injection of interest antigen or its derivative, and such animals include, but are not limited to, rabbits, mice, rats and Guinea pigs. To increase the immune response can be used in a variety of adjuvants, depending on the species of host, and such adjuvant who we are, but not limited to, beta-blockers (complete or incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols-pluronic, polyanion, peptides, oil emulsions, hemocyanine lymph snails, dinitrophenol, and potentially useful for human adjuvants such as BCG (Bacillus Calmette-Guerin) andCorynebacterium parvum. Such adjuvants are well known to specialists.

Suitable for use monoclonal antibodies are homogeneous populations of antibodies directed against a specific antigenic determinants (e.g., an antigen of the cancer cells, viral antigen, a microbial antigen, a protein, peptide, carbohydrate, chemicals, nucleic acids or their fragments). Monoclonal antibody (mAb) against the interest of the antigen can be obtained by any known method for the production of antibody molecules stable cell lines in culture. Such methods include, but are not limited to, hybridoma technology, first described by Köhler &Milstein (1975, Nature 256, 495-497), the technology of In-cell human hybrid (Kozbor et al., 1983, Immunology Today 4:72) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p.77-96). Such antibodies may be immunoglobulins of any class, including IgG, IgM, IgE, IgA and IgD and any what about the subclass. Hybridoma producing monoclonal antibodies (mAb)used in the present invention, can be culturedin vitroorin vivo.

Suitable for use monoclonal antibodies include, but are not limited to, human monoclonal antibodies, gumanitarnye monoclonal antibodies, fragments of antibodies or chimeric monoclonal antibodies human-mouse (or other species). Human monoclonal antibodies can be obtained by any of various methods known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci., USA, 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79 & Olsson et al., 1982, Meth Enzymol. 92, 3-16).

The specified antibody can also be bespecifically antibody. Methods of obtaining bespecifically antibodies known in the art. Traditional production of full-size bespecifically antibodies based on co-expression of two pairs of heavy chain-light chain immunoglobulin, where the two chains have different specificnosti (Milstein et al., 1983, Nature 305:537-539). Because of the presence of a randomized set of heavy and light chains of immunoglobulin these hybridoma (quadroma) produce a potential mixture of ten different antibody molecules, of which only one molecule has the “right” bespecifically structure. Similar procedures are described in the International publication of statements and no WO 93/08829 and in Traunecker et al. EMBO J. 10:3655-3659 (1991).

In accordance with another approach, the variable domains of the antibodies with the desired binding specificity (binding sites of the antibody-antigen) is added to the sequence of the constant domain of immunoglobulin. Preferred hybrid is a hybrid with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, WithN2 - and CN3-sphere. Thus preferably, the hydride had the first constant region of the heavy chain (CN1)containing the site necessary for binding to the light chain and is present at least in one of the hybrids. Nucleic acid sequences encoding the hybrid heavy chain immunoglobulin, and if necessary, the light chain immunoglobulin is inserted into separate expression vectors and cotransfected in a suitable organism, the host. This allows high flexibility in the adjustment of the ratios of the three polypeptide fragments in those embodiments of the invention, in which there is an unequal relationship of the three polypeptide chains used in this design, and get the best outputs. However, the coding sequences for two or all three polypeptide chains can be built in one expression vector when the expression of at IU is e, the two polypeptide chains in equal relationships give high outputs or if these relationships are not significant.

In one variation of this approach bespecifically antibodies have a hybrid heavy chain immunoglobulin with a first binding specificity in one branch and a hybrid pair of heavy chain-light chain immunoglobulin (providing a second binding specificity) in the other branch. This asymmetric structure facilitates the separation of the desired especifismo connections from unwanted combinations of chains of immunoglobulin, such as facilitating the separation due to the presence of the light chain of immunoglobulin in only one half of bespecifically molecules (see publication of the International application No. WO94/04690, which in its entirety is introduced into the present description by reference).

A more detailed description of obtaining bespecifically antibodies can be found, for example, Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. With the use of such methods can be obtained bespecifically antibodies intended for the treatment or prevention of these diseases.

Bifunctional antibodies are also described in published European patent application No. EPA 0105360. As the op is Sano in this publication hybrid or bifunctional antibodies can be obtained by biological methods, i.e. methods merge cells, or by chemical methods, and in particular with the use of cross-linking agents or reagents, forming disulfide bridges, and these antibodies may represent a whole antibodies or fragments thereof. Methods of obtaining such hybrid antibodies are described, for example, published International application no WO 83/03679 and in the publication of European patent application No. EPA 0217577, each of which is introduced into the present description by reference.

The antibody may be a functionally active fragment, derivative or analog of an antibody that immunospecificity contact with antigens on cancer cells, viral antigens, microbial antigens or with other antibodies associated with tumor cells or matrix. Accordingly, the term “functionally active” means that the fragment, derivative or analogue have the ability to produce anti-anti-idiotypic antibodies that recognize the same antigen as the antibody from which originate the data fragment, derivative or analog. In particular, in the representative embodiment of the invention, the antigenicity of the idiotype of the immunoglobulin molecules may be improved by the deletion of the frame on which sledovatelnot and CDR sequences, located With-end with respect to the CDR sequence that specifically recognizes the antigen. In order to determine which sequences of CDRs associated with the antigen can be carried out analyses on binding to the antigen, synthetic peptides containing the CDR sequence, by any known method, typically used by experts for analysis on the binding (for example, analysis using BIA CORE) (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat, E. et al., 1980, J. of Immunology 125(3):961-969).

Other suitable antibodies include fragments of antibodies such as, but not limited to, F(ab')2fragments that contain the variable region, constant region light chain and SN-domain heavy chain and which can be produced by hydrolysis of the molecule pepsin antibodies, and Fab fragments that can be produced by restoring the disulfide bridges of F(ab')2-fragments. Other suitable antibodies are dimers of the heavy and light antibody chains, or any minimal fragment such as Fv, or single-chain antibodies (scAb) (for example as described in U.S. patent No. 4946778; Bird, 1988, Science 242:423-42; Huston et al., 1998, Proc. Natl. Acad. Sci., USA, 85:5879-5883, and Ward et al., 1989, Nature 334:544-54), or any other molecule with the so is th same specificity, as this antibody.

In addition, suitable antibodies are recombinant antibodies such as chimeric and gumanitarnye monoclonal antibody containing human and non-human parts, which can be obtained using standard methods of recombinant DNA. A chimeric antibody is a molecule in which different portions are descended from animals of different species, namely the part that contains the variable region derived from constant regions of mouse monoclonal antibody and a human immunoglobulin (see, e.g., Cabilly et al., U.S. patent No. 4816567 and Boss et al., U.S. patent No. 4816397, which in its entirety are introduced in the present description by reference). Gumanitarnye antibodies are antibody molecules originating from animals that are not human, and having one or more hypervariable regions (complementarity-determining regions, CDRs)derived from an animal, not a man, and a frame region, originating from molecules to human immunoglobulin (see, e.g., Queen, U.S. patent No. 5585089, which in its entirety is introduced into the present description by reference). Such chimeric and gumanitarnye monoclonal antibodies can be produced by the methods of recombinant DNA, known special there, for example the methods described in published International application no WO 87/02671; in the publication of European patent application No. 184187; in the publication of European patent application No. 171496; in the publication of European patent application No. 173494; publication of the International application no WO 86/01533; U.S. patent No. 4816567; in the publication of European patent application No. 12023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci., USA, 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci., USA 84:214-218; Nishimura et al., 1987, Cancer Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechnology 4:214; U.S. patent No. 5225539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534 and Beidler et al., 1988, J. Immunol. 141:4053-4060, each of which in its entirety is introduced into the present description by reference.

A full-sized human antibodies are particularly preferred, and they can be produced in transgenic mice, which are unable to Express the endogenous genes of the heavy and light chains of immunoglobulin, but is able to Express the genes of the heavy and light chains of human immunoglobulin. Transgenic mice subjected to immunization with a selected antigen, for example a whole polypeptide according to the invention or part thereof, in the usual way. Monoclonal antibodies directed against this antigen can be obtained with the use of the em standard hybridoma technology. The transgenic human immunoglobulin present in transgenic mice, rearranged in the process of differentiation of b-cells, and then switch class of immunoglobulins and somatic mutation. Thus, the use of this technique allows to produce therapeutically effective antibodies IgG, IgA, IgM and IgE. A General overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93).

A detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies can be found, for example, in U.S. patents№ 5625126, 5633425, 5569825, 5661016, 5545806, each of which in its entirety is introduced into the present description by reference. Other human antibodies can be supplied, for example, firms Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

A full-sized human antibodies that recognize a selected epitope can be produced by a method called “directional selection”. In this method, the selected non-human monoclonal antibody, such as a mouse antibody, is used for directional selection full-sized human antibody recognizing the same epitope (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be produced different is diversified known methods, including libraries of phage view (Hoogenboom &Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991); Quan M.P. & Carter P. 2002.The rise of monoclonal antibodies as therapeutics.In Anti-IgE and Allergic Disease, Jardieu P.M. & Fick Jr. R.B. eds, Marcel Dekker, New York, NY, Chapter 20, p.427-469).

In other embodiments of the invention the antibody is a hybrid protein antibody or functionally active fragment, in which, for example, the antibody is linked via a covalent bond (e.g., a peptide bond) or N-Terminus or at the C-end, with the amino acid sequence of another protein (or portion thereof, preferably having at least 10, 20 or 50 amino acids), which is not an antibody. When the antibody or fragment, preferably, covalently linked to other proteins at the N-Terminus of the constant domain.

The term “antibody” includes analogs and derivatives that are modified, i.e, by the covalent binding molecule of any type, provided that such covalent binding allows the antibody to retain its immunospecificity binding to the antigen. So, for example, derivatives and analogs of the antibodies include, but are not limited to, derivatives and analogues, which were further modified, for example by glycosylation, acetylation, paglierani, phosphorylation, amidation, derivatization is known defensive/blocking groups, proteolytic cleavage, linkage to a cellular antibody or other protein, etc. of Any of numerous chemical modifications may be carried out with known methods, including, but not limited to, specific chemical cleavage, acetylation, formirovanie, metabolic synthesis in the presence of tunicamycin, etc. in Addition, such an analogue or derivative may contain one or more non-natural amino acids.

The term “antibody” includes antibodies having modifications (e.g., substitutions, deletions or additions in the amino acid residues that interact with Fc receptors. In particular, antibodies are antibodies with modifications in amino acid residues identified as the residues involved in the interaction between the antibody against the Fc domain and the FcRn receptor (see, for example, the publication of International application no WO 97/34631, which in its entirety is introduced into the present description by reference). Antibodies with immunospecificity to the antigen of the cancer cells, can be supplied, for example, the firm Genentech (San Francisco, CA), or they can be produced by any method known in the art, such as, for example, a method of chemical synthesis or by expression of recombinant DNA. The nucleotide sequence encoding the antibody, obladaushi what immunospecificity to the antigen of a cancer cell, can be obtained, for example, from the Genbank database or similar database methods described in the literature, or by routine cloning and sequencing.

In a specific embodiment of the invention for the treatment or prevention of cancer can be used known antibodies. Antibodies with immunospecificity to the antigen of a cancer cell, can be obtained from commercial sources, or they can be produced by any methods known in the art, such as, for example, methods expression of recombinant DNA. The nucleotide sequence encoding the antibody, with immunospecificity to the antigen of a cancer cell, can be obtained, for example, from the Genbank database or similar database methods described in the literature, or by routine cloning and sequencing. Examples of antibodies that can be used for the treatment of cancer include, but are not limited to, gumanitarnoe monoclonal antibody against HER2, Herceptin® (trastuzumab; Genentech) for the treatment of patients with breast cancer, giving metastases; antibody Rituxan® (rituximab; Genentech)which is a chimeric monoclonal antibody against CD20, intended for the treatment of patients with non-Hodgkin's lymphoma; antibody OvaRex (AltaRex Corporation, MA), which is a murine antibody, the art is meant for the treatment of ovarian cancer; antibody Panorex (Glaxo Wellcome, NC)which is a murine antibody IgG2adesigned for the treatment of colon cancer; and antibody Cetuximab Erbitux (Imclone Systems Inc., NY)which is a chimeric IgG antibody against EGFR, intended for the treatment of cancer, positive for expression of epidermal growth factor, such as head and neck cancer; the antibody of Vitaxin (MedImmune, Inc., MD), which is gumanitarnoe antibody for the treatment of sarcoma; antibody Campath I/H (Leukosite, MA)which is gumanitarnoe IgG1 antibody designed for the treatment of chronic lymphocytic leukemia (CLL); antibody Smart MI95 (Protein Design Labs, Inc., CA), which is an anti-CD33 IgG antibody designed for the treatment of acute myeloid leukemia (AML); antibody LymphoCide (Immunomedics, Inc., NJ), which is gumanitarnoe anti-CD22 IgG antibody designed for the treatment of non-jackinsky lymphoma; antibody Smart ID10 (Protein Design Labs, Inc., CA), which is an anti-HLA-DR antibody designed for the treatment of non-jackinsky lymphoma; antibody Oncolym (Techniclone, Inc., CA), which is a radioactively labeled anti-HLA-Dr10 antibody designed for the treatment of non-jackinsky lymphoma; antibody Allomune (BioTransplant, CA)which is gumanitarnoe anti-CD2 mAb, intended for the treatment of diseases Khojki is a or not-jackinsky lymphoma; antibody Avastin (Genentech, Inc., CA), which is gumanitarnoe anti-VEGF antibody designed for the treatment of lung cancer and colon cancer; and antibody Epratuzumab (Immunomedics, Inc., NJ and Amgen, CA)which is an anti-CD22 antibody designed for the treatment of non-jackinsky lymphoma; and antibody CEAcide (Immunomedics, NJ)which is an anti-CEA antibody designed for the treatment of colon cancer.

Other antibodies that can be used for the treatment of cancer include, but are not limited to, antibodies against the following antigens, such as: SA (ovarian cancer), CA-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA (cancer of the colon), alkaline phosphatase placental (carcinoma), antigen specific for prostate cancer (prostate cancer), acid phosphatase prostate (prostate cancer), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinoma), antitranspirants receptor (carcinoma), R (melanoma), MUC1-KLH (breast cancer), CEA (cancer of the colon), gp100 (melanoma), MART1 (melanoma), PSA (prostate cancer), the receptor for IL-2 (T-cell leukemia and T-cell lymphoma), CD20 (non-jackinsky lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human is a mini-human chorionic gonadotropin (HCC), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma) and oncogenic Neu product (carcinoma). Some specific and effective antibodies include, but are not limited to, mAb BR96 (Trail P.A., Willner D. Lasch S.J. Henderson A.J. Hofstead, S.J. Casazza A.M. Firestone, R. A. Hellstrom, I., Hellstrom, K.E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail P.A., Willner D. Knipe J. Henderson A.J. Lasch, S.J. Zoeckler, M.E., Trailsmith M.D. Doyle, T.W. King, H.D., Casazza, A.M. Braslawsky, G.R. Brown J.P. Hofstead, S.J. Greenfield R.S. Firestone, R.A. Mosure K. Kadow, D.F., Yang, M. Hellstrom, K.E. & Hellstrom, I. “Effect of Linker Variation on the Stability, Potency and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research, 1997, 57, 100-105), mAbs against CD40 antigen, such as mAb S2C6 (Francisco, J.A., K.L. Donaldson Chace D. Siegall, C.B. & A.F. Wahl “Agonistic properties and in vivo antitumor activity of the anti-CD40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), mAbs against the CD70 antigen, such as mAb 1F6 and mAb 2F2, and mAb against the CD30 antigen, such as AS (Bowen M.A., Olsen K.J. Cheng, L. Avilla, D., & Podack, E.R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993; Wahl et al., 2002 Cancer Res. 62(13):3736-42). Can also be used and many other internalize antibodies that bind to tumor-associated antigens, and these antibodies are described in the literature (Franke, A.E., Sievers E.L. & D.A. Scheinberg, “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review, “Cancer Biother Radiopharm. 2000, 15, 459-76; J.L. Murray “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; F. Breitling & Dubel S. Recombinant Antibodies, John Wiley & Sons, New York, 1998).

In kotoryj embodiments of the invention, the specified antibody is trastuzumab (full gumanitarnoe antibody against HER2 (MW 145167)), Herceptin-F(ab')2(originating from enzymatic split the anti-HER2 antibodies, MW 100000), 4D5 (full-size mouse anti-HER2 antibody produced by hybridomas), rhu4D5 (temporarily expressed full gumanitarnoe antibody), rhuFab4D5 (recombinant gumanitarnye Fab, MW 47738), 4D5Fc8 (full-size mouse anti-HER2 antibody with mutated FcRn-binding domain) or Hg (“hingeless” full gumanitarnoe antibody 4D5, in which cysteine the hinge region of the heavy chain were motivovany in serine and which was expressed inE. coli(and so it is deglycosylation)).

In another specific embodiment of the invention known antibodies intended for the treatment or prevention of autoimmune diseases, are used in accordance with the compositions and methods according to the invention. Antibodies with immunospecificity to the antigen, the cells responsible for the production of autoimmune antibodies can be obtained from any organization (for example, research Institute or company), or they can be produced by any known method, such as, for example, chemical synthesis or by expression of recombinant DNA. In another embodiment of the invention suitable antibodies are immunospecific antibody designed for the treatment of autoim unnuh diseases, such antibodies include, but are not limited to, antinuclear antibody, anti-dzanc antibody, anti-IDNC antibody, antibody IgM and IgG against cardiolipin, antibody IgM and IgG against phospholipid, anti-SM antibody, antimitochondrial antibody, the antibody is specific for thyroid microsomal antibody, antibody against thyroglobulin, anti-SCL-70 antibody, anti-Jo antibody; anti-U1RNP antibody; anti-La/SSB antibody; anti-SSA antibody; anti-SSB antibody; antibody against paritally cells; antibody against histone; anti-RNP antibody; antibody against C-ANCA; antibody against P-ANCA; antibody against centromere; antibody against fibrillarin and anti-GBM antibody.

In some embodiments of the invention suitable for use the antibody can be contacted with the receptor and receptor complex expressed on activated lymphocytes. The receptor or receptor complex may contain a member of the superfamily of immunoglobulin genes, a member of the superfamily of TNF receptors, integrin, a cytokine receptor, chemokinesis receptor protein major histocompatibility complex, lectin, or a complement-regulatory protein. Non-limiting examples of suitable members of the immunoglobulin superfamily, are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1 and ICOS. Non-limiting examples of suitable members of the soup is a family of TNF receptors are CD27, CD40, CD95/Fas, CD134/OH, CD137/4-IBB, TNF-R1, TNFR-2, RANK, TACI, SMA, osteoprotegerin, Aro/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, and CD103 CD104. Non-limiting examples of suitable lectins are lectin C-type, S-type and I-type.

In one embodiment of the invention, the specified ligand binds to activated lymphocytes, associated with autoimmune disease.

In another specific embodiment of the invention suitable ligands, immunospecificity to viral or microbial antigen, are monoclonal antibodies. Such antibodies can be chimeric, gumanitarnye or human monoclonal antibodies. Used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide, protein (e.g., HIV gp120,nefHIV glycoprotein RSV F, neuraminidase of influenza virus a hemagglutinin of influenza virus, HTLV tax, glycoprotein of herpes simplex virus (e.g., gB, gC, gD and gE) and surface antigen of hepatitis b virus), which are capable of producing an immune response. Used herein, the term “microbial antigen” includes, but is not limited to, any molecule microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid (e.g., the polypeptide of bacteria, fungi, pathogen the x protozoa and yeast, including, for example, LPS and capsular polysaccharide 5/8), which was capable of producing an immune response.

Antibodies that are immunospecificity to viral or microbial antigen, can be supplied from commercial firms, for example, from BD Bioscience (San Francisco, CA), Chemicon International Inc. (Temecula, CA) or Vector Laboratories, Inc. (Burlingame, CA), or can be obtained by any known method, such as, for example, a method of chemical synthesis, or expression of the recombinant DNA. The nucleotide sequence encoding the antibodies, which are immunospecificity to viral or microbial antigen can be obtained, for example, from the Genbank database or similar database, and the method described in the literature, or it can be obtained by routine cloning and sequencing.

In a specific embodiment of the invention suitable ligands are ligands that may be used to treat or prevent viral or microbial infection in accordance with the ways described here. Examples of antibodies that can be used to treat viral infections or microbial infections include, but are not limited to, antibody SYNAGIS (MedImmune, Inc., MD), which is gumanitarnoe monoclonal antibody against respiratory syncytial virus (RSV), designed to treat the patients with RSV infection; antibody PRO542 (Progenics), which is a hybrid anti-CD4 antibody designed for the treatment of HIV infection; antibody OSTAVIR (Protein Design Labs., Inc., CA), which is a human antibody designed for the treatment of infections caused by hepatitis b virus; antibody PROTOVIR (Protein Design Labs., Inc. CA), which is gumanitarnoe IgG1 antibody designed for the treatment of infections caused by cytomegalovirus (CMV), and antibodies against LPS.

Other antibodies suitable for the treatment of infectious diseases include, but are not limited to, antibodies against antigens derived from pathogenic strains of bacteria (Streptococcus pyogenes,Streptococcus pneumoniae,Neisseria gonorrhoeae,Neisseria meningitidis,Corynebacterium diphtheriae,Clostridiumbotulinum,Clostridiumperfringens,Clostridium tetani,Hemophilus influenzae,Klebsiella pneumoniae,Klebsiella ozaenas,Klebsiella rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagie, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.);pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histolasma capsulatum); the simplest (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria);or helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobiumand hookworms).

Other antibodies that can be used in the present invention for the treatment of viral diseases include, but are not limited to, antibodies against antigens of pathogenic viruses, including, for example, but not limited to, poxviruses, herpes viruses, herpes simplex virus 1, herpes simplex virus 2, adenoviruses, papovaviruses, enteroviruses, picornaviruses, parvoviruses, reoviruses, retroviruses, influenza viruses, parainfluenza viruses, mumps virus mumps virus, measles virus, respiratory syncytial virus, rubella virus, arbovirus, rhabdovirus, arenavirus, hepatitis a virus, hepatitis In, hepatitis C virus, hepatitis E virus, the virus non-a/non-b hepatitis, rhinoviruses, coronaviruses, rotavirus and human immunodeficiency virus.

To identify effective cellular targets for diagnosis and therapy of cancer researchers attempted to identify transmembrane or any other tumor-associated polypeptides that are specifically xpresroute on the surface of one or more cancer cells of a particular(s) type(s) compared with one or more normal non-cancerous cells. In most cases, these tumor-associated polypeptides abundantly expressed on the surface of cancer cells, and not on the surface of the non-cancerous cells. The identification of such tumor-associated polypeptide antigen on the cell surface will allow for the specific destruction of cancer cells targeted by therapy using antibodies.

Antibodies, which are Ab conjugates of the antibody-drug formula Ic (ADC) and which can be used for the treatment of cancer include, but are not limited to, antibodies against tumor-associated antigens (TAA). Such tumor-associated antigens are known in the art and can be obtained for producing antibodies using methods and data, are well known in the art. Examples of TAA are (1)to(35), but are not limited to, TAA (1)-(35)listed below. For convenience, the data relating to all these well-known antigens presented below and include the name, alternative names, registration numbers Genbank and initial(s) source(s). Tumor-associated antigens, which are directed antibodies include all variants of amino acid sequences of isoforms having a sequence that is at least about 70%, 80%, 85%, 90% or 95% identical posledovatel the follow identified in the list of sequences (SEQ ID NO:1-35), or sequences identified in the cited works. In some embodiments of the invention TAA with variants of amino acid sequences that have essentially the same biological properties or characteristics, as TAA, having the sequence represented in the list of sequences (SEQ ID NO:1-35). For example, TAA, having a modified sequence, essentially, can specifically bind with an antibody that specifically binds to TAA, with corresponding sequence specified in the list of sequences. These sequences and specifically cited here describe in its entirety introduced into the present description by reference.

Tumor-associated antigens (1)-(35)

(1) BMPR1B (the receptor protein of bone morphogenesis type IB, Genbank reg. No. NM_001203, ten Dijke, P. et al., Science 264 (5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004063362 (claim 2); WO2003042661 (12 claims); US2003134790-AI (pages 38-39); WO2002102235 (item 13 claims; page 296); WO2003055443 (pages 91-92); WO200299122 (example 2; page 528-530); WO2003029421 (p.6 claims); WO2003024392 (claim 2; Fig); WO200298358 (claim 1 of the formula of the invention; page 183); WO200254940 (page 100-101); WO20259377 (page 349-350); WO200230268 (item 27 claims; page 376); WO200148204 (example; figure 4); NP_001194 receptor protein of bone morphogenesis of type IB/pid =NP_001194.1. Cross-references: MIM:603248; NP_001194.1; NM_001203_1; 502 amino acids:

(2) e (LAT1, SLC7A5, Genbank reg. No. NM_003486); Biochem. Biophys. Res. Commun. 255(2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch H.W. et al., (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (example 2); WO2004032842 (example IV); WO2003042661 (12 claims); WO2003016475 (claim 1 of the patent claims); WO200278524 (example 2); WO200299074 (p.19 claims; pages 127-129); WO200286443 (item 27 claims, page 222, 393); WO2003003906 (paragraph 10 of the claims; page 293); WO200264798 (p claims; pages 93-95); WO200014228 (p.5 claims; pages 133-136); US2003224454 (figure 3); WO2003025138 (para.12 claims; page 150); NP_003477 member of family 5 soluble media 7 (a Transporter of cationic amino acids, y+system), member 5/pid=NP_003477.3-Homo sapiens. Cross-references: MIM:600182; NP_03477.3; NM_015923; NM_003486_1, 507 amino acids:

(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate, Genbank reg. No. NM_012449; Cancer Res. 61(15), 5857-5860 (2001) Hubert RS, et al. (1999) Proc. Natl. Acad. Sci., USA, 96(25):14523-14528); WO2004065577 (p.6 claims); WO2004027049 (Fig.1L); ER (example 11); WO2004016225 (claim 2); WO20030442661 (12 claims); US200357089 (example 5); US2003185830 (example 5); US2003064397 (figure 2); WO200289747 (example 5; pages 618-619); WO2003022995 (example 9; figa, example 53, page 173, example 2; figa); NP_036581, testimony epithelial antigen of the prostate. Cross-references: MIM:604415; NP_036581.1; NM_012449_1, 339 amino acids:

(4) R (SA, MUC16, Genbank reg. No. AF361486 J. Biol. Chem. 276(29):27371-27375 (2001)); WO2004045553 (14 claims); WO200292836 (p.6 claims; Fig); WO200283866 (§15 claims; pages 116-121); US2003124140 (example 16); US2003091580 (p.6 claims); WO200206317 (claim 6; pages 400-408). Cross-references: GI:34501467; AAK74120.3; AF361486_1, 6995 amino acids:

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank reg. No. NM_005823, N. Yamaguchi et al., Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci., USA, 96(20):11531-11536 (1999) Proc. Natl. Acad. Sci., USA, 93(1):136-140 (1996), J. Biol. Chem. 270(37):21984-21990 (1995)); WO2003101283 (14 claims); WO2002102235 (item 13 claims; pages 287-288); WO2002101075 (p.4 claims; pages 308-309); WO200271928 (pages 320-321); WO9410312 (pages 52-57). Cross-references: MIM:601051; NP_005814.2; NM_005823_1; 622 amino acids:

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, member 2 family soluble carriers 4 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II, Genbank reg. No. NM_006424; J. Biol. Chem. 277(22):19665-19672 (2002), Genomics 62(2):281-284 (1999), Feild J.A. et al. (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (claim 2); ER (example 11); WO2002102235 (item 13 claims; page 326); IR (claim 1 of the formula of the invention; pages 17-19); WO200157188 (claim 20 claims; page 329); WO2004032842 (example IV); WO200175177 (paragraph 24 claims; pages 139-140). Cross-references: MIM:604217; NP_006415.1; NM_006424_1, 690 amino acids:

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5B, Genbank reg. No. AW, T. Nagase et al., (2000) DNA Res. 7(2):143-150); WO2004000997 (claim 1 of the patent claims); WO2003003984 (claim 1 of the patent claims); WO200206339 (claim 1 of the formula of the invention; page 50); WO200188133 (claim 1 of the formula of the invention; pages 41-43, 48-58); WO2003054152 (claim 20 claims); WO2003101400 (claim 1 of the patent claims); reg. No. Q9P283; EMBL; AB040878; BAA95969.1 Genew; HGNC:10737; 1093 amino acids:

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, cDNA gene RIKEN 2700050C12, Genbank reg. No. AY358628); US2003129192 (claim 2); US2004044180 (12 claims); US2004044179 (11 claims); US2003096961 (claim 11 of the formula izaberete the ia); US2003232056 (example 5); WO2003105758 (12 claims); US2003206918 (example 5); IR (claim 1 of the patent claims); WO2003025148 (claim 20 claims). Cross-references: GI:37182378; AAQ88991.1; AY358628_1; 141 amino acid:

(9) ETBR (endothelin receptor type b, Genbank reg. No. AY275463; Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39,1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai, H., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai, H., et al. J. Biol. Chem. 268, 3463-3470,1993; Sakamoto, A., Yanagisawa, M., et al. Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy, N.A., et al. J. Biol. Chem. 268, 3873-3879, 1993; Haendler C., et al. J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg R.L., et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; Bourgeois C, et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij JB, et al. Am. J. Med. Genet. 108, 223-225, 2002; R.M.W. Hofstra, et al. Eur. J. Hum. Genet. 5, 180-185,1997; Puffenberger EG., et al. Cell 79, 1257-1266, 1994; Attie T., et al. Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio, A., et al. Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; R.M.W. Hofstra, et al. Nat. Genet. 12, 445-447,1996; Svensson, P.J., et al. Hum. Genet. 103, 145-148, 1998; Fuchs, S., et al. Mol. Med. 7, 115-124, 2001; Pingault V, et al. (2002) Hum. Genet. Ill, 198-206; WO2004045516 (claim 1 of the patent claims); WO2004048938 (example 2); WO2004040000 (p claims); WO2003087768 (claim 1 of the patent claims); WO2003016475 (claim 1 of the patent claims); WO2003016475 (claim 1 of the patent claims); WO200261087 (1); WO2003016494 (6); WO2003025138 (clause 12 claims; page 144); WO200198351 (claim 1 of the formula of the invention; pages 124-125); IR (Fig.8 formula of the invention; figure 2); WO200177172 (claim 1 of the formula izobreteny is; pages 297-299); US2003109676; US6518404 (figure 3); US5773223 (PA claims; column 31-34); WO2004001004, 442 amino acids:

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763); WO2003104275 (claim 1 of the patent claims); WO2004046342 (example 2); WO20030442661 (12 claims); WO2003083074 (14 claims; page 61); WO2003018621 (claim 1 of the patent claims); WO2003024392 (claim 2; Fig); WO200166689 (example 6). Cross-references: LocusID:54894; NP_060233.2; NM_017763_1, 783 amino acids:

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of prostate cancer, Genbank reg. No. AF455138; Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (claim 1 of the formula of the invention; Fig 1); WO200272596 (item 13 claims; pages 54-55); WO200172962 (claim 1 of the formula of the invention; figv); WO2003104270 (11 claims); WO2003104270 (item 16 claims); US2004005598 (p.22 claims); WO2003042661 (12 claims); US2003060612 (para.12 formula of the invention; figure 10); WO200226822 (item 23 claims; 2); WO200216429 (para.12 formula of the invention; figure 10). Cross-references: GI:22655488, AAN04080.1; AF455138_1, 490 amino acids:

(12) TrpM4 (BR22450, FLJ2041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M, Genbank reg. No. NM_017636; X.Z. Xu et al., Proc. Natl. Acad. Sci., USA, 98(19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (4 claims); WO200040614 (14 claims; pages 100-103); WO200210382 (claim 1 of the formula of the invention; figa); WO2003042661 (para.12); WO200230268 (item 27 claims; page 391); US2003219806 (4 claims); WO200162794 (14 claims; figa-D). Cross-references: MIM:606936; NP_060106.2; NM_017636_1, 1214 amino acids:

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma, Genbank reg. No. NP_003203 or NM_003212, Ciccodicola A. et al., EMBO J. 8(7):1987-1991 (1989), Am. J. Hum. Genet. 49(3):555-565 (1991)); US2003224411 (claim 1 of the patent claims); WO2003083041 (example 1); WO2003034984 (12 claims); WO200288170 (claim 2; page 52-53); WO2003024392 (claim 2; Fig); WO200216413 (claim 1 of the formula of the invention; pages 94-95, 105); WO200222808 (claim 2; Fig 1); US5854399 (example 2; columns 17-18); US5792616 (figure 2). Cross-references: MIM:187395; NP_003203.1; NM_003212_1, 188 amino acids:

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/receptor of the virus of Epstein-Barr) or Hs.73792, Genbank reg. No. M26004; Fujisaku et al. (1989) J. Biol. Chem. 264(4):2118-2125); J.J. Weis et al., J. Exp. Med. 167, 1047-1066, 1988; Moore, M., et al., Proc. Natl. Acad. Sci., USA, 84, 91949198, 1987; Barel M., et al., Mol. Immunol. 35, 1025-1031, 1998; J.J. Weis et al. Proc. Natl. Acad. Sci., USA, 83, 5639-5643, 1986; Sihna S.K. et al. (1993) J. Immunol. 150, 5311-5320; WO2004045520 (example 4); US2004005538 (example 1); WO2003062401 (9 claims); WO2004045520 (example 4); WO9102536 (Fig-9.9); WO2004020595 (claim 1 of the patent claims); registration number: P20023; Q13866; Q14212, EMBL; M26004; AAA35786.1, 1033 amino acids:

(15) CD79b (CD79B, CD79β, IGb (beta protein associated with immunoglobulin), V, Genbank reg. No. NM_000626 or 11038674, Proc. Natl. Acad. Sci., USA, (2003) 100(7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al., (1992) Eur. J. Immunol. 22(6):1621-1625); WO2004016225 (claim 2; Fig); WO2003087768, US2004101874 (claim 1 of the claims, page 102); WO2003062401 (9 claims); WO200278524 (example 2); US2002150573 (claim 1 of the claims, page 15); US5644033, WO2003048202 (claim 1 of the claims, page 306 and 309); WO99/558658, US6534482 (item 13 claims; figa/); WO200055351 (§11 claims, pages 1145-1146). Cross-references: MIM:147245; NP_000617.1; NM_000626_1; 229 amino acids:

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C, Genbank reg. No. NM_030764, Genome Res. 13(10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002)Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci., USA, 98(17):9772-9777 (2001), Xu M.J. et al. (2001) Biochem. Biophys. Res. Commun. 280(3):768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (p.5 claims; fig.18D-1-18D-2); WO2003097803 (12 claims); WO2003089624 (A.25 claims). Cross-ssy is key: MIM:606509; NP_110391.2; NM_030764_1, 508 amino acids:

(17) HER2 (ErbB2, Genbank reg. No. M11730, Coussens L. et al. Science (1985) 230(4730):1132-1139); T. Yamamoto et al., Nature 319, 230-234, 1986; K. Semba et al., Proc. Natl. Acad. Sci., USA, 82, 6497-6501, 1985; Swiercz J.M. et al. J. Cell. Biol. 165, 869-880, 2004; Kuhns J.J. et al., J. Biol. Chem. 274, 36422-36427, 1999; Cho H.S. et al. Nature 421, 756-760, 2003; A. Ehsani et al., (1993) Genomics 15, 426-429; WO2004048938 (example 2); WO2004027049 (Fig); WO2004009622; WO2003081210; WO2003089904 (9 claims); WO2003016475 (claim 1 of the patent claims); US2003118592; WO2003008537 (claim 1 of the patent claims); WO2003055439 (phormula invention 29; figa-IN); WO2003025228 (clause 37 claims; figs); WO200222636 (example 13; pages 95-107); WO200212341 (p claims; 7); WO200213847 (pages 71-74); WO200214503 (pages 114-117); WO200153463 (claim 2 of the formula of the invention; pages 41-46); WO200141787 (page 15); WO200044899 (paragraph 52 of the claims; 7); WO200020579 (p.3 formula of the invention; figure 2); US5869445 (p.3 claims; columns 31-38); WO9630514 (claim 2; page 56-61); OR (7 claims); WO2004043361 (7 claims); WO2004022709; WO200100244 (example 3; figure 4); reg. No. PO4626; EMBL; M11767; AAA35808.1. EMBL; M; AOA. 1255 amino acids:

(18) NCA (CEACAM6, Genbank reg. No. m); Barnett, T., et al., Genomics 3, 59-66, 1988; Tawaragi Y. et al., Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L. et al. Proc. Natl. Acad. Sci., USA, 99:16899-16903, 2002; WO2004063709, EP1439393 (7 claims); WO2004044178 (example 4); WO2004031238; WO20030442661 (12 claims); WO00278524 (example 2); WO200286443 (item 27 claims; page 427); WO200260317 (claim 2); reg. No. P40199; Q14920; EMBL; M; AOA, EMBL; M; 344 amino acids:

(19) MDP (DPEP1, Genbank reg. No. BC017023, Proc. Natl. Acad. Sci., USA, 99(26):16899-16903 (2002)); WO2003016475 (claim 1 of the patent claims); WO200264798 (p claims; pages 85-87); JPO5003790 (Fig.6-8); WO9946284 (Fig.9). Cross-references: MIM:179780; AN; BC017023_1, 411 amino acid:

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank reg. No. AF184971); H.F. Clark et al., Genome Res. 13, 2265-2270, 2203; Mungall AJ, et al., Nature 425, 805-811, 2003; Blumberg H. et al., Cell 104, 9-19, 2001; Dumoutier L. et al., J. Immunol. 167, 3545-3549, 2001; Parrish-Novak, J. et al., J. Biol. Chem. 277, 47517-47523, 2002; Pletnev, S. et al., (2003) Biochemistry 42:12617-12624; Sheikh F. et al. (2004) J. Immunol. 172, 2006-2010; ER (example 11); US2004005320 (example 5); WO2003029262 (pages 74-75); WO2003002717 (claim 2; page 63); WO200222153 (pages 45-47); WO2003002717 (2 formulas), US2002042366 (pages 20-21); WO200146261 (pages 57-59); WO200146232 (pages 63-65); WO9837193 (claim 1 of the formula of the invention; pages 55-59); reg. No. Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1, 553 amino acids:

(21) Brevican (BCAN, BEHAB, Genbank reg. No. AF229053); Gary S.C. et al., Gene 256, 139-147, 2000; Clark H.F. et al. Genome Res. 13, 2265-2270, 2203; Strausberg R.L. et al. Proc. Natl. Acad. Sci., USA, 99, 16899-16903, 2002; US2003186372 (11 claims); US2003186373 (11 claims); US2003119131 (claim 1 of the formula of the invention; Fig); US2003119122 (claim 1 of the formula of the invention; Fig); US2003119126 (claim 1 of the patent claims); US2003119121 (claim 1 of the formula the invention; Fig); US2003119129 (claim 1 of the patent claims); US2003119130 (claim 1 of the patent claims); US2003119128 (claim 1 of the formula of the invention; Fig); US2003119125 (claim 1 of the patent claims); WO2003016475 (claim 1 of the patent claims); WO200202634 (claim 1 of the patent claims); 911 amino acids:

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank reg. No. NM_004442); Chan, J. & Watt V.M. Oncogene 6(6), 1057-1061 (1991) Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (12 claims); WO200053216 (claim 1 of the formula of the invention; page 41); WO2004065576 (claim 1 of the patent claims); WO2004020583 (9 claims); WO2003004529 (pages 128-132); WO200053216 (claim 1 of the formula of the invention; page 42). Cross-references: MIM:600997; NP_004433.2; NM_004442_1, 987 amino acids:

(23) ASLG659 (B7h, Genbank reg. No. AX092328), US20040101899 (claim 2); WO2003104399 (11 claims); WO2004000221 (figure 3); US2003165504 (claim 1 of the patent claims); US2003124140 (example 2); US2003065143 (Fig); WO2002102235 (item 13 claims; page 299); US2003091580 (example 2); WO200210187 (p.6 formula of the invention; figure 10); WO2001194641 (clause 12 claims; fig.7b); WO200202624 (item 13 claims, figa-1B); US2002034749 (item 54 of the claims; pages 45-46); WO200206317 (example 2; pages 320-321; 34 claims; page 321-322); WO200271928 (pages 468-469); WO200202587 (example 1, figure 1); WO200140269 (example 3; pages 190-192); WO200036107 (example 2; with the pages 205-207); WO2004053079 (12 claims); WO2003004989 (claim 1 of the patent claims); WO200271928 (pages 233-234, 452-453); WO0116318; 282 amino acids:

(24) PSCA (antigen precursor stem cells of the prostate gland, Genbank reg. No. AJ297436); Reiter R.E. et al., Proc. Natl. Acad. Sci., USA, 95, 1735-1740, 1998; Gu Z., et al., Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000)275(3):783-788; WO2004022709; ER (example 11); US2004018553 (17 claims); WO2003008537 (claim 1 of the patent claims); WO200281646 (claim 1 of the formula of the invention; page 164); WO2003003906 (paragraph 10 of the claims; page 288); WO200140309 (example 1; Fig); US2001055751 (example 1; fig.1b); WO20032752 (p formula of the invention; Fig 1); WO9851805 (17 claims; page 97); WO9851824 (paragraph 10 of the claims; page 94); WO98440403 (p.2 claims; figv); reg. No. O43653; EMBL; AF043498; AAC39607.1, 123 amino acids

(25) GEDA (Genbank reg. No. AY260763); a protein such partner “hybrid lipoma HMGIC AAP14954-Homo sapiens: Species: Homo sapiens (human) WO2003054152 (claim 20 claims); WO2003000842 (claim 1 of the patent claims); WO2003023013 (example 3, claim 20 of the claims); US2003194704 (§45 of the claims). Cross-references: GI:30102449; AAP14954.1; AY260763_1, 236 amino acids

(26) BAFF-R (factor receptor activation In cells, BLyS receptor 3, BR3, Genbank reg. No. NP_443177.1); NP_443177 BAFF receptor/pid=NP_443177.1-Homo sapiens J.S. Thompson et al. Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (example; pages 32-3); WO2003014294 (p claims; figv); WO2003035846 (item 70 of the claims; pages 615-616); WO200294852 (columns 136-137); WO200238766 (p.3 claims; page 133); WO200224909 (example 3; Fig 3). Cross-references: MIM:606269; NP_443177.1; NM_052945_1, 184 amino acids

(27) CD22 (b-cell receptor CD22, isoform B, Genbank reg. No. NP-001762.1); Stamenkovic I. and Seed, B., Nature 345 (6270), 74-77 (1990); US2003157113; US2003118592; WO2003062401 (9 claims); WO2003072036 (claim 1 of the formula of the invention; Fig 1); WO200278524 (example 2). Cross-references: MIM:107266; NP_001762.1; NM_001771_1, 847 amino acids:

(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha-protein-klecko-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with molecules of IgM on the cell surface, passing the signal that is involved in the differentiation of b-cells) PROTEIN SEQUENCE Full mpggpgv...dvqlekp (1..226; 226 amino acids), pI: 4.84, MW:25028 TM: 2[P] Gene Chromosome: 19q13.2, reg. No. NP_001774.1; WO2003088808, US20030228319, WO2003062401 (9 claims); US2002150573 (p.4 claims, pages 13-14); WO99558658 (item 13 claims, Fig); WO9207574 (1); US5644033; Ha et al., (1992) J. Immunol. 148(5):1526-1531; Mueller et al., (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al., (1994) Immunogenetics 40(4):287-295; Preud''homme et al., (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464; 226 amino acids:

(29) CXCR5 (receptor 1 lymphoma Burkitt, the receptor-associated G-protein and activated by the chemokine CXCL13, provides migration of lymphocytes and humoral defense, plays a role in infection by HIV-2 and probably in the development SPEED and, lymphoma, myeloma, and leukemia) PROTEIN SEQUENCE Full mnypltl...atslttf (1..372, 372 amino acids), pI:8.54 MW:41959 TM: 7[P] Gene Chromosome: 11q23.3, Genbank reg. No. NP_001707.1, WO200404000; WO2004015426; US2003105292 (example 2); US6555339 (example 2); WO200261087 (1); WO200157188 (claim 20 claims; page 269); WO200172830 (pages 12-13); WO200022129 (example 1, page 152-153, example 2, pages 254-256); WO9928468 (claim 1 of the claims, page 38); US5440021 (example 2, columns 49-52); WO9428931 (pages 56-58); WO9217497 7 claims; figure 5); Dobner et al. (1992) Eur. J. Immunol. 22:2795-2799; Barella et al., (1995) Biochem. J. 309:773-779; 372 amino acids:

(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes) PROTEIN SEQUENCE Full mgsgwvp...vllpqsc (1...273; 273 amino acids, pI:6.56 MW:30820 TM:1[P] Gene Chromosome: R, Genbank reg. No. NP_002111.1; Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al., (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci., USA, 99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al., (1996) J. Mol. Biol. 255:1-13; Naruse et al., (2002) Tissue Antigens 59:512-519; WO9958658 (item 13 claims; Fig); US6153408 (columns 35-38); US5976551 (columns 168-170); US6011146 (columns 145-146); Kashara et al., (1989) Immunoenetics 30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119; 273 amino acids:

(31) RG (ion channel -5 opened ligand purinergic receptor RH; ion channel opening extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may play a role in the pathophysiology of idiopathic bladder dysfunction) PROTEIN SEQUENCE Full mgqagck...lephrst (1..422; 422 amino acids), pI:7.63; MW 47206 TM:1[P] Gene Chromosome: R, Reg. No. NP_002552.2; Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035 (10 claims); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20 claims); WO2003093444 (claim 1 of the patent claims); WO2003087768 (claim 1 of the patent claims); WO2003029277 (page 82); 422 amino acids:

(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2) PROTEIN SEQUENCE Full maeaity...tafrfpd (1..359; 359 amino acids), pI: 8.66, MW:40255 TM:1[P] Gene Chromosome: R, reg. No. NP_001773.1; WO2004042346 (p claims); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci., USA, 99:16899-16903; 359 amino acids:

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I, which regulates the activation and apoptosis of b-cells, and sweat the OC function of this protein is associated with progression in patients of systemic lupus erythematosus) PROTEIN SEQUENCES Full mafdvsc...rwkyqhi (1..661; 661 amino acid), pI:6.20, MW:74147 TM 1[P] Gene Chromosome 5q12, Genbank reg. No. NP_005573.1; US2002193567; WO9707198 (§11 claims; pages 39-42); Miura et al. (1996) Genomic 38(3):299-304; Miura et al., (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (item 8 claims; pages 57-61); WO200012130 (pages 24-26); 661 amino acid:

(34) FCRH1 (Fc receptor-like protein 1, the presumed receptor for the Fc domain of immunoglobulin, which contains Ig-like domains of type C2 and ITAM domains and may play a role in the differentiation of b-lymphocytes) PROTEIN SEQUENCE Full mlprlll...vdyedam (1..429; 429 amino acids), pI:5.28, MW:46925 TM:1[P] Gene Chromosome: 1q21-1q22, Genbank reg. No. NP_443170.1); WO2003077836; WO2001384090 (item 6 claims, five-1-18-E2); Davis et al. (2001) Proc. Natl. Acad. Sci., USA, 98(17):9772-9777; WO2003089624 (8 claims); IR (claim 1 of the patent claims); WO2003089624 (7 claims); 429 amino acids:

(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily 2, the estimated immunoreceptor, which might play a role in the development of b cells and lymphomagenesis; and in some malignant b cells observed dysregulation of the gene by translocation) PROTEIN SEQUENCE Full mllwvil...assaphr (1..977; 977 amino acids), pI:6.88 MW:106468 TM:1[P] Gene Chromosome: 1q21, Genbank reg. No. NP_112571.1; WO2003024392 (claim 2; Fig); Nakayama et al., (2000) Biochem. Biophs. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (p.3 claims; figv-1-18V-2); 977 amino acids:

Cm. also: WO04/045516 (3 June 2004); WO03/000113 (3 January 2003); WO02/016429 (28 February 2002); WO02/16581 (28 February 2002); WO03/024392 (27 March 2003); WO04/016225 (26 February 2004); WO01/40309 (7 June 2001) and the provisional patent application U.S. reg. No. 60/520842 “COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN”, filed November 17, 2003, each of which in its entirety is introduced into the present description by reference.

In one embodiment of the invention, the conjugate of the ligand-linker-drug” has the formula IIIa, where the ligand is an antibody Ab, including antibody that binds at least one antigen CD30, CD40, CD70, Lewis Y, w=0, y=0, and D has formula Ib. Representative conjugates of formula IIIa are conjugates in which R17represents -(CH2)5-. The present invention also includes such conjugates of formula IIIa, in which D has the structure of compound 2 in example 3 and its esters. The present invention also includes conjugates of formula IIIa, containing from about 3 to 8, and in one aspect, from about 3 to 5 molecules of the drug D, i.e. the conjugates of formula Ia, where R is the amount in the range of about 3 to 8, such as about 3-5. The compounds according to izobreteny which also includes conjugates, having a combination of structural characteristics specified in this paragraph.

In another embodiment of the invention, the conjugate of the ligand-linker-drug” has the formula IIIa, where the ligand is an antibody Ab, including antibody that binds to one antigen CD30, CD40, CD70, Lewis Y, w=1, y=0, and D has formula Ib. The present invention also includes such conjugates of formula IIIa, in which R17represents -(CH2)5-. The present invention also includes such conjugates of formula IIIa, in which W is a Val-Cit and/or in which D has the structure of compound 2 described in example 3, and its esters. The present invention also includes conjugates of formula IIIa, containing from about 3 to 8, and preferably from about 3 to 5 molecules of the drug D, i.e. the conjugates of formula Ia, where R is the amount in the range of about 3 to 8, such as about 3-5. Representative conjugates are also compared with the combination of structural characteristics specified in this paragraph.

In another embodiment of the invention, the conjugate of the ligand-linker-drug” has the formula IIIa, where the ligand is an antibody Ab, including antibody that binds to one antigen CD30, CD40, CD70, Lewis Y, w=1, y=1, and D has formula Ib. The present invention also in the cancel such conjugates of formula IIIa, in which R17represents -(CH2)5-. The present invention also includes such conjugates of formula IIIa, in which W is a Val-Cit, Y has the formula X; D has the structure of compound 2 described in example 3, and its esters; p is from about 3 to 8, and preferably from about 3 to 5 molecules of the drug D. the scope of the compounds according to the invention also includes conjugates having a combination of structural characteristics specified in this paragraph.

In another embodiment, the present invention relates to a conjugate of the antibody-drug” (ADC) or its pharmaceutically acceptable salt or MES, where Ab is an antibody that binds to one of tumor-associated antigens (1)to(35)above (“connection TAA”).

In another embodiment, the present invention relates to the connection TAA or its pharmaceutically acceptable salt or MES that are in isolated and purified form.

In another embodiment, the present invention relates to a method of preventing or inhibiting the multiplication of tumor or cancer cells comprising the administration to a patient, such as person suffering from a hyperproliferative disorder, the compounds of the TAA or its pharmaceutically acceptable salt or MES is in the quantity effective for preventing or inhibiting the multiplication of tumor or cancer cells.

In another embodiment, the present invention relates to a method of treatment of cancer, comprising the administration to a patient, such as person suffering from a hyperproliferative disorder, the compounds of the TAA or its pharmaceutically acceptable salt or MES in amounts effective for the treatment of cancer, alone or in combination with an effective amount of another anticancer tools.

In another embodiment, the present invention relates to a method of treating an autoimmune disease comprising the administration to a patient, such as person suffering from a hyperproliferative disorder, the compounds of the TAA or its pharmaceutically acceptable salt or MES in amounts effective for the treatment of autoimmune diseases.

Antibodies suitable for use in the present invention can be obtained by any known method for the synthesis of antibodies, and in particular by the method of chemical synthesis or by expression of recombinant DNA, and preferably by the method of expression of recombinant DNA.

4.5.1. Production of recombinant antibodies

Antibodies according to the invention can be obtained by any known method for the synthesis of antibodies, and in particular by the method of chemical synthesis or expression recombine the different DNA.

For recombinant expression of antibodies or their fragments, derivatives or analogs, you must construct a nucleic acid encoding such an antibody. If the nucleotide sequence for this antibody is known, then the Assembly of nucleic acid encoding such an antibody can be made from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), and this Assembly includes the synthesis of overlapping oligonucleotides containing part of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification legirovannykh oligonucleotides, for example, by using PCR.

Alternatively, a nucleic acid molecule encoding the antibody can be obtained from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not commercially available, but the known sequence of the antibody, the nucleic acid encoding the antibody can be obtained from a suitable source (e.g., from a cDNA library of antibodies or from a cDNA library produced from any tissue or other cells expressing immunoglobulin, for example, by PCR amplification using synthetic primers, hybridizers with 3'- and 5'-ends of placentas the work, or by cloning using oligonucleotide probe specific for the particular gene sequence.

If the antibody that specifically recognizes a particular antigen (or the source of the cDNA library for cloning a nucleic acid encoding such immunoglobulins), is not commercially available, antibodies that are specific to a given antibody can be obtained by any known method, for example by immunization of the patient or a suitable animal model, such as a rabbit or mouse, for producing polyclonal antibodies or, more preferably by generating monoclonal antibodies, for example, as described by Kohler and Milstein (1975, Nature 256:495-497) or as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. in 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p.77-96). Alternatively, the clone that encodes at least the Fab-fragment of the antibody, can be obtained by screening the Fab-expressing libraries (for example, as described by Huse et al., 1989, Science 246:1275-1281) to clone Fab fragments that bind with a specific antigen, or by screening libraries of antibodies (see, for example, Clackson et al., 1991, Nature 352:624; Hane et al. 1997, Proc. Natl. Acad. Sci., USA, 94:4937).

After obtaining nucleic acid sequence that encodes at least the variable domain of the antibody, it can be introduced into a vector containing a nucleotide is the selected, encoding the constant region of the antibody (see, for example, the publication of International applications No. WO86/05807; WO89/01036 and U.S. patent No. 5122464). Vectors containing full-sized light or heavy chain and allows to Express the full-sized molecule antibodies are commercially available. Then the nucleic acid encoding the antibody can be used for introduction of nucleotide substitutions or deletions needed to replace (or deletion) of one or more cysteine residues of the variable region involved in the formation of noticeplease disulfide bond at amino acid residue that does not contain a sulfhydryl group. Such modifications can be carried out by any known methods of introducing specific mutations or deletions in the nucleotide sequence, and such methods include, but are not limited to, for example, a method of chemical mutagenesis and site-directed mutagenesisin vitro(Hutchinson et al. 1978, J. Biol. Chem. 253:6551).

In addition, can be applied techniques developed for the production of “chimeric antibodies” (Morrison et al. 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes of a mouse antibody molecule with the appropriate specificity to the antigen, together with the genes of molecules of human antibodies with the corresponding the overall biological activity. A chimeric antibody is a molecule in which different parts come from different animal species, such as parts having a variable region derived from murine monoclonal antibody and a constant region of human immunoglobulin, for example gumanitarnoe antibody.

Alternatively, for producing single-chain antibodies can be adapted to known methods used to obtain such antibodies (U.S. patent No. 4694778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci., USA, 85:5879-5883, and Ward et al., 1989, Nature 334:544-54). Single-chain antibodies produced by binding fragments of the Fv region of the heavy and light chains by amino acid bridge, resulting in a single-chain polypeptide. Can also be applied to methods for the Assembly of functional Fv fragments inE. coli(Skerra et al., 1988, Science 242:1038-1041).

Antibody fragments that recognize specific epitopes may be produced by the known methods. For example, such fragments include, but are not limited to, F(ab')2-fragments that can be produced by hydrolysis of the molecule pepsin antibodies, and Fab fragments that can be produced by restoring the disulfide bridges of F(ab')2-fragments.

After obtaining nucleic acid sequence that encodes the antibodies is about, can be obtained, the vector for producing antibodies are well known methods of recombinant DNA. To construct expression vectors containing sequences encoding antibodies and the corresponding signals in the regulation of transcription and translation, can be applied by methods well known in the art. Such methods are, for example, methods of recombinant DNAin vitromethods of synthesis and methods of genetic recombinationin vivo. See, for example, methods described in Sambrook et al. (1990, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

The expression vector containing the nucleotide sequence of the antibody or the nucleotide sequence of the antibody can be transferred into the cell host by standard methods (e.g., by electroporation, liposomal transfection, and precipitation of calcium phosphate), and then transfected cells can be cultured by standard methods of producing antibodies. In specific embodiments of the invention the expression of antibodies is regulated by a constitutive, inducible or tissue-specific promoter.

The cells of the host used for expression of recombinant antibodies can be either bacterial cells such kak> Escherichia colior preferably eukaryotic cells, in particular cells suitable for expression of the full-size recombinant molecules of the immunoglobulin. In particular, an efficient expression system of immunoglobulins are mammalian cells, such as cells of the Chinese hamster ovary (Cho) in combination with a vector, such as a promoter element of the main pretannage gene, derived from the human cytomegalovirus (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2).

For the expression of immunoglobulins (antibodies) can be used in different systems of host-expression vector. Such systems are “host-expression vector” are the media through which the sequence encoding the antibody can be produced, and then cleared, however, such systems may also be cells that, in their transformation or transfection with the appropriate nucleotide coding sequences, Express molecule of the immunoglobulin (antibody)in situ. Such cells include, but are not limited to, microorganisms such as bacteria (for example,E. coliandB. subtilis), transformed with vectors expressing recombinant bacteriophagous DNA, plasmid DNA or kosmidou DNA containing the sequence encoding immunoglob is lean; yeast (for example,Saccharomyces Pichia)transformed with recombinant vectors for expression in yeast containing sequences encoding immunoglobulin; cellular system of insects infected with recombinant virus expression vectors (e.g. baculovirus)containing the sequence encoding the immunoglobulin; cellular system of insects infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)containing sequences encoding immunoglobulin; or cellular system mammalian (e.g., COS cells, Cho, NR, 293, T, T)containing a recombinant expression constructs comprising the promoters derived from the genome of mammalian cells (for example, the promoter metallothionein) or from mammalian viruses (e.g., the late promoter of adenovirus; promoter C cowpox virus).

In bacterial systems a number of expression vectors may be selected mainly depending on the purpose of application of the expressed antibody. For example, if produced large quantities of this protein, it is desirable that the vectors that directed the expression of high levels of products of the hybrid protein, could be easily cleaned. Such vectors include, but are not limited to, the expression vector pUR278E. coli(Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be separately Legerova in the vector, in the same reading frame with the coding region of thelacZ, so that this results in producyrovtsa hybrid protein; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster 1989, J. Biol. Chem. 24:5503-5509), etc. For the expression of foreign polypeptides in the form of a hybrid protein with glutathione-S-transferase (GST) can be used pGEX vectors. In General these hybrid proteins are soluble and can easily be purified from lysed cells by adsorption and binding to the areas of sensors coated with glutathione-agarose, followed by elution in the presence of free glutathione. The pGEX vectors are manufactured so that they include the sites of cleavage by thrombin or by the protease factor XA, resulting in the desired cloned gene product may be released from the molecule GST.

In the system of insects as vectors for expression of foreign genes used nuclear polyhedrosis virusAutographa californica(AcNPV) or a similar virusDrosophila Melanogaster.This virus is produced in the cells ofSpodoptera frugiperda. The sequence encoding the antibody can be kleinova is and separately in the secondary region (for example, in the region of the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In the cells of the host mammal can be used a number of expression systems based viruses. In cases when the expression vector is adenovirus of interest antibody-encoding sequence can be Legerova with complex, which regulates transcription/translation of adenovirus, for example, the late promoter and three of the leader sequence. Then this chimeric gene may be integrated into the genome of adenovirus byin vitroorin vivorecombination. Embedding in the secondary region of the viral genome (e.g., region E1 or E3) leads to the formation of a recombinant virus that is viable and can Express the immunoglobulin molecule in infected hosts (see, e.g., Logan &Shenk, 1984,Proc. Natl. Acad. Sci.USA, 81:355-359). For efficient broadcast of built-antibody-coding sequences may also require specific initiation signals. Such signals are the initiating ATG codon and adjacent sequences. In addition, to ensure translation of the entire insert the initiating codon must be in the same reading frame that encodes a follower of the awn. These exogenous signals regulation of translation initiator codons can occur from various sources, i.e. they can be natural or synthetic. The efficiency of expression can be increased by enabling the respective enhancer elements, transcription, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, to modulate the expression of the built-in sequences or for the modification and the specific processing of the desired gene product can be selected strain host cell. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may play an important role in the functioning of the protein. Different cell owners have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure that the desired modification and processing of the expressed foreign protein can be selected from appropriate cell lines or system owners. To achieve this goal can be used eukaryotic cell hosts with cellular mechanism for the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such cells mammalian hosts I have are, but not limited to, Cho, VERY, NR, HeLa, COS, MDCK, 293, T, T, WI38, BT483, Hs578T, NTV, BT20 and T47D, CRL7030 and Hs578Bst.

For continuous highly efficient production of recombinant proteins, it is preferable for stable expression. So, for example, can be constructed cell lines, stably expressing the antibody. To do this, instead of expression vectors that contain viral origin replication can be used cell host transformed with DNA controlled by appropriate elements of the regulation of expression (for example, promoter sequences, enhancer, transcription terminators, polyadenylation sites, and the like) and a selective marker. After the introduction of foreign DNA, engineered cells may be placed for 1-2 days in an enriched environment for the growth, and then the environment can be replaced with selective medium. Selective marker in the recombinant plasmid reported resistance to the agent selected for the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form loci, which in turn can be cloned and amplified in cell lines. This method can preferably be used for the construction of cell lines expressing the antibody. Such engineered cell lines m which may be particularly effective for screening and assessment of tumor antigens which directly or indirectly interact with the antibody.

Different sampling systems, including, but not limited to, gene thymidine kinase of herpes simplex virus (Wigler et al., 1977, Cell 11:223), gipoksantin-guanine-phosphoribosyltransferase (Szybalska & Szybalska, 192, Proc. Natl. Acad. Sci. USA, 48:202), and adenine-phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817), can be used in tk-, hgprt - or aprt-cells, respectively. In addition, resistance to antimetabolites can be used as the basis for selection for the following genes: DHFR, which tells resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci., USA, 77:357; O'hare et al., 1981, Proc. Natl. Acad. Sci., USA 78:1527); gpt, which tells resistance to mycophenolate acid (Mulligan &Berg, 1981, Proc. Natl. Acad. Sci., USA, 78:2072); neo, which tells resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu &Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932 & Morgan & Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-217), and hygro, which tells resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Well-known methods used in the technique of recombinant DNA described in Ausubel et al. (eds. 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY and in chapters 12 and 13 of the Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

The levels of expression of antibodies can is to be increased by amplification of the vector (for an overview see, for example, Bebbington &Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing antibody is amplificare, as the increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Because amplificatory region is associated with the nucleotide sequence of the antibody will also increase the level of production of this antibody (Crouse et al., Mol. Cell. Biol. 3:257).

A host cell may be co-transfected with two expression vectors, the first vector encoding a polypeptide heavy chain and a second vector encoding a light chain polypeptide. These two vectors may contain identical selective markers that allow for the expression of polypeptides of the heavy and light chains. Alternatively, for encoding the polypeptides of the heavy and light chains can be used the same vector. In these cases, in order to avoid excessive production of toxic free heavy chain, the light chain should be placed before the heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci., USA, 77:2197). Coding sequences of the heavy and light chains may contain cDNA or genomic DNA.

After recombinant expression of antibodies it is may be purified by any known method, used for purification of antibodies, for example, by chromatography (ion exchange, affinity, particularly by chromatography on affinity for the specific antigen on a column of protein A, and exclusion chromatography), centrifugation, separation based on the different solubility, or by any other standard methods of protein purification.

In another representative example, the antibody is a monoclonal antibody.

In any case, hybrid antibodies may have a dual specificity preferably by the presence of one or more binding sites that are specific to the desired hapten, or one or more binding sites that are specific to the target antigen, such as an antigen associated with a tumor, autoimmune disease, infectious microorganism or other pathological condition.

4.5.2. The production of antibodies

The production of antibodies will be illustrated below, the antibodies against CD30, but for every person it is obvious that the same image can be produced and modified antibodies against other members of the family of TNF receptors. Using CD30 for the production of antibodies is provided only for illustrative purposes and has no specific limitations.

The CD30 antigen used for production of antibodies may be, is for example, to have a soluble form of the extracellular domain of CD30 or part thereof, containing the desired epitope. Alternatively, to generate antibodies can be used in cells expressing CD30 on its surface (for example, L540 (cell line, derived from jackinsky lymphoma, T-cell phenotype) and L428, (cell line, derived from jackinsky lymphoma, b-cell phenotype)). Specialists and other known forms of CD30, which can be used for producing antibodies.

In another representative embodiment of the invention the ErbB2 antigen used for production of antibodies may, for example, be soluble form of the extracellular domain of ErbB2 or part thereof, containing the desired epitope. Alternatively, to generate antibodies can be used in cells expressing ErbB2 on their surface (e.g., transformed cells NIH-T expressing excessive levels of ErbB2; or cell line carcinoma, such as cells SK-BR-3, see Stancovski et al., Proc. Natl. Acad. Sci. USA, 88:8691-8695 (1991)). Specialists and other known forms of ErbB2, which can be used for producing antibodies.

(i) polyclonal antibodies

Polyclonal antibodies are preferably produced in animals after multiple subcutaneous (s.c.) or intraperitoneal (I.P. Pavlova.) injections of the relevant antigen and an adjuvant. It is can be used for conjugation of the corresponding antigen with protein, which is immunogenic for species subjected to immunization, for example, hemocyanine lymph snails, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, such as maleimidophenylmethacrylates (conjugated through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic acid anhydride, SOCl2or R1N=C=NR, where R and R1represent different alkyl groups.

Animals subjected to immunization with the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant's adjuvant and by subcutaneous injection of a solution in many areas. A month later the animals repeatedly subjected to immunization in many areas by subcutaneous injection 1/5-1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant. After 7-14 days in animals blood sample and analyze the serum for antibody titer. Then animals again subjected to immunization before reaching the plateau of the title. Repeated immunization of animals preferably carried out by injection of the conjugate of the same antigen, but conjugated to a different protein and/or the through a different cross-linking reagent. The conjugates can also be obtained in recombinant cell culture as hybrid proteins. In addition, to enhance the immune response can also be used aggregating agents such as alum.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from populations of essentially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for antibodies that have a natural mutation that may be present in small quantities. Thus, the adjective “monoclonal” indicates the type of antibody, which is not a discrete mixture of antibodies.

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

To obtain a hybrid mouse or other suitable animal host, such as a hamster, subjected to immunization, as described above, for generating them lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes may be immunizedin vitro. Then lymphocytes subjected to fusion with myeloma cells using a suitable agent for the merger, such to the to the glycol, resulting in formation of hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)).

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

Preferred myeloma cells are cells that are able to be effective with the merger, to maintain a stable production of high levels of antibodies selected antibody-producing cells, and are sensitive to the environment, such as a NAT environment. Of these cells, preferred myeloma cell lines are murine myeloma lines, such as cell lines, derived from cells of murine tumors MORSE-21 and MPC-11, available at the Institute Salk Institute Cell Distribution Center, San Diego, California USA, and SP cells-2 or H-Ag8-653, available in the American type culture collection, Rockville, Maryland USA. For producing human monoclonal anti who ate also used human myeloma cell lines and heteromyinae cell line mouse-human” (Kozbor J. Immunol., 133:3001 (1984) & Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p.51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium for the growth of hybridoma cells examined for the production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells, determined preferably by thus or by holding thein vitroanalysis of the binding, such as radioimmunoassay (RIA) or solid-phase immunofermentnyi assay (ELISA). The binding affinity of the monoclonal antibody can be, for example, identified through analysis of Scatchard described by Munson et al., Anal. Biochem., 107:220 (1980).

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

Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascitic fluid, or is varodi by carrying out standard procedures for the purification of antibodies, such as, for example, chromatography on protein a-sepharose, chromatography on hydroxiapatite, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies can be readily isolated and sequenced according to standard procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). A preferred source of such DNA are hybridoma cells. After selecting this DNA can be incorporated into expression vectors, which are then transferout in cell host, such as cellsE. coli, monkey COS cells, cells of the Chinese hamster ovary (Cho or myeloma cells that in other cases not produce protein antibodies, resulting in these recombinant cells masters synthesized monoclonal antibodies. Discussion recombinant expression of the antibody-coding DNA in bacteria can be found in articles Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In another embodiment of the invention, monoclonal antibodies or antibody fragments can be isolated from ragovoy library of antibodies generated by the methods described by McCafferty et al., Nature, 348:552-554 (1990). In the work of Clackson et al. Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) described the selection we are the other and human antibodies, respectively, using phage libraries. In more recent publications describe the production of high affinity (nm) human antibodies by replacing genes chains antibody (Marks et al. Bio/Technology, 10:779-783 (1992)), and by combined infection and recombinationin vivoused as a strategy for constructing very large phage libraries (Waterhouse et al. Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these methods are an acceptable alternative to the traditional hybridoma technology used for selection of monoclonal antibodies.

DNA can also be modified, for example, by replacing the sequence encoding the constant domains of the heavy chain and light chain of a human antibody, homologous sequences of mouse antibodies (U.S. patent No. 4816567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851) or by covalent joining the coding sequence for the immunoglobulin of all or part of the coding sequence for a non-immunoglobulinemia polypeptide.

Typically, such non-immunoglobulinemia polypeptides are used to replace the constant domains of an antibody, or they are used to replace the variable domains of one antigennegative site, to obtain a chimeric bivalent antibody, having specificity for a single antigen, or other antigennegative site with what pecifically to another antigen.

(iii) Gumanitarnye antibodies

Gumanitarnoe antibody may have one or more of the included amino acid residues originating from a source, not a person. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be performed mainly by the method of winter (Winter) and employees (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoyen et al., Science 239:1534-1536 (1988)) by replacing sequences of the hypervariable region corresponding sequences of a human antibody. Accordingly, such “gumanitarnye” antibodies are chimeric antibodies (U.S. patent No. 4816567), in which, basically, the smaller part compared to the variable domain of the intact human antibody are replaced by the corresponding sequence from a non-human antibody. Actually gumanitarnye antibodies are typically human antibodies in which some hypervariable residues region and possibly some remnants of the framework region (FR) residues replaced, derived from similar areas of rodent antibodies.

To reduce the antigenicity when creating gumanitarnyh antibodies is very important to choose the variable domains of both egcoa, and heavy chains of human antibodies. In accordance with the so-called method of “fitting” the sequence of the variable domain of the antibody rodent sceneroot entire library of known sequences of the variable domains of a human antibody. Then the human sequence that is most similar to the sequence of rodents, take as the human framework region (FR) for creating gumanitarnogo antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). In another method uses a particular framework region derived from a consensus sequence of all human antibodies of a particular subgroup of light and heavy chains. This same frame area can be used for several different gumanitarnyh antibodies (Carter et al., Proc. Natl. Acad. Sci., USA, 89:4285 (1992); Presta et al., J. Immunol, 151:2623 (1993)).

In another embodiment, antibodies of the invention can be Humanitary with retention of high affinity for a given antigen and other favorable biological properties. Gumanitarnye antibodies can be obtained by analyzing the original sequences and various conceptual gumanitarnyh products using three-dimensional models of the parental and gumanitarnyh sequences. Three-dimensional immunoglobulin model is Vlada publicly available and well known in the art. There are computer programs that illustrate and represent probable three-dimensional conformational structures of selected sequences of the candidate immunoglobulin. The study of these views allows to consider the likely role of these residues in the sequences of immunoglobulin candidate, i.e. residues that influence the ability of the immunoglobulin candidate to communicate with its antigen. This way, FR residues can be selected from the recipient and import” sequences and merged, the result can be obtained the desired antibody with the desired properties, such as increased affinity to the target antigen(s). In General terms, the remains of the hypervariable region directly affect the binding of antigen, and mainly involved in this binding.

Reviewed various forms gumanitarnogo antibodies. For example, gumanitarnym antibody may be an antibody fragment, such as Fab. Alternatively, gumanitarnoe antibody can be an intact antibody, such as an intact IgG1 antibody.

In the examples described the production of a representative gumanitarnogo antibodies against ErbB2. This gumanitarnoe antibody may, for example, contain the remains of a hypervariable region of a non-human is about antibodies, part of the variable domain of the heavy chain of a human antibody, and may optionally contain substitution in the framework region (FR) in a position selected from the group consisting of N, N and N, according to the numbering system variable domains described by Cabaton (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). In one embodiment of the invention gumanitarnoe antibody includes replacement FR in two or all of the provisions N, N and N. In another example described obtaining purified antibody trastuzumab of the drug HERCEPTIN®.

(iv) Human antibodies

Alternatively, humanization can be produced human antibodies. So, for example, at the present time can be obtained from transgenic animals (e.g. mice)that are capable of after immunization, of producing a full repertoire of human antibodies without producing endogenous immunoglobulin. For example, it was reported that the homozygous deletion of the gene region of the junction in the heavy chain (JH) antibodies in chimeric and mutant mice germ line leads to a complete inhibition of the production of endogenous antibodies. The transfer of genes of human immunoglobulin germline this mutant mice germ line can lead to the production of human antibodies after stimulating the AI antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci., USA, 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggerman et al., Year in Measurement, 7:33 (1993) and U.S. patent No. 5591669, 5589369 and 5545807.

Alternatively, for producing human antibodies and fragments of antibodiesin vitrofrom a set of genes variable domain (V) of immunoglobulin derived from maimonidean donors, may be applied the technology of phage view (McCafferty et al. Nature 348:552-553 (1990)). In accordance with this technology, the genes of domain V antibody clone with preservation of the reading frame in the gene of the main or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and are as functional fragments of the antibodies on the surface ragovoy particles. Because threadlike particle contains a copy of the single-stranded DNA phage genome, the selection based on the functional properties of antibodies also allows the selection of the gene encoding the antibody possessing these properties. Thus, the phage mimics some properties of b-cells. Phage representation can be implemented in a variety of formats; see, for example, the review in the work of Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). For the implementation of the phage representation can be used several sources of V-gene segments. Clackson et al., Nature, 352:624-628 (1991) have identified another set of different antibodies against about is catalona from a small random combinatorial library of V genes, originating from the spleen immunogenic mice. This can be constructed set of genes V maimonidean people-donors, and can then be selected antibodies against a variety of antigens, including autoantigens), mainly in accordance with the methods described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). Cm. also U.S. patent No. 5565332 and 5573905. As discussed above, human antibodies can also be producedin vitroactivated b-cells (see U.S. patent No. 5567610 and 5229275). Human anti-CD30 antibodies described in application for U.S. patent, reg. No. 10/338366.

(v) antibody Fragments

To obtain fragments of antibodies, various techniques have been developed. Traditionally, these fragments are formed as a result of proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can be produced directly by recombinant cell host. For example, antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternatively, Fab'-SH fragments can be directly isolated fromE. coliand chemically bonded with the formation of F(ab')2fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, F(ab')2fragments can be allocated is orestano of culture recombinant host cell. Specialists and other known methods of obtaining fragments of antibodies. In other embodiments of the invention selected antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185, U.S. patent No. 5571894 and U.S. patent No. 5587458. The antibody fragment may also be “single-chain antibody, such as antibody described in U.S. patent No. 5641870. Such single-chain antibody fragments can be monospecific or bespecifically.

(vi) Bespecifically antibodies

Bespecifically antibodies are antibodies that have binding specificity for at least two different epitopes. Representative bespecifically antibodies can bind to two different epitopes of the protein CD30. Alternatively, the branch antibodies against CD30 can be combined with the branch that is associated with Fc-receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so that the cellular protective mechanisms were focused in cells expressing CD30. Bespecifically antibodies can also be used for the localization of cytotoxic agents to cells expressing CD30.

Traditional production of full-size bespecifically antibodies is based on the co-expression of two pairs of heavy chain-light chain immunoglobulin, where the two chains have different specificity (Millstein et al., Nature, 30:537-539 (1983)). These hybridoma (quadroma), due to the randomized set of heavy and light chains of immunoglobulin, produce a potential mixture of 10 different antibody molecules, of which only one molecule has the “right” bespecifically structure. Cleaning such a “right” of the molecule, which is usually carried out by stepwise carrying out affinity chromatography, quite difficult and gives a low yield of product. A similar procedure described in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 (1991). In accordance with another approach, the variable domains of the antibodies with the desired specificnosti binding (combined sites of the antibody-antigen”) is attached to the sequence of the constant domain of immunoglobulin. This fusion is preferably carried out with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, CH2 and CH3. While it is preferable that this hybrid was first constant region of the heavy chain (SN)containing the site necessary for binding to the light chain is present at least in one of the hybrids. DNA encoding the hybrid heavy chain immunoglobulin and, if necessary, the light chain immunoglobulin is inserted into separate expression vectors and co-transferout in a suitable organism, the host. This ensures a high grade is flexibility in the correction ratios of the three polypeptide fragments in those embodiments of the invention, in which unequal content of the three polypeptide chains used in the construction provide the optimum outputs. However, you can embed the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal proportions produces high outputs or if such ratio is not decisive.

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

In accordance with the second approach, described in U.S. patent No. 5731168, the boundary between a pair of antibody molecules can be engineered to maximize the% of the NTA of heterodimers, isolated from recombinant cell culture. This boundary preferably contains at least part of the domain CH3 constant domain of the antibody. In this method, small side chains of one or several amino acids in the edge region of the first antibody molecules replace the larger side chains (e.g. tyrosine or tryptophan). In the edge region of the second antibody molecules create compensatory “cavities”, having a size that is identical or similar size larger(s) side(s) chain(s), by replacing large side chains of amino acids smaller side chains (e.g., alanine or threonine). This allows to increase the output of heterodimers in relation to other unwanted end-products such as homodimers.

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

Recent advances in this field allow direct selection of theE. colifragments, Fab'-SH, which can be chemically related to education bespecifically antibodies. In the work Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of fully gumanitarnoi molecule F(ab')2especifismo antibodies. Each Fab'fragment was separately secreted fromE. coliand subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies.

Were also described other methods of acquisition and allocation of fragments especifismo antibodies directly from recombinant cell culture. For example, bespecifically antibodies were produced using “latinovich lightning”. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Peptides latinboy lightning originating from proteins Fos and Jun were added to the Fab'-parts of two other antibodies PU is eat ligating genes. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used for the production of homodimeric antibodies. Technology “datel”described by Hollinger et al., Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993), provides an alternative mechanism for obtaining fragments especifismo antibodies. These fragments contain the variable domain of the heavy chain (VH)connected to the variable domain light chain (VL) through a linker that is too short to create a pairing between the two domains on the same chain. In accordance with this VHand VLdomains of one fragment are forced to mate with a complementary VLand VH-domains of another fragment, thereby forming two antigenspecific site. Was also described another strategy to obtain fragments especifismo antibodies using single-chain Fv(sFv)dimers. Cm. Gruber et al., J. Immunol., 152:5368 (1994).

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

(vii) Other modifications of the amino acid sequence

In this application discusses the modification of amino acid sequences is of NITEL. For example, it may be desirable to increase the affinity of binding and/or other biological properties of this antibody. Variants of the amino acid sequences of these antibodies are produced by introducing appropriate nucleotide substitutions in the nucleic acid antibodies, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antibodies. In the final design can be any combination of deletions, insertions and substitutions, provided that the final design will have the desired properties. Amino acid substitutions can also affect post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

Method used for identification of certain residues or regions of the antibody and which allows to determine the localization of mutagenesis is called “alanine scanning mutagenesis” and described Cunningham & Wells, Science 244:1081-1085 (1989). In this method, a residue or group needs identify residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) for the effect on the interaction of these amino acids with antigen. ZAT is m the localization of amino acids, demonstrating functional sensitivity to such changes, clarify by introducing additional or other variants at this position change, or instead of this replacement. For example, the introduction of amino acid substitutions may be determined in advance, while the nature of the mutationper sedoes not have to be defined in advance. For example, to analyze the performance of a mutation at a given site, Ala scanning or non-specific mutagenesis is carried out in the correct codon or region and the options expressed antibodies sceneroot on the desired activity.

Insertions in the amino acid sequences are inserted at the amino and/or carboxy-ends, forming hybrids, having a length from one residue to polypeptides containing a hundred or more residues, as well as to insert into the sequence, consisting of one or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal nationalsim residue or the antibody attached to a cytotoxic polypeptide. Other insertional variants of the antibody molecules are hybrids N - or C-ends of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the half-life of this antibody in the serum.

Another option is the option of replacing amino acids. These VA is ianti have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for mutagenesis carried out by replacing are hypervariable region, but can also be viewed and replace FR.

Significant changes in the biological properties of the antibody are accomplished by selecting substitutions that have a significant impact on (a) the structure of the polypeptide backbone in the area of the substitution, for example folded and helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (C) the volume of the side chain. Natural remnants are divided into the following groups according to common properties of the side chains:

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

(2) neutral hydrophilic residues: Cys, Ser, Thr;

(3) acid residues: Asp, Glu;

(4) basic residues: Asn, Gln, His, Lys, Arg;

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

(6) aromatic residues: Trp, Tyr, Phe.

Non-conservative substitutions lead to the replacement of a member of one of these classes with a member of another class.

Particularly preferred variant type replacement is the replacement of one or more residues of the hypervariable region of the parent antibody (e.g., gumanitarnogo or human antibody). In General received options selected to give the further development will contribute to improved biological properties compared to the parent antibody from which they originate. A standard way of generating such variants with substitutions provides for the affinity maturation using the method of phage view. Several provisions of the hypervariable region (e.g., 6-7 provisions) is subjected to mutation to generate amino acid substitutions at each position. Generated thus variants of the antibodies will be presented as a monovalent molecules on the particles of filamentous phage in the form hybrids with the product of the gene III, Packed in every part of the phage M13. Then the options presented on the phage, sceneroot on their biological activity (e.g. binding affinity of), as described in this application. To identify sites in hypervariable region that are candidates for modification may be performed alanine-scanning mutagenesis, which allows to identify the remains of the hypervariable region, which play an essential role in binding to the antigen. Alternative or additionally, it may be beneficial to analyze a crystal structure of the complex antigen-antibody for the identification of contact sites between antibody and antigen. Such contact residues and neighboring residues are Kandy is Atami replacement carried out in accordance with the method developed here. After generating these options panel of these variants is subjected to screening as described in this application, and antibodies that detect superior properties in one or more relevant assays may be selected for further investigation.

It may be desirable to modify the antibody of the present invention from the point of view of their effector functions, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of antibodies. Alternative or additionally, in this Fc region may(may) be entered(s) to cysteine(C) residue(s)that will contribute to the formation of messagewall disulfide bond in this area. Generated thus homodimeric antibody may have an enhanced ability to internalize and/or increased complement-dependent cytotoxicity (ADCC). Cm. Caron et al. J. Exp. Med. 176:1191-1195 (1992) and Shopes, B.J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained using heterobifunctional cross-linking agents described in Wolff et al. Cancer Research 53:560-2565 (1993). Alternatively, it may be engineered antibody that has two Fc-region, and therefore may have an enhanced ability to complement-dependent lysis and ADCC. Cm. Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the half-life of antibodies in the serum of this antibody (in particular, the antibody fragment) can be entered receptor-svyazyami epitope of salvation, as described, for example, in U.S. patent No. 5739277. Used herein, the term “receptor-svyazyami epitope salvation” means an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the half-life of the IgG molecule in the serum ofin vivo.

(viii) Options glycosylation

Antibodies in the ADC according to the invention can be glycosylated in conservative positions in their constant regions (Jefferis & Lund (1997) Chem. Immunol. 65:111-128; Wright & Morrison (1997) TibTECH 15:26-32). Oligosaccharide side chains of immunoglobulins affect the function of a protein (Boyd et al. (1996) Mol. Immunol. 32:1311-1318; Wittwe & Howard (1990) Biochem. 29:4175-4180) and intramolecular interactions between parts of the glycoprotein, which may affect the conformation and presented three-dimensional surface of the glycoprotein (Hefferis & Lund, see above; Wyss & Wagner (1996) Current Opin. Biotech. 7:409-416). Oligosaccharides can also be used for this glycoprotein to some molecules with specificity the ski recognize patterns. For example, it was reported that in neglikozilirovanny IgG oligosaccharide part of the “falls“ of space between the CH2 and terminal N-acetylglucosamine remains available for binding to protein, to bind to the mannose (Malhorta et al. (1995) Nature Med. 1:237-243). Destruction under the action of glycopeptides, oligosaccharides from SAMRAT-1H (recombinant gumanitarnogo mouse monoclonal IgG1 antibodies that recognize the antigen CDw52 human lymphocytes)produced in the cells of the Chinese hamster ovary (Cho), led to complete cessation of the complement-dependent lysis (CMCL) (Boyd et al. (1996) Mol. Immunol. 32:1311-1318), whereas the selective removal of the residues of sialic acid under the action of neuraminidase did not lead to the loss of DMCL. It was also reported that glycosylation of antibodies affect antibody-dependent cellular cytotoxicity (ADCC). In particular, it was reported that cells SNO with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), i.e. the glycosyltransferases that catalyze the formation of GlcNAc, dividing the sequence in half, has an increased ADCC activity (Umana et al. (1999) Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation means attaching carbohydrate group to the side chain of aspartic ostad is. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X denotes any amino acid except Proline, are sequences of recognition for enzymatic joining carbohydrate portion to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide promotes the creation of a potential site of glycosylation. O-linked glycosylation means joining one of the sugars, such as N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate mainly to serine or threonine, although they may also be 5-hydroxyproline or 5-hydroxylysine.

Variant glycosylation of antibodies are ways in which altered type of glycosylation of antibodies. Under this “change” refers to the deletion of one or more carbohydrate groups present in the antibody, the addition of one or more carbohydrate groups to the antibody, the changing composition of glycosylation (such as glycosylation), degree of glycosylation, etc.

The accession of glycosylation sites to the antibody is usually carried out by modification of the amino acid sequence in which this amino acid sequence will contain one or more of the above what's Tripeptide sequences (for sites of N-linked glycosylation). This modification can also be carried out by adding one or more serine or treoninove residues to the sequence or replacement in the sequence of the original antibody (for sites of O-linked glycosylation). Similarly, deletion of glycosylation sites can be carried out by modification of amino acids in the natural sites of glycosylation of antibodies.

Amino acid sequence usually modify by changing the coding of its nucleic acid sequence. Such methods include, but are not limited to, isolation from a natural source (in the case of a natural amino acid sequence variants) or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis previously received options or unmodified variant antibodies.

Glycosylation (including type glycosylation) antibodies can also be changed without carrying out modifications of the amino acid sequence or the coding of its nucleotide sequence. Glycosylation mainly depends on the host cell used for expression of the antibody. Because the cells used for expression of recombinant glycoproteins, such as antibodies, which potential is different therapies rarely are natural cells, one can expect significant changes in the type of glycosylation of antibodies. See, for example, Hse et al., (1997) J. Biol. Chem. 272:9062-9070. In addition to selecting host cells factors affecting glycosylation during recombinant production of antibodies, the character growth, the composition of the medium, the density of the culture, oksigenirovannym, pH, purification, etc. To change the type of glycosylation achieved in a particular organism, the host, different methods have been proposed, including the introduction or overexpression of some enzymes involved in the production of oligosaccharides (U.S. patent No. 5047335; 5510261; 5278299). Glycosylation (or glycosylation of a certain type) glycoprotein can be prevented, for example, using endoglycosidase H (endo-H). In addition, recombinant a host cell can be genetically engineered, for example it can be made defective processing of polysaccharides of some types. These and similar methods well known to specialists.

The structure of the glycosylation of antibodies can be easily analyzed by standard methods of carbohydrate analysis, including chromatography lectin, NMR, mass spectrometry, HPLC, GFH, analysis of the monosaccharide composition of sequential enzymatic hydrolysis and NRAES-PAD, which is used anyoneon the military chromatography with high pH for the separation of oligosaccharides on the basis of their charge. Methods of release of oligosaccharides used for analytical purposes are also known, and such methods include, but are not limited to, enzymatic treatment (usually performed using peptide-N-glycosidase F/endo-β-galactosidase), elimination, mainly O-linked structures in hard alkaline conditions and chemical methods of release of N - and O-linked oligosaccharides using anhydrous hydrazine.

A. Screening for the conjugates of the antibody-drug” (ADC)

Transgenic animals and cell lines are particularly suitable for screening of the conjugates of the antibody-drug” (ADC), which can be used for prophylactic or therapeutic treatment of diseases or disorders associated with overexpression of proteins, including the Lewis Y antigens, CD30, CD40 and CD70. Transgenic animals and cell lines are particularly suitable for screening of the conjugates of the antibody-drug” (ADC), which can be used for prophylactic or therapeutic treatment of diseases or disorders associated with overexpression of HER2 (US6632979). Screening for effective ADC may include the introduction of a transgenic animal of the ADC candidate at various intervals doses and analysis of efficiency(s) of the ADC in the treatment of rasf is travelago diseases and disorders in different periods of time. Alternative or additionally, the drug can be introduced before treatment or simultaneously with the processing of inducing the disease factor, if it applies. ADC-candidate can be siniawan Paladino and separately or simultaneously in the environment or in high-throughput format. The speed with which it can be siniawan ADC on the possibility of its effective use for prophylactic or therapeutic treatment of diseases or disorders, limited only by the rate of synthesis or screening method, including detection/measurement/data analysis.

In one embodiment of the invention, the screening method includes (a) transplantation of cells from a stable cell line kidney cancer animal, not a person, (b) the introduction of the ADC candidate to an animal, not a person, and (C) determining the ability of the specified candidate to inhibit the formation of tumors from the transplanted cell line.

Another variant of the invention is a screening method comprising (a) contacting cells, derived from stable cell lines Hodgkin's disease, with the ADC candidate and (b) the ability of such ADC candidate to block the activation of CD40 ligand.

Another variant of the invention is a screening method comprising (a) contacting cells occurring on the stable cell lines Hodgkin's disease, with the ADC candidate and (b) the ability of such ADC candidate to induce cell death. In one embodiment of the invention assessing the ability of the ADC candidate to induce apoptosis.

One variant of the invention is a screening method comprising (a) transplantation of cells from a stable cancer cell line animal, not a person, (b) the introduction of the ADC candidate to an animal, not a person, and (C) determining the ability of the specified candidate to inhibit the formation of tumors in transplanted cell lines. The present invention also relates to a method of screening ADC candidates for the possibility of their use for the treatment of a disease or disorder characterized by overexpression of HER2, where the method includes (a) contacting cells, derived from stable cell lines breast cancer, with a candidate drug; and (b) the ability of such ADC candidate to inhibit the growth of a stable cell line.

Another variant of the invention is a screening method comprising (a) contacting cells, derived from a stable cancer cell lines, with the ADC candidate and (b) the ability of such ADC candidate to block ligand activation of HER2. In one embodiment of the invention assessing the ability of the ADC-Candida is and to block the binding of heregulin. In another embodiment of the invention assessing the ability of the ADC candidate to block ligand-stimulated tyrosine phosphorylation.

Another variant of the invention is a screening method comprising (a) contacting cells, derived from a stable cancer cell lines, with the ADC candidate and (b) the ability of such ADC candidate to induce cell death. In one embodiment of the invention assessing the ability of the ADC candidate to induce apoptosis.

Another variant of the invention is a screening method comprising (a) introduction the ADC candidate for transgenic mammal that is not a man in whose breast cells observed overexpression of native human HER2 protein or its fragment, where the transgenic animal is stable integration into the genome nucleic acid sequence that encodes a native human HER2 protein or a fragment having biological activity of native human HER2, and operatively linked to sequences regulating transcription, directing its expression in the mammary gland and the tumor develops breast cancer, not susceptible or poorly susceptible to treatment with anti-HER2 antibody; or the introduction of this candidate a mammal which is not human is kOhm, which has a tumor developed from the transplanted cells taken from the indicated transgenic mammal that is not a person; and (b) assess the impact of the specified ADC candidate on a given disease or disorder. These diseases or disorders can be, but are not limited to, a cancer associated with overexpression of HER2, such as breast cancer, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. The specified cancer is preferably breast cancer in which HER2 is expressed in the amount of at least about 500000 copies per cell, and more preferably at least about 2000000 copies per cell. ADC candidates can be assessed on their ability to induce cell death and/or apoptosis analytical methods, well known in the art and described below.

In one embodiment of the invention the ADC candidate sceneroot by introducing a transgenic animal in different doses and assess the physiological response of the animal to the data connection depending on time. The introduction can be carried out orally or by a suitable injection, depending on the chemical nature of the evaluated compounds. In some SL is the teas may be desirable to introduce this compound in combination with cofactors, which should improve the efficiency of this connection. If for screening compounds that can be used to treat a variety of disorders, used cell lines originating from a given transgenic animals, the test compound is added to the medium for culturing cells through an appropriate period of time and assess the cellular response to the connection within a certain time interval using the appropriate biochemical and/or histological analysis. In some cases, it may be desirable to add interest compounds into the culture medium in combination with cofactors, which should improve the efficiency of the connection.

Thus, the present invention describes the analyses undertaken to identify the ADC that specifically targets the protein-target and associated with, the presence of this protein correlates with abnormal cellular function and in the pathogenesis of cell proliferation and/or differentiation of cells his presence causative associated with tumor development.

To identify the ADC, which blocks ligand activation of an ErbB receptor (e.g., ErbB2), may be assessed the ability of the compounds to block the binding of ErbB ligand with cells, the former is residuosity the ErbB receptor (ErbB2) (for example, when conjugation with another ErbB receptor, which represents the interest of the ErbB receptor forms heterooligomeric ErbB). For example, cells isolated from a transgenic animal, which sverkhekspressiya HER2, and transfected for the expression of other ErbB receptor (which HER2 forms heterooligomeric), can be incubated, that is cultivated with ADC and then processed labeled ErbB ligand. After that may be done to assess the ability of the compounds to block the binding of ligand to the receptor ErbB in heterooligomeric ErbB.

For example, inhibition of binding heregulin (HRG) with cell lines of breast cancer, sverkhekspressiya HER2 and evolved from non-human transgenic mammals (e.g. mice) under the action of the ADC candidate according to the invention can be implemented using monolayer cultures on ice in the format of 24-well plates. Monoclonal antibodies against ErbB2 can be added to each well and incubated for 30 minutes. Can then be added125I-labeled rHRGβ1177-224(25000 pulses/min), and the incubation may be continued for 4-16 hours. After this can be constructed curves dose-response and interest compounds can be computed the value of the IC50(cytotoxicities).

Alternative or additionally, may be assessed the ability of the ADC to block ligand-stimulated ErbB tyrosine phosphorylation of ErbB receptor, present in heterooligomeric ErbB. So, for example, cell lines developed from transgenic animals according to the invention can be incubated with the tested ADC, and then they can be analysed on the dependent ErbB ligand activity of tyrosine phosphorylation using monoclonal antibodies against phosphotyrosine (which is, but not necessarily, conjugated with a detectable label). To determine activation of the ErbB receptor and blocking the activation of this connection can also be applied to the analysis of the activation of kinase receptor, is described in U.S. patent No. 5766863.

In one embodiment of the invention can be conducted by screening for the ADC, which inhibits HRG-stimulation of the phosphorylation of tyrosine R in MCF7 cells, mainly, as described below. For example, a cell line produced from HER2-transgenic animal, can be cultured in 24-hole tablets, and each well can be added in connection with subsequent incubation for 30 minutes at room temperature, after which each well can be added rHRGβ1177-224to a final concentration of 0.2 nm, and the incubation may be the ü continued for about 8 minutes. Then from each hole sucked off Wednesday, after which the reaction can be terminated by adding 100 ál of the LTO-buffer for sample (5% LTOs, 25 mm DTT, and 25 mm Tris-HCl, pH 6,8). Each sample (25 μl) may be subjected to electrophoresis in 4-12% gradient gel (Novex)and then it can be electrophoretic transferred to polyvinylidenedifluoride membrane. After that, immunoblot antibodies against phosphotyrosine (at 1 μg/ml) can be taken and the intensity of the predominant reactive band at Mr~180000 can be quantified using reflection densitometry. An alternative method for assessing the inhibition of phosphorylation of the receptor is the analysis KIRA (to activate kinase receptor), described Sadick et al. (1998) Jour. of Pharm. and Biomed. Anal. Some of the well identified monoclonal antibodies against HER2, which are known to inhibit HRG-stimulation of the phosphorylation of tyrosine R, can be used in this assay as a positive control. Can then be constructed curve dose-response for inhibition of HRG-stimulation of the phosphorylation of tyrosine R, which can be estimated using reflection densitometry, and then can be calculated IC50for interest connection.

Can also be evaluated on the tumor-inhibitory effect in the ADC has been created on the cell line, derived from HER2-transgenic animal, for example, essentially as described by Schaefer et al. (1997) Oncogene 15:1385-1394. In accordance with this analysis, the cells can be treated with test compound at various concentrations for 4 days and stained crystal violet or dye Almoravid blue with redox activity. By incubating with this connection you can demonstrate the tumor-inhibitory effect on this cell line, similar to the action of monoclonal antibodies T cells MDA-MB-175 (Schaefer et al., see above). In another embodiment of the invention exogenous HRG does not significantly reverse this inhibition.

To identify tumor-inhibiting compounds, which are specifically aimed at representing the interest of the antigen can be carried out by screening for compounds that inhibit the growth of cancer cells, sverkhekspressiya interest antigen and derived from transgenic animals, and such screening analysis described in U.S. patent No. 5677171. In accordance with this analysis of cancer cells, sverkhekspressiya interest antigen, cultivated in a mixture of 1:1 F12 medium and DMEM, to which were added 10% fetal bovine serum, glutamine and penicillin-streptomycin. The cells are then seeded at a density ITC 35 mm Cup for cell culture (2 ml/35 mm Cup) and add a test compound in various concentrations. Six days count the number of cells compared to untreated cells using electronic counter cells COULTERTM. Compounds that inhibit cell growth by about 20-100%, or about 50-100%, can be selected as compounds inhibiting the growth of cells.

For selection of compounds that induce cell death can be assessed on the loss of membrane integrity, as indicated, for example, the uptake of PI, Trypanosoma blue or 7AAD compared with the control. In the analysis on the uptake of PI used cells isolated from the interest of the tumor tissue of the transgenic animal. In accordance with this analysis cells were cultured in a mixture of modified according to the method of Dulbecco eagle medium (D-MEM):environment Hams F-12 (50:50), to which were added 10% thermoinactivation FBS (Hyclone) and 2 mm L-glutamine. Thus, this analysis is carried out in the absence of complement and immune effector cells. The cells are then seeded in Cup size 100 × 20 mm at a density of 3 × 106the Cup and leave overnight to merge. After that, the medium removed and replaced with only one fresh medium or medium containing various concentrations of compounds. Cells are incubated for 3 days. After each treatment the monolayers washed with PBS and separated by trypsinization. After that lecitotrophic at 1200 rpm for 5 minutes at 4°C and the precipitate resuspended in 3 ml of cold Ca 2+binding buffer (10 mm Hepes, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and is divided into 35 mm aliquots, which are distributed in 12 × 75 ml test tubes with mesh filter (1 ml per tube, 3 tubes per group processing) to remove accumulations of cells. Then in a test tube add PI (10 μg/ml). Samples can be analyzed on a flow cytometer FACSCANTMand using computer programs FACSCONVERTTMCellQuest (Becton Dickinson). Compounds that induce statistically significant levels of cell death as determined by PI uptake may be selected as compounds that induce cell death.

For selection of compounds that induce apoptosis, carry out analysis on the binding of annexin using cells derived from interest neoplastic tissue of transgenic animals. The cells are then cultivated and sown in the Cup, as described in the previous paragraph. Then, the medium removed and replaced with only one fresh medium or medium containing 10 μg/ml conjugate the antibody-drug” (ADC). After incubation for 3 days, the monolayers are washed with PBS and separated by trypsinization. The cells are then centrifuged, resuspended in Ca2+binding buffer and divided into aliquots, as discussed above in the description of the analysis on cell death. Then in a test tube add the keys labeled annexin (for example, annexin V-FITZ) (1 μg/ml). Samples can be analyzed on a flow cytometer FACSCANTMand using computer programs FACSCONVERTTMCellQuest (Becton Dickinson). Compounds that induce statistically significant levels of binding to annexin, compared with the control, taken as compounds that induce apoptosis.

4.5.3. Analyses on cell proliferationin vitro

In General, the cytotoxic or cytostatic activity of the conjugate the antibody-drug” (ADC) measured by treatment of mammalian cells with a receptor protein, the antibody conjugate (ADC environment for culturing cells; culturing the cells for a period of time from about 6 hours to 5 days; and determining the viability of the cells. Suchin vitroanalyses using cells used to assess viability (proliferation), cytotoxicity and induction of apoptosis (activated caspase) cells under the action of the ADC according to the invention.

In vitro-the efficiency of the conjugates of the antibody-drug” is measured by analysis of cell proliferation (example 18, Fig.7-10). Analysis of cell viability based on luminescence (CellTiter-Glo®) is commercially available (Promega Corp., Madison, WI) and represents a homogeneous analysis conducted by recombinant EC is pressie luciferase Coleoptera(U.S. patent No. 5583024; 5674713 and 5700670). This analysis on the proliferation of cells makes it possible to determine the number of viable cells in culture based on a quantitative assessment present the APR, which is an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. patent No. 6602677). Analysis of the CellTiter-Glo® was held in the format of a 96-hole tablet that allows automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398-404). Homogeneous analytical procedure involves adding a single reagent (reagent CellTiter-Glo®) directly to cells cultured in medium with the addition of serum. This does not require the stages of washing the cells, remove the medium and the many stages of pipetting. This system allows the detection of at least 15 cells/well in 384-well the tablet after 10 minutes after adding the reagent and mixing. These cells can be continuously processed ADC, or they can be processed and separated from the ADC. In General, treatment of cells within a short period of time, i.e. within 3 hours, gives the same effect as continuous processing cells.

Homogeneous analysis carried out in the form of “add-mix-measure”, leads to the lysis of the cells and the generation of a fluorescent signal, proporti the national number is present APR. The amount of ATP is directly proportional to the number of cells present in the culture. In the analysis of the CellTiter-Glo® generated fluorescent signal type “glow”produced by the luciferase reaction, which has a half-life of more than five hours, depending on cell type and your environment. Viable cells determined in relative luminescence units (RLU). The substrate, luciferin beetles, undergoes oxidative decarboxylation under the action of recombinant luciferase fireflies with simultaneous conversion of ATP to AMR and the generation of photons. Long half-life of luciferase allows you to avoid having to use injectors to enter reagents and provides the possibility of continuous or batch processing of multiple plates. Such analysis on cell proliferation can be conducted in various advance tablets, for example in 96-well or 384-well pad. Data can be recorded using a luminometer or imaging camera based on a CCD. The output luminescence are presented in relative light units (RLU)measured depending on time.

Antiproliferative effects of conjugates of the antibody-drug” measure for cell proliferation is described in which use in vitrothe analysis on the cytolysis of cells of four different cell lines of breast tumors (Fig.7-10). For cells SK-BR-3 and BT-474, which, as you know, there is overexpression of its protein receptor HER2, were determined IC50. In table 2A presents performance measurement (IC50) representative conjugates of the antibody-drug” analysis on cell proliferation of SK-BR-3. In table 2b presents performance measurement (IC50) representative conjugates of the antibody-drug” analysis on the proliferation of cells BT-474.

The conjugates of the antibody-drug: trastuzumab-MC-vc-RAV-MMAF, 3.8 MMAF/Ab, trastuzumab-MC-(N-Me)vc-RAV-MMAF, 3.9 MMAF/Ab, trastuzumab-MC-MMAF, 4.1 MMAF/Ab; trastuzumab-MC-vc-RAV-MMAE, 4.1 MMAE/Ab; trastuzumab-MC-vc-RAV-MMAE, 3.3 MMAE/Ab; trastuzumab-MC-vc-RAV-MMAF, 3.7 MMAF/Ab inhibit the proliferation of MCF-7 cells (Fig.9).

The conjugates of the antibody-drug: trastuzumab-MC-vc-RAV-MMAE, 4.1 MMAE/Ab, trastuzumab-MC-vc-RAV-MMAE, 3.3 MMAE/Ab; trastuzumab-MC-vc-RAV-MMAF, 3.7 MMAF/Ab; trastuzumab-MC-vc-RAV-MMAF, 3.8 MMAF/Ab; trastuzumab-MC-(N-Me)vc-RAV-MMAF, 3.9 MMAF/Ab; trastuzumab-MC-MMAF, 4.1 MMAF/Ab inhibit cell proliferation of MDA-MB-468 (figure 10).

Cells MCF-7 and MDA-MB-468 not sverkhekspressiya protein receptor HER2. It follows that the conjugates of anti-HER2 antibody-drug” according to the invention have sat what aktivnosti in relation to inhibition of the cells, which Express HER2.

Table 2A

Cells SK-BR-3

Table 2b

Cells BT474

H=trastuzumab.

S=mouse antibody against HER2, which binds to the epitope different from the epitope is associated with trastuzumab.

Fc8=mutant that does not bind to FcRn.

Hg=“does Not contain a hinge region” full gumanitarnoe antibody 4D5, in which cysteine the hinge region of the heavy chain is replaced by Suriname. This antibody was expressed inE. coli(and so it is deglycosylation).

Anti-TF Fab=fragment antibody against tissue factor.

* activity against cells MDA-MB-468.

Amazing and unexpected were the results obtained for the ADC activity against cell proliferationin vitroand are presented in tables 2A and 2b, which in General showed that the ADC with low average content of drug molecules in relation to the antibody had a significant efficiency, such as IC50<0.1 ág ADC/ml These results suggest that at least for the ADC with trastuzumab, the optimal ratio of molecules of the drug to antibody molecules may be less than 8 or even ome 2-5.

4.5.4.In vivoexcretion ADC from plasma and its stability

Pharmacokinetics elimination ADC from plasma and its stability was investigated in rats and abacadabra monkeys. Concentration in plasma was determined over a certain period of time. In table 2C presents pharmacokinetic data for conjugates of the antibody-drug” and other doses samples in rats. Rats are nonspecific models for generating antibodies against ErbB receptor, as yet unknown, are expressed whether they have protein receptor HER2.

Table 2C

Pharmacokinetics in rats

H=trastuzumab linked through cysteine [Cys], if it is not specifically mentioned; dose 2 mg/kg, unless specifically

AUCinf represents the area under the curve plasma concentration from time dose to infinity and is a measure of the overall impact of the measured molecules (drugs, ADC). CL is defined as the volume of plasma cleared of the measured molecules per unit time and is expressed as a value normalized to body weight. T1/2period means the half-life of drugs in the body, measured in the AZE its elimination. % conjugate indicates the relative number of ADC compared with the total number of detected antibodies in the individual tests on immunoaffinity using ELISA (“Analytical Methods for Biotechnology Products”, Ferraiolo et al, p.85-98 in Pharmacokinetics of Drugs (1994) P.G. Welling & L.P. Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110, Springer-Verlag). % conjugate is calculated by the formula: AUCinf ADC : AUCinf General Ab, and this % is a General indicator of the stability of the linker, although, in fact, such indicators may be other factors and mechanisms.

Figure 11 shows a graph of the removal of the investigated concentrations of plasma after administration to rats Sprague-Dawley conjugates of the antibody-drug”: H-MC-vc-RAV-MMAF-TEG and H-MC-vc-RAV-MMAF. Concentrations of total antibody and ADC was measured in dependence on time.

On Fig shows a graph of the two-stage removal of the investigated concentration of the plasma, where the ADC was administered at different doses and concentrations of total antibodies and measured depending on time.

The effectiveness ofin vivo

In vivothe efficiency of the ADC according to the invention was measured in the mouse model with transgenic Explant expressing high levels of HER2. Allograft cultivated transgenic, MMTV-infected mice Fo5 that are unresponsive or poorly susceptible to treatment with Herceptin®. Mice once processed ADC and held Moni the o-ring within 3-6 weeks to determine the period of time, passing to the increase of tumor volume in twice the log of the number of destroyed cells; and the degree of reduction of tumor volume. Then build a graph of the dose-response” and conducted experiments using a variety of doses.

Tumor developed in transgenic mice that expressed activated by mutation of the formneu, the rat homolog of HER2, however, HER2, which sverkhekspressiya in cancer of the breast, was not mutated, and therefore the formation of tumors proceeded much less intensively in transgenic mice, which was observed overexpression of neutropenia HER2 (Webster et al., (1994) Semin. Cancer Biol. 5:69-76).

To accelerate the formation of tumors with nemotional HER2 received transgenic mice using plasmid cDNA encoding HER2, in which the upstream ATG was deleterous to prevent initiation of translation in such upstream ATG codons, which should reduce the frequency of translation initiation from the downstream authentic initiation codon HER2 (see, for example, Child et al. (1999) J. Biol. Chem. 274:24335-24341). In addition, the 5'-end was added to the chimeric intron, which should also increase the level of expression, as reported previously (Neuberger & Williams (1998) Nucleic Acids Res. 16:6713; Buchman & Berg (1988) Mol. Cell. Biol. 8:4395; Brinster et al. (1988) Proc. Natl. Acad. Sci., USA, 85:836). This chimeric intron happens is the vector Promega, vector pCI-neoused for expression in mammals (890-1022 P.N.). 3'-end cDNA flanked by exons 4 and 5 of human growth hormone and polyadenylation sequences. In addition, were used FVB mouse because this strain is more susceptible to tumor development. To ensure tissue-specific expression of HER2 in the breast was used promoter, derived from the MMTV-LTR. To increase susceptibility to the formation of tumors, the animals were given food AIN 76A (Rao et al. (1997) Breast Cancer Res. and Treatment 45:149-158).

Table 2d

Measurement of tumor in Nude mice model of allograft tumors of the breast MMTV-HER2 Fo5;

one dose on day 1 (T=0), if it is not mentioned explicitly;

H=trastuzumab linked through cysteine [Cys], if it is not specifically mentioned.

S=mouse antibody against HER2, which binds to the epitope different from the epitope is associated with trastuzumab.

Fc8=mutant that does not bind to FcRn.

Hg=“does Not contain a hinge region” full gumanitarnoe antibody 4D5, in which cysteine the hinge region of the heavy chain is replaced by Suriname. This antibody was expressed inE. coli(and so it is deglycosylation).

N=ant the-EphB2R antibody

11D10=anti-R antibody.

The term “Ti” means the number of animals with tumors in the study group at T=0 : the total number of animals in the group. The term HR means the number of animals who have achieved partial remission of the tumor : the number of animals with tumors in the group at T=0. The term CR represents the number of animals who have achieved complete remission of the tumor : the number of animals with tumors in the group at T=0. The term log of the number of destroyed cells mean time in days for which the volume of the tumor increases in two times : the time, in days, for which the volume of the tumor increases in two times for the control group, divided by 3.32 × time during which the volume of the tumor increases in two times that of the control animals (which was introduced dose of media). The value of the log of the number of destroyed cells is calculated taking into account the time slowing tumor growth in processing time and increasing the volume of the tumor doubled in the control group. Antitumor activity of ADC classified by the values of the log of the number of destroyed cells, namely:

++++≥3,4 (high activity)
+++= 2.5 and 3.4
++= 1,7-2,4
+ = 1,0-1,6

No activity=0

On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER1 Fo5 that on day 0 was introduced: the media, trastuzumab-MC-vc-RAV-MAE (1250 mg/m2) and trastuzumab-MC-vc-RAV-MAAF (555 mg/m2) (H=trastuzumab). Tumor growth was slowed after processing ADC compared with the level of growth in the control group (medium). On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5 that on day 0 were injected: 10 mg/kg (660 g/m2) conjugate trastuzumab-MC-MMAE and 1250 mg/m2conjugate trastuzumab-MC-vc-RAV-MMAE. On Fig illustrates the change in average tumor volume depending on the time in Nude “Nude” mice with allografts of breast tumors MMTV-HER2 Fo5, which was introduced 650 g/m2trastuzumab-MC-MMAF. In table 2d and Fig-15 shows that the ADC has a strong antitumor activity in allograft HER2-positive tumors (Fo5), which was originally developed in MMTV-HER2-transgenic mice. In this model, a single antibody (e.g., trastuzumab) did not possess significant antitumor activity (Ericksn et al., U.S. patent No. 6632979). As illustrated in Fig-15, after processing the ADC tumor growth was slowed compared with the level of growth in the control group (medium).

Amazing and unexpected were the results obtained for the antitumorin vivoactivity of the ADC illustrated in table 2d, which in General showed that the ADC with low average content of drug molecules in relation to the antibody had a noticeable efficiency, for example, after processing the ADC, the ramp time of tumor volume doubling accounted for >15 days, and the average value of the log of the number of destroyed cells is >1.0 in. On Fig illustrated that in order to conjugate the antibody-drug”, that is, trastuzumab-MC-vc-RAV-MMAF, the average tumor volume was decreased and not increased when the ratio MMAF:trastuzumab was 2 and 4, whereas when the ratio of 5.9 and 6, the tumor progressed, but the speed of its progression was lower than the group that was injected media (buffer). The rate of progression of tumors in these mice with xenograft model in the group, which has introduced the media, and in the group which was administered trastuzumab, was about the same, there was 3 days. These results suggest that, at least for the ADC with trastuzumab, the optimal ratio of drug molecules is the means to the antibody should be less than 8 and can range from about 2 to 4.

4.5.5. Toxicity in rodents

The conjugates of the antibody-drug” and ADC negative control (“media”) was evaluated for acute toxicity to the rat model. Toxicity ADC was assessed by treatment of male and female rats, Sprague-Dawley, which was introduced by the ADC, and subsequent research and analysis of its effect on various organs. Empirical observations included an assessment of changes in body weight and signs of infection and bleeding. Evaluation of clinical parameters pathology (chemical and hematological analysis of serum), histopathological analysis and autopsy was performed on animals after administration of the dose.

It is believed that weight loss or weight change of the animals, which were injected ADC, compared to animals that were injected only media is approximate and General indicator of systemic or local toxicity. On Fig-19 illustrates the effect of different ADC and control (media) on body weight of rats.

Hepatotoxicity was measured by increased levels of enzymes in the liver, the increase in the number of mitotic and apoptotic characteristics and degree of necrosis of hepatocytes. Presence of blasts toxicity was observed upon depletion of leukocytes, primary granulocytes (neutrophils and/or platelets and defeat lymphoid organs, i.e. atrophy or apoptotic activity. On the sicnosti can also specify lesions of the gastrointestinal tract, such as increasing the number of mitotic and apoptotic signs and degenerative enterocolitis.

The enzymes are indicators of the studied lesions of the liver are:

AST (aspartate aminotransferase):

- localization: cytoplasm, liver, heart, skeletal muscle, kidney;

- the ratio of the liver:plasma=7000:1;

- T1/2: 17 hours;

ALT (alanine aminotransferase):

-localization: cytoplasm, liver, kidney, heart, skeletal muscle;

- the ratio of the liver:plasma=3000:1;

- T1/2: 42 hours; diurnal variation;

GGT (gamma-glutamyltransferase):

- localization: plasma membrane of cells with high secretory or absorptive capacity; liver, kidney, small intestine;

- poor prognosis of the liver; the contents of this enzyme usually rises in diseases of the bile ducts.

The toxicity profiles of the conjugates of trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-RAV-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-RAV-MMAF was investigated in female rats, Sprague-Dawley (example 19). Gumanitarnoe antibody trastuzumab did not show appreciable binding to rat tissue, and any of its toxicity should be considered as non-specific. Options MMAF at doses of 840 and 2105 g/m2were comparable to conjugate trastuzumab-MC-val-cit-RAV-MMAF with the dose 2105 g/m2.

Animals in groups 1, 2, 3, 4, 6 and 7 (wear the e l e C 9,94 and of 24.90 mg/kg trastuzumab-MC-val-cit-MMAF, 10,69 mg/kg trastuzumab-MC(Me)-val-cit-RAV-MMAF and 10,17 and 25.50 mg/kg trastuzumab-MC-MMAF, respectively), there was an increase of body weight during the study. Animals in groups 5 and 8 (26,78 mg/kg trastuzumab-MC(Me)-val-cit-PAB-MMAF and to 21.15 mg/kg trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight during the study. On day 5 of the study the change of body weight of the animals in groups 2, 6 and 7 was not significantly different from the changes in the body weight of animals of group 1. Change of body weight of the animals in groups 3, 4, 5, and 8 are statistically distinguishable from changes in body weight of animals in group 1 (example 19).

Rats treated with conjugate trastuzumab-MC-MMAF (groups 6 and 7), at both doses did not differ from control animals treated with media, that is, in this model, this conjugate was found the most high profile security. Rats treated with conjugate trastuzumab-MC-val-cit-MMAF” (without carolinerileyt RAV group; groups 2 and 3), was found to dose-dependent changes typical of MMAF-conjugates; the degree of change was compared with the effect of full-conjugate “MC-val-cit-RAV-MMAF” (group 8). The number of platelets on day 5 was approximately 30% from baseline values for animals of group 3 (high dose “trastuzumab-MC-val-cit-MMAF) in contrast to animals, group 8, Ko is that the number of platelets was 15% (high dose “trastuzumab-MC-val-cit-RAV-MMAF”). Dose-dependent increase in the level of enzymes AST and ALT or bilirubin in the liver and the degree of thrombocytopenia were the most noticeable in animals treated with conjugate trastuzumab-MC(Me)-val-cit-RAV-MMAF” (groups 4 and 5), and in animals of group 5 (group, which was administered high-dose)on day 5, the ALT levels approximately 10 times higher than baseline values, and the number of platelets at the time of autopsy were reduced by approximately 90%.

The female rats of Sprague-Dawley was also introduced high-dose (example 19, the experiment with high doses: group 2, 3, 4) conjugate “trastuzumab-MC-MMAF and media (control group 1). In this case, the observed weak signals of toxicity, including dose-dependent increase in the levels of the enzymes ALT, AST and GGT in the liver. On the 5th day of the animals in the group that was administered the highest dose, was observed 2-fold increase in ALT levels and 5-fold increase in AST level; it also increased levels of GGT (6 units/l). On the 12th day of enzymes tended to normalize. On the 5th day the groups for all three doses was observed weak granulocytes, but in all animals the number of platelets remained almost unchanged. Morphological changes were weak, i.e. histological analysis of liver, spleen, thymus, small intestine and bone marrow of animals treated with a dose 4210 mg/m2(group 2)did not reveal significant changes. After the CA increased apoptotic and mitotic activity was observed in the thymus and liver, respectively, in animals, treated with dose of 5500 mg/m2(group 3). The bone marrow was normal cells, but was found granulocytic hyperplasia, which corresponded to an absolute granulocytes observed when calculating formula of blood cells in these animals. In animals of group 4, which was administered the highest dose, was observed the same qualitative characteristics, however, compared with the animals of group 3, mitotic activity in the liver was slightly increased. In addition, in the spleen and in the liver of these animals were observed extramedullary haematopoiesis.

EphB2R is tyrosinekinase receptor TM type 1, and mouse and human receptors EphB2R have a high degree of homology, and this receptor sverkhekspressiya in colon cancer cells. N is an antibody against EphB2R. Taken separately, the antibody does not have any impact on tumor growth, whereas conjugate “N-val-cit-MMAE” destroys EphB2R-expressing cells and detects efficacy in mice with xenograft model of human tumor colon CXF1103 (Mao et al. (2004) Cancer Res. 64:781-788). N and S are mouse IgG1 antibodies against HER2. Conjugates N-MC-val-cit-PAB-MMAF (3.7 MMAF/Ab), S-MC-val-cit-RAV-MMAF (4 MMAF/Ab) and trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) have comparable profiles of toxicity. Differences in the structures of each immunoconjugates medicinal component such immunoconjugate may affect the pharmacokinetics and ultimately to the security profile. Gumanitarnoe antibody trastuzumab does not detect any measurable binding to rat tissue, and therefore any of its toxicity should be considered as non-specific.

The toxicity level/degree of security, studied at abacadabra monkeys

Similarly, the study of the toxicity level of/degree of safety in rats were investigated on abacadabra monkeys, which included treatment of animals ADC with subsequent measurement of the level of enzymes in the liver, as well as evaluation and analysis of their impact on different organs. General observations included an assessment of changes in body weight and signs of infection and bleeding. Evaluation of clinical parameters pathology (chemical and hematological analysis of serum), histopathological analysis and autopsy was performed on animals after administration of the dose (example 19).

Conjugate the antibody-drug”, H-MC-vc-RAV-MMAE (H=trastuzumab, attached via a cysteine) did not find evidence of any toxicity in the liver, with any of the tested doses. After a single dose of 1100 mg/m2observed depletion of granulocytes in the peripheral blood, and 14 days after administration of this dose was observed to their full recovery. Conjugate the antibody-drug”, H-MC-vc-RAV-MAE, was found to increase in level, the second enzyme in the liver at doses of 550 (short-term) and 880 mg/m 2while it was not observed any signs granulotsitopenii and dose-dependent short-term (groups 2 and 3) reduction in the number of platelets.

4.6. The synthesis of compounds according to the invention

Representative compounds and conjugates can be obtained by the procedure of synthesis described below in schemes 5-16. As will be described in more detail below, representative compounds or conjugates can be easily obtained using a linker having a reactive binding site with drug and ligand. In one aspect of the invention, the linker has a reactive site which contains an electrophilic group that interacts with the nucleophilic group present on the ligand, such as, but not limited to, antibody. Suitable nucleophilic groups on the antibody include, but are not limited to, sulfhydryl, hydroxyl, and amino groups. Heteroatom nucleophilic group of an antibody interacts with an electrophilic group on the linker and forms a covalent bond with the linker component. Suitable electrophilic groups include, but are not limited to, maleimide and halogenated group. Specified electrophilic group provides a suitable site for binding with the antibody.

In another embodiment of the invention, the linker has a reactive sa is t, which contains a nucleophilic group that interacts with an electrophilic group present on the antibody. Suitable electrophilic groups on the antibody include, but are not limited to, the carbonyl group of aldehyde and ketone. Nucleophilic heteroatom linker group can interact with an electrophilic group on the antibody and forms a covalent bond with the antibody. Suitable nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and originated. Specified electrophilic group on the antibody provides a suitable site for binding to the linker.

The functional group of carboxylic acid functional groups chloroformiate are also suitable reactive sites for bonding with the linker, because they can interact with the secondary amine groups of the medicinal product and to form an amide bond. As a reactive site can also be used carbonate functional group on the linker, such as, but not limited to it, p-nitrophenylarsonic, which can interact with the amino group of the medicinal product, such as, but not limited to it, N-methylvaline, and to form a urethane linkage. Typically, peptide drug substances is and can be obtained through the formation of the peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, by the method of synthesis in solution (see E. Schröder & K. Lübke, “The Peptides”, volume 1, p.76-136, 1965, Academic Press)that is well-known method used in the field of peptide synthesis.

A representative synthesis of the extension component having electrophilic maleimide group is illustrated below in schemes 8-9. General methods of synthesis used for the synthesis of the linker described in scheme 10. Figure 11 shows the design of the linker component having a group of Val-Cit, electrophilic maleimide group and somalimerirosvot spacer elements RAV group. Figure 12 illustrates the synthesis of the linker with the group Phe-Lys, electrophilic maleimide group carolinerileyt spacer elements RAV group without it. Figure 13 illustrates the General method for the synthesis of compounds of “a drug-linker”, as in figure 14 presents an alternative method for obtaining compounds of “drug-linker”. In figure 15 presents the synthesis of the branched linker containing a group BHMS. Scheme 16 illustrates the method of attachment of antibodies to the connection “drug-linker” with the formation of conjugate “drug-linker-antibody”, as in figure 14 illustrates the synthesis of conjugates “drug-linker-antibody”, with, for example the EP, but not limited to, 2 or 4 molecules of the drug to the antibody.

As will be described in more detail below, a representative conjugates are usually obtained by using a linker having two or more reactive site for binding with the drug and ligand. In one aspect of the invention, the linker has a reactive site which contains an electrophilic group that interacts with the nucleophilic group present on the ligand, such as an antibody. Suitable nucleophilic groups on the antibody include, but are not limited to, sulfhydryl, hydroxyl and aminogroup. Heteroatom nucleophilic group of an antibody interacts with an electrophilic group on the linker and forms a covalent bond with the linker component. Suitable electrophilic groups include, but are not limited to, maleimide and halogenated group. Specified electrophilic group provides a suitable site for binding with the antibody.

In another embodiment of the invention, the linker has a reactive site which contains a nucleophilic group that interacts with an electrophilic group present on the ligand, such as an antibody. Suitable electrophilic groups on the antibody include, but are not limited to, the carbonyl group of aldehyde and ketone. Heteroatom well leofiles group of the linker may interact with an electrophilic group on the antibody and forms a covalent bond with the antibody. Suitable nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arithematic. Specified electrophilic group on the antibody provides a suitable site for binding to the linker.

4.6.1 Synthesis of the medicinal product

Typically, peptide drugs can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, by the method of synthesis in solution (see E. Schröder & K. Lübke, “The Peptides”, volume 1, p.76-136, 1965, Academic Press)that is well-known method used in the field of peptide synthesis.

Drug auristatin/dolastatin can be obtained by the General methods described in U.S. patent No. 5635483, in 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 and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863.

In one of the embodiments of the invention, the drug is produced by combining approximately stoichiometric equivalent of the dipeptide and Tripeptide, and preferably by conducting the reaction in the same vessel in suitable conditions of condensation. This method is illustrated below in schemes 5-7.

Scheme 5 illustrates the synthesis of N-to Navoi Tripeptide molecules F, which is appropriate intermediate compound for the synthesis of medicinal compounds of formula 1b.

Scheme 5

As illustrated in scheme 5, the protected amino acid And (where PG is an amine protective group, R4selected from hydrogen, C1-C8of alkyl, C3-C8carbocycle, -O-(C1-C8of alkyl, aryl, alkylaryl, alkyl-(C3-C8carbocycle)3-C8heterocycle, alkyl(C3-C8heterocycle), where R5selected from H and methyl; or R4and R5taken together with the carbon atom to which they are linked, form a ring having the formula -(CRaRb)n-where Raand Rbindependently selected from hydrogen, C1-C8the alkyl and C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6) is subjected to reaction combination with tert-butyl ester (where R6selected from-H and-C1-C8of alkyl, and R7selected from hydrogen, C1-C8of alkyl, C3-C8carbocycle, -O-(C1-C8of alkyl, aryl, alkylaryl, alkyl-(C3-C8carbocycle)3-C8heterocycle and alkyl-(C3-C8heterocycle)) in a suitable reaction conditions, the combination of, for example, in the presence of PyBrop and diisopropylethylamine or using DCC (see, for example, Miyazki K. et al., Chem. Pharm. Bull. 1995, 43(10), 1706-1718).

Suitable protective group PG and suitable methods of synthesis for the protection of the amino protecting group well known in the art. See, for example, T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Representative protected amino acids And are PG-Ile, and in particular PG-Val, and other appropriate protected amino acids include, but are not limited to, the PG-cyclohexylglycine, PG-cyclohexylamin, PG-aminocyclopropane-1-carboxylic acid, PG-aminoadamantane acid, PG-phenylalanine, PG-phenylglycine and PG-tert-butylglycol. Z is a representative of the protective group. Fmoc is another representative of the protective group. Representative tert-butyl ether is tert-butyl ether daisosasen.

The dipeptide can be purified, for example, using chromatography, and then it can be subjected to reaction unprotect, for example, using H2and 10% Pd-C in ethanol, if PG is benzyloxycarbonyl, or using diethylamine for removal of the protective Fmoc group. Received Amin D readily forms a peptide bond with the amino acid CENTURIES (where R1selected from-H, -C1-C8the alkyl and-C3-C8carbocycle, R2selected from-H and-C1-C8of alkyl; or R1and R2taken together with the atom is m nitrogen, with which they are linked, form a ring of formula -(CRaRb)nwhere Raand Rbindependently selected from-H, -C1-C8the alkyl and-C3-C8carbocycle, and n is selected from 2, 3, 4, 5 and 6, and R3selected from hydrogen, C1-C8of alkyl, C3-C8carbocycle, -O-(C1-C8of alkyl, aryl, alkylaryl, alkyl-(C3-C8carbocycle)3-C8heterocycle and alkyl-(C3-C8heterocycle)). Representative N,N-dialkylaminoalkyl are amino acids CENTURIES, such as the commercially available N,N-dimethylamine. Other N,N-dialkylaminoalkyl can be obtained by reductive bis-alkylation in accordance with standard procedures (see, for example, R.E. Bowman Stroud H.H. J. Chem. Soc. 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two representative amino acids CENTURIES, which can be used for the synthesis of N-monoalkyl derivatives. Amin D and amino acid CENTURIES subjected to interaction in the presence of the reagent combination of DEPC and triethylamine as a base and get a Tripeptide that is Then C-terminal protective group is removed using HCl, resulting in a gain Tripeptide compound of formula F.

An illustrative method of conducting the reaction mix using DEPC and reaction combinations using PyBrop presented on CX is IU 5, described in General procedure a and General procedure V, respectively. Illustrative methods of removing protection from Z-protected amine by catalytic hydrogenation described in General procedure C.

General procedure A: Peptide synthesis using DEPC. N-protected or N,N-disubstituted amino acid or peptide D (1.0 EQ.) and Amin BB (1.1 EQ.) diluted with an aprotic organic solvent, such as dichloromethane (0.1-0.5 M). Then add organic base such as triethylamine or diisopropylethylamine (1.5 equiv.) then add DEPC (1.1 EQ.). The resulting solution is stirred, preferably in an argon atmosphere over a period of time up to 12 hours and monitor using HPLC or TLC. The solvent is removed in vacuum at room temperature and the crude product was then purified using, for example, HPLC or column flash chromatography (on a column of silica gel). The appropriate fractions are combined and concentrated in vacuo to obtain Tripeptide E, which is dried in vacuum over night.

General procedure Q: Peptide synthesis using PyBrop. Amino acid (1.0 EQ.), not necessarily having a carboxyl protective group, is diluted with an aprotic organic solvent, such as dichloromethane or DME, and get a solution with a concentration from 0.5 to 1.0 mm, after h is th add diisopropylethylamine (1.5 EQ.) Then one portion add Fmoc - or Z-protected amino acid (1.1 EQ.) in the form of solids, then to the resulting mixture addPyBrop(1.2 EQ.). Reaction monitoring performed by TLC or HPLC, followed by processing similar to the processing described in General procedure A.

General procedure: remove the Z protective group by catalytic hydrogenation. Z-protected amino acid or Z-protected peptide With dilute ethanol and receive a solution with a concentration of 0.5 to 1.0 mm in a suitable vessel, such as a round-bottom flask with thick walls. Then add 10%palladium on coal (5-10% wt./wt.) and the reaction mixture is placed in an atmosphere of hydrogen. The reaction monitoring is carried out using HPLC, and the reaction usually completes in 1-2 hours, the Reaction mixture was filtered through pre-washed layer celite, and then, after filtration, celite again washed with a polar organic solvent, such as methanol. The eluent solution was concentrated in vacuo to obtain a residue which is diluted with an organic solvent, preferably toluene. Then the organic solvent is removed in vacuum and receive Amin With no protective group.

Figure 6 illustrates the method used to obtain the C-terminal dipeptide of the formula For, and method used in the reaction of a combination of the dipeptide of formula K with the Tripeptide of formula F with obtaining compounds of medicinal product fo the mules 1b.

Scheme 6

Dipeptide It can be easily obtained by condensation of the modified amino acid BOC-delaplaine G (see, for example, Pettit, G.R., et al., Synthesis, 1996, 719-725) with an amine of formula H using condensing agents, well known to experts in the field of peptide chemistry, such as, for example,DEPCin the presence of triethylamine, as shown in figure 5.

Dipeptide of formula K can then be subjected to reaction combination with the Tripeptide of formula F according to General procedure D with obtaining Fmoc-protected compounds of the medicinal product formula L, which according to General procedure E can then be removed protective group, the result can be obtained compound medicines of the formula (Ib).

General procedure D: Synthesis of the medicinal product. A mixture of dipeptide (1.0 EQ.) and Tripeptide F (1 EQ.) diluted with an aprotic organic solvent, such as dichloromethane, to obtain 0,1M solution, and then add a strong acid, such as triperoxonane acid (1/2,./vol.), and the resulting mixture was stirred in nitrogen atmosphere for two hours at 0°C. the reaction Monitoring can be performed using TLC or preferably HPLC. The solvent is removed in vacuo and the resulting residue is dried by double azeotropic distillation before occhialino using toluene. The resulting residue is dried under high vacuum for 12 hours and then diluted with an aprotic organic solvent, such as dichloromethane. After that add an organic base such as triethylamine or diisopropylethylamine (1.5 equiv.) and then, depending on the chemical functional groups of the residue, add or PyBrop (1.2 equiv.) either DEPC (1.2 EQ.). Reaction monitoring performed by TLC or HPLC and after completion of the reaction, the reaction mixture is subjected to processing similar to or identical to the processing described in General procedure A.

General procedure E: Removing Fmoc using diethylamine. Fmoc-protected drug L is diluted with an aprotic organic solvent, such as dichloromethane, and to the resulting solution add diethylamine (1/2,./vol.). Monitoring completion of the reaction performed by TLC or HPLC, and the reaction usually completes in 2 hours. The reaction mixture was concentrated in vacuo and the resulting residue dried by azeotropic distillation preferably using toluene and then dried under high vacuum to obtain medicines 1b having the amino group is removed from protection.

Figure 7 illustrates the method used to obtain MMAF-derivatives of the formula (Ib).

Scheme 7

Dipeptide About can be easily obtained by condensation of the modified amino acid BOC-delaplaine G (see, for example, Pettit, G.R., et al., Synthesis, 1996, 719-725) with a protected amino acid of formula M using condensing agents, well known to experts in the field of peptide chemistry, such as, for example,DEPCin the presence of triethylamine, as shown in schemes 5 and 6.

Dipeptide of the formula O can then be subjected to reaction combination with the Tripeptide of formula F according to General procedure D with obtaining Fmoc-protected MMAF-compounds of the formula R, which according to General procedure E can then be removed protective group, the result can be obtained MMAF-link medicines of the formula (Ib).

For example, the above methods are suitable for obtaining medicines that can be used in the present invention.

4.6.2. Synthesis of conjugate “drug-linker”

For connection “drug-linker” according to the invention, the drug is subjected to interaction with a reactive site on the linker. In General, the linker may have the structure:

The reactive site 2 - Aa-Ww-Yy-The reactive site 1

where there are spacer elements component (Y) and extending component (-And-). Alternatively, the linker may have the structure:

The reactive site 2- Aa-Ww-The reactive site 1

where there is no spacer elements component (-Y-).

The linker may also have the structure:

The reactive site 2-Ww-The reactive site 1

where no extension component (-A-) and spacer elements component (-Y-).

The linker may also have the structure:

The reactive site 2- Aa-The reactive site 1

where there is no amino acid component (W) and spacer elements component (Y).

In General terms, a suitable linker has the amino acid component is attached to an optional extension component and an optional component spacer elements. Jet is AIT 1 is present at the end of the spacer, and the reactive site 2 is present at the end of the extension component. If the spacer elements component is absent, the reactive site 1 is present at the C-end amino acid feature.

In a representative embodiment of the present invention, reactive site No. 1 is subjected to reaction with the nitrogen atom of the medicinal product, and the reactive site No. 2 is subjected to reaction with a sulfhydryl group on the ligand. Reactive sites 1 and 2 can react with various functional groups.

In one aspect of the invention, reactive site No. 1 is a.

In another aspect of the invention, reactive site No. 1 is:

In another aspect of the invention, reactive site No. 1 is a p-nitrophenylarsonic having the formula:

In one aspect of the invention, reactive site No. 2 is a thiol-accepteras group. Suitable thiol-aktsepteerimine groups are halogenated group having the formula:

where X represents a leaving group, preferably O-mesyl, O-tosyl, -Cl, -Br or-I; or maleimido group having the formula:

Used linkers can be obtained from commercially available sources is of IKI, such as Molecular Biosciences Inc. (Boulder, CO), or they can be obtained as described below in schemes 8-10.

Scheme 8

where X represents-CH2- or-CH2Och2-; and n is an integer from 0 to 10, if X represents-CH2-; or from 1 to 10, if X represents-CH2Och2-.

In the method illustrated in scheme 9, maleimide subjected to interaction with the glycol in the reaction conditions, Mitsunobu, resulting in lengthening get the glycol-maleimide” (see, for example, Walker M.A. J. Org. Chem. 1995, 60, 5352-5), and then in the reactive site enter s-nitrophenylarsonic group.

Scheme 9

where E represents-CH2- or-CH2Och2-; and e is an integer from 0 to 8.

Alternatively, PEG-maleimide and PEG-halogenated extension components can be obtained as described Frisch et al. Bioconjugate Chem. 1996, 7, 180-186. Figure 10 illustrates the General synthesis of a representative of the linker component containing maleimido extension group and, optionally, carolinensis the spacer, representing p-aminobenzoyl ether.

Scheme 10

where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-is iano; m is an integer from 0 to 4, and n is an integer from 0 to 10.

Used extension components can be introduced into the linker using commercially available intermediates supplied Molecular Biosciences (Boulder, CO), as described below in accordance with known methods of organic synthesis. Extending components of the formula (IIIa) can be introduced into a linker by reacting the following intermediates with the N-end amino acid component, as shown in schemes 11 and 12.

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

where n is an integer from 0 to 3;

and

.

Extending components of the formula (IIIb) can be introduced into a linker by reacting the following intermediates with the N-end amino acid component:

where X represents Br or I; and

Extending components of the formula (IV) can be introduced into a linker by reacting the following intermediates with the N-end amino acid component:

Extending components of the formula (Va) can be entered in the link is R by reacting the following intermediates with the N-end amino acid component:

Other extension components can be synthesized by known methods. Lengthening aminooxy components the following formula can be obtained by processing alkylhalogenide N-BOC-hydroxylamine in accordance with the procedures described D.S. Jones et al. Tetrahedron Letters, 2000, 41(10), 1531-1533 and C. Gilon et al., Tetrahedron, 1967, 23(11), 4441-4447.

where R17selected from-C1-C10alkylene, -C3-C8carbocycle, -O-(C1-C8of alkyl, arylene, -C1-C10alkylenediamine, Allen-C1-C10alkylene, -C1-C10alkylene-(C3-C8carbocycle)-, -(C3-C8carbocycle)-C1-C10alkylene, -C3-C8heterocycle, -C1-C10alkylene-(C3-C8heterocycle)-, -(C3-C8heterocycle)-C1-C10alkylene, -(CH2CH2O)r-, -(CH2CH2O)r-CH2-; and r is an integer from 1 to 10.

Isothiocyanate extension components the following formula can be obtained from acid chlorides isothiocyanatobenzene acid, as described in Angew. Chem. 1975, 87(14):517.

where R17is such as defined in the description.

Figure 11 illustrates a method of obtaining the dip is pignolo linker val-cit, having maleimide extension component and, optionally, carolinensis the spacer, representing p-aminobenzyl.

Scheme 11

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

Figure 12 illustrates the synthesis of Phe-Lys(Mtr)-dipeptide linker component having maleimide extension component and p-aminobenzoyl carolinensis spacer elements component. Source material AD (Lys(Mtr)) is commercially available (Bachem, Torrance, CA), or it can be obtained as described Dubowchik et al. Tetrahedron Letters, (1997) 38:5257-60.

Scheme 12

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

As shown in Scheme 13, the linker may be subjected to interaction with the amino group of compound medicines of the formula (Ib) with education connection “drug-linker”, which contains amide or urethane group, a linking component “drug” component “linker”. If the reactive site No. 1 is carbonisation group as the linker AJ, who eacce combinations can be made using HATU or PyBrop and the appropriate amine base to obtain compound “drug-linker AK, containing amide bond between the component “drug” and component “linker”. If the reactive site No. 1 is a carbonate, as in the linker AL, the linker may be attached to drug using HOBt in a mixture of DMF/pyridine getting connection “drug-linker” S containing a urethane bond between the component “drug” and component “linker”.

Alternatively, if the reactive site No. 1 is a good leaving group, such as in the linker'AN, the linker can be attached to the amino group of the medicinal product by the reaction of nucleophilic substitution, in which results can be obtained connection “drug-linker”having amine linkage (AO) between the component “drug” and component “linker”.

Representative methods used to attach the drug to the ligand, to obtain the connection “drug-linker as described in scheme 13 and illustrated in the General procedures G-H.

Scheme 13

General procedure G: amide Formation using HATU. Drug (Ib) (1.0 EQ.) and N-protected linker containing carbonisation reactive site (1.0 EQ.), dilute the appropriate organic is named solvent, such as dichloromethane, and the resulting solution was treated with HATU (1.5 EQ.) and an organic base, preferably pyridine (1.5 EQ). The reaction mixture is stirred in an inert atmosphere, preferably argon atmosphere, 6 hours, during which monitor the reaction mixture by HPLC. The reaction mixture was concentrated and the resulting residue purified by HPLC to obtain the amide of formula AK.

Procedure H: Formation of carbamate using HOBt. The mixture linker AL with p-nitrophenylarsonic reactive site (1.1 EQ.) and drug (1b) (1.0 EQ.), diluted with an aprotic organic solvent, such as DMF, to obtain a solution having a concentration of 50-100 mm, and the resulting solution was treated with HOBt (2.0 EQ.) and placed in an inert atmosphere, preferably in an atmosphere of argon. The reaction mixture is stirred for 15 minutes, and then add an organic base, such as pyridine (1/4, vol./vol.), and monitor the reaction by HPLC. The absorption of the linker typically occurs for 16 hours. Then the reaction mixture was concentrated in vacuo and the resulting residue is purified, for example, by HPLC, to obtain the carbamate AM.

An alternative method of preparing compounds “drug-linker” is illustrated in figure 14. In accordance with the method, proell is reported in figure 14, the drug is attached to a part of the linker component (e.g., ZA), which is not attached extension component. The result can be obtained intermediate compound AR, which has the amino acid component containing Fmoc-protected N-terminal group. Then the Fmoc group is removed, after which the amine intermediate connection AQ attached to the extension component by means of combination reaction catalyzed using PyBrop or DEPC. Constructing compounds “drug-linker containing either bromoacetamide extension component AR, or PEG-maleimide extension component AS illustrated in figure 14.

Scheme 14

M!

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

The methodology used to obtain the linker component containing the spacer branched chain, shown in figure 15.

Scheme 15

Scheme 15 illustrates the synthesis of a dipeptide linker val-cit, has maleimides extension component and bis(4-hydroxymethyl)styrene (BHMS) component. Synthesis of intermediate compound BHMS (AW) was improved as compared with the synthesis, opisaniya in the literature (see publication of International application no WO 9813059, Firestone et al. & Crozet M.P.; Archaimbault G.; Vanelle, P.; Nouguier R. Tetrahedron Lett. (1985) 26:5133-5134), and in this synthesis as a source of materials used commercially available diethyl-(4-nitrobenzyl)phosphonate (at) and commercially available 2,2-dimethyl-1,3-dioxane-5-he (AU). Linkers AY and BA can be obtained from the intermediate AW in accordance with the methodology illustrated in scheme 9.

4.6.3. Dendritic linkers

The linker may be a linker dendritic type used for covalent binding of more than one molecule drugs with a ligand such as, but not limited to, antibody, through a branching, multifunctional linker molecule (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers allow you to increase the molar ratio of drug to antibody, i.e. the load that corresponds to the efficiency of conjugate “drug-linker-ligand”. Thus, if constructed on the basis of cysteine antibody contains only one reactive thiol group of cysteine, by dendritic linker can be attached to a large number of molecules of the drug.

In the following representative embodiments, the reagents dendritic is incera allow to form conjugates with nine nucleophilic reagent molecule drugs through interaction with chlorethylene functional groups of the nitrogen analogue of mustard gas:

4.6.4. Conjugation of drug molecules with antibodies

Scheme 16 illustrates the methodology used to produce conjugates “drug-linker-ligand, having from about 2 to 4 molecules of the drug to the antibody. The antibody is treated with a reducing agent, such as dithiothreitol (DTT) to restore some or all of the cysteine disulfide residues with formation of highly nucleophilic thiol groups of cysteine (-CH2SH). This partially restored the antibody is subjected to reaction with compounds “drug-linker or the linker reagents, i.e. their electrophilic functional groups, such as maleimide or α-halogencarbonic, in accordance with the method of conjugation described in str Klussman et al. (2004) Bioconjugate Chemistry 15(4):765-773.

Scheme 16

For example, the antibody, for example AS, dissolved in 500 mm sodium borate and 500 mm sodium chloride at pH 8.0, is treated with an excess of 100 mm dithiothreitol (DTT). After incubation at 37º within 30 minutes, the buffer is replaced by elution of the resin Sephadex G25 and elute PBS with 1 mm DTPA. The ratio of thiol/Ab is calculated by determining the concentration of the recovered antibodies on the basis of optical density PR is 280 nm of the solution and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the optical density at 412 nm. The recovered antibody dissolved in PBS and cooled on ice. Conjugate “drug-linker”, for example MC-val-cit-RAV-MMAE in DMSO, dissolved in acetonitrile and water at known concentration, is added to the cooled recovered antibody in PBS. After approximately one hour to extinguish the reaction and kupirovaniya” unreacted thiol group of the antibody add excess maleimide. The reaction mixture is concentrated by ultrafiltration on a centrifuge and the ADC, for example AS-MC-vc-RAV-MMAE, clear, absoluut by elution through G25 resin in PBS, filtered through 0.2 μm filters under sterile conditions, and frozen for storage.

Received various conjugates of the antibody-drug” (ADC) with different linkers and molecules of the drug, MMAE and MMAF. The table below presents representative group of the ADC, which were obtained in accordance with the Protocol described in example 27, and characterized by HPLC and analysis by loading the drug.

4.7. Compositions and methods introduction

In another embodiment, the present invention relates to compositions, including the non effective number of representative compounds and/or representative of the conjugate and a pharmaceutically acceptable carrier or excipient. For convenience, components of the “drug” and connection “drug-linker” will be called representative compounds and conjugates “drug-ligand” and “drug-linker-ligand” will be called representative conjugates. These compositions can be used for the introduction of animals and man.

Compositions according to the invention can be obtained in any form, which allows you to enter these compositions to a patient. For example, the composition may be in solid, liquid or gas (aerosol) form. Typical routes of administration include, but are not limited to, oral, local, parenteral, sublingual, rectal, vaginal, intraocular, intratumoral, and intranasal administration. Parenteral administration includes subcutaneous, intravenous, intramuscular and intrasternal injection or infusion. In one aspect of the invention the composition is administered parenterally. In another aspect of the invention, representative compounds and/or representative conjugates or compositions are administered intravenously.

The pharmaceutical compositions can be prepared so that after the introduction of the patient representative compounds and/or representative conjugates was bioavailable. The composition can be wearable is prepared since the one or more uniform dosage forms, for example in the form of pills, which can be a single unified dosage form, and the container containing a representative compound and/or a representative conjugate in the form of an aerosol, which can contain many standardized dosage forms.

The materials used to obtain pharmaceutical compositions may be non-toxic in the quantities used. For every person it is obvious that the optimal dose of the active(s) ingredient(s) in the pharmaceutical composition depends on a number of factors. These factors include, but are not limited to, the type of animal (such as man), a particular form of representative compound or conjugate, route of administration and composition used.

Pharmaceutically acceptable carrier or excipient may be a solid substance, which allows to obtain the composition, for example, in the form of tablets or powder. The carrier may be a liquid, and therefore containing compositions can be, for example, prepared in the form of a syrup for oral administration or injection. In addition, the medium may be gaseous or powdered and used for the preparation of aerosol compositions used, for example, for administration by inhalation.

For oral administration composition predpochtitelnei in solid or liquid form, which can be semi-solid, semi-solid, suspension and gel forms, which are included here in the forms considered as solid or liquid.

Solid composition for oral administration can be prepared in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers or other Such solid composition typically contains one or more inert diluents. In addition, this composition can be one or more of the following agents: binding agents, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins; dezintegriruetsja agents such as alginic acid, sodium alginate, primogel, corn starch and the like; oil, such as magnesium stearate or sarotex; substances that increase the slip, such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring, such as peppermint, methyl salicylate or orange flavoring; and dye.

If the composition is prepared in the form of capsules, for example in the form of gelatin capsules, it is, in addition to the above materials may contain a liquid carrier such as polyethylene glycol, cyclodextrin or fatty oil.

The composition may be in preparation, is in the form of liquid, for example, elixir, syrup, solution, emulsion or suspension. Such a fluid may be used for oral administration or for delivery by injection. Composition for oral administration may contain one or more such agents, sweetening agents, preservatives, dye/coloring matter and an intensifier of taste and aroma. In a composition intended for administration by injection may be included one or more of such agents, as surfactant, preservative, wetting agent, dispersing agent, suspendisse agent, buffer, stabilizer and the agent, giving isotonicity.

Liquid composition, regardless of whether they are solutions, suspensions or have a different shape, may also include one or more of the following agents, namely sterile diluents such as water for injection, saline solution, preferably physiological solution, ringer's solution, isotonic sodium chloride, fatty oils such as synthetic mono or diglycerides which may serve as a solvent or suspendida environment, glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid is or sodium bisulfite; hepatoblastoma agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of toychest, such as sodium chloride or dextrose. The composition for parenteral administration can be enclosed in ampoules, disposable syringes or in a vessel for repeated administration, made of glass, plastic or other material. Representative adjuvant is saline. Composition for injection is preferably sterile.

The number of representative compounds and/or representative conjugate, which is effective for the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, can be performed, but not necessarily,in vitroorin vivoanalyses to determine the optimal dose levels. The exact dose to be used in these compositions, also depends on the method of administration, the severity of the disease or disorder, and should be prescribed by the attending physician for each patient individually.

Songs contain a certain number of representative compounds and/or representative conjugate effective to receive the appropriate dose. Usually this number is, m is Nisha least approximately 0.01% of the representative compounds and/or representative of the conjugate by weight of this composition. For oral administration, it can vary in the range of from about 0.1% to 80% by weight of the composition. In one aspect of the invention, compositions for oral administration may contain from about 4% to 50% of the representative compounds and/or representative of the conjugate by weight of the composition. In yet another aspect, the compositions according to the invention are prepared so that a uniform dosage form for parenteral administration contain from about 0.01 to 2 wt.% representative compounds and/or representative conjugate.

Composition for intravenous infusion may contain from about 0.01 to 100 mg of representative compounds and/or representative conjugate per kg of animal body weight. In one aspect of the invention, this composition may contain from about 1 to 100 mg of representative compounds and/or representative conjugate per kg of animal body weight. In another aspect of the invention introduce a number of representative compounds and/or representative of the conjugate is in the range from about 0.1 to 25 mg/kg of body weight of the animal.

The usual dose of the representative compounds and/or representative conjugate, administered to the patient is the ome from 0.01 to 2000 mg/kg animal body weight. In one aspect of the invention, the dose, administered to the patient is from about 0.01 to 10 mg/kg animal body weight, in another aspect of the invention, the dose, administered to the patient is from about 0.1 to 250 mg/kg animal body weight, In yet another aspect of the invention, the dose, administered to the patient is from about 0.1 to 20 mg/kg animal body weight, In yet another aspect of the invention injected dose is from about 0.1 to 10 mg/kg animal body weight, and in another aspect of the invention injected dose is from about 1 to 10 mg/kg animal body weight.

Representative compounds and/or representative conjugate or composition can be introduced by standard methods, for example by infusion or injection loading dose, by absorption through epithelial or the lining of the mucous membrane (for example, through the oral mucosa, rectum, small intestine and the like). This introduction can be systemic or local. There are various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, capsules and the like, and these systems can be used for the introduction of representative compounds and/or representative conjugate or composition. In some embodiments of the invention, the patient can be entered more than one representative compound and/or a representative conjugate or to the position.

In specific embodiments of the invention may be desirable local introduction of one or more representative compounds and/or representative conjugates or compositions in the area of the body that requires treatment. This can be achieved by such methods, but not limited to, as, for example, local infusion during surgery; topical, for example, in the form of a bandage on the wound after surgery; injection; introduction through the catheter; introduction by suppository or injection of the implant, where the specified implant consists of a porous, non-porous, or gelatinous material, including membranes, such as SelectionKey membranes or fibers. In one embodiment of the invention, the introduction can be effected by direct injection into a given area (or in the primary region) localization of cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment of the invention, the introduction can be effected by direct injection into a given area (or in the primary region) the manifestation of symptoms of autoimmune disease.

In some embodiments of the invention may be desirable introduction of one or more representative compounds and/or representative conjugates or compositions in the Central nervous system Luba the appropriate way including intraventricular and intrathecal injection. Intraventricularly injection can be more easily introduced through intraventricularly catheter, for example, is attached to the tank, Ommaya.

Can also be implemented pulmonary introduction, for example, using the inhaler or nebulizer, or by obtaining the drug, using the spray agent, or by perfusion in a fluorocarbon or synthetic surface-active substance, which is suitable for introduction into the lungs.

In yet another variant of the invention, representative compounds and/or representative conjugates or compositions can be entered using the system for the controlled delivery, such as, but not limited to, a pump or different polymeric materials. In another embodiment of the invention, the system for the controlled delivery may be placed in the immediate vicinity of the target, which aimed representative compounds and/or representative conjugates or compositions, for example in the brain, this may require only part of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol.2, p.115-138 (1984)). Can be used and other systems for the controlled delivery discussed Langer (Science 249:1527-1533 (1990)).

The term “carrier” means a diluent, adjuvant or order the tel, in combination with which is injected representative compound and/or a representative conjugate. Such pharmaceutical carriers can be liquids, such as water and oils, including liquid paraffin, animal and vegetable oil or synthetic oil, such as peanut oil, soybean oil, mineral oil, sesame oil and other Media can be saline, Arabian gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. can also be used additives, stabilizing agents, thickeners, oil and dyes. In one embodiment of the invention of a representative compound and/or a representative conjugate or composition, and pharmaceutically acceptable carriers injected to the patient, are sterile. At intravenous introduction of representative compounds and/or representative conjugates suitable carrier is water. As liquid carriers, particularly for obtaining solutions for injection, can also be used saline solutions and aqueous dextrose and glycerol. Suitable pharmaceutical carriers are also fillers, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, x is oral sodium, dry the separated milk, glycerol, propylene glycol, water, ethanol, etc. of the Composition according to the invention, if necessary, may also contain small amounts of wetting or emulsifying agents or pH-tabularasa agents.

Compositions according to the invention can be prepared in the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained release formulations release, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for this application. Other examples of suitable pharmaceutical carriers are described in the manual E.W. Martin “Remington''s Pharmaceutical Sciences”.

In one embodiment of the invention, representative compounds and/or representative conjugates prepared in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to animals, particularly humans. Usually carriers or fillers for intravenous injection is a sterile isotonic aqueous buffer solutions. If necessary, such compositions can also include solubilizers agent. Compositions for intravenous administration can, but not necessarily, contain a local anesthetic agent, such as lignocaine, to weaken Bo and at the injection site. Usually the ingredients are added either individually or in a mixture in the form of a standardized dosage forms, for example in the form of dry liofilizirovannogo powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet, which shows the amount of active substance. If a representative compound and/or a representative conjugate is administered by infusion, they can be poured, for example, vials containing sterile water or saline pharmaceutical purity. If a representative compound and/or a representative conjugate is administered by injection, it can be obtained vial with sterile water for injection, or can be prepared saline solution for injection so that the ingredients could be mixed prior to their introduction.

Compositions for oral delivery can be obtained, for example, in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Oral input compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; fragrances such as peppermint, Wintergreen oil, cherry oil; colorants; preservatives, for giving such pharmaceuticals pickup is acceptable taste. In addition, if the compositions are in the form of tablets or pills, they can be coated to delay disintegration and absorption in the gastrointestinal tract and, thereby, to ensure their actions over a long period of time. For oral insertion compounds also are suitable selectively permeable membranes surrounding osmotically active stimulating compounds. In these membranes fluid from the environment surrounding the capsule, impregnate stimulating compound, which swells and displaces the agent or the composition through the membrane opening. These membranes to delivery can provide, basically, the profile delivery zero order, unlike the pulse profile of drugs immediate release. To slow down the delivery time may be used such a substance, such as glycerol monostearate or glycerol stearate.

The composition can be intended for local use, and in this case can be used as a carrier in the form of a solution, emulsion, ointment or gel base. Composition for percutaneous introduction can be obtained in the form of a transdermal patch or device for iontophoresis. Compositions for topical application may contain concentrations of representative compounds and/or representative conjugate comprising primer is from 0.05% to 50% wt./about. (mass per unit volume of the composition), and in another aspect from 0.1% to 10% wt./about.

The composition may be introduced rectally, for example in the form of suppositories, which melt in the rectum and release the representative compound and/or a representative conjugate.

This composition may include various materials which modify the physical form of a solid or liquid unified drug. For example, the composition may include materials that form a shell covering the active ingredients. The materials that form the coating are usually inert and can be selected, for example, of sugar, shellac, or other agents for Intercollege coverage. Alternatively, the active ingredients can be enclosed in a gelatin capsule.

The composition may consist of gaseous uniform dosage forms, for example they can be an aerosol. The term “aerosol” is used to describe a variety of systems, from colloidal systems to systems consisting of sealed packages. Shipping can be carried out using a liquefied or compressed gas or by using a suitable system of pumps, metering release of the active ingredients.

Compositions according to the invention, regardless of the presence of Lyon in solid, liquid or gaseous form, may include a pharmacological agent used for the treatment of cancer, autoimmune disease or infectious disease.

4.8. Therapeutic application of representative conjugates

Representative compounds and/or representative conjugates can be used for the treatment of cancer, autoimmune disease or infectious disease in a patient.

4.8.1. Cancer treatment

Representative compounds and/or representative conjugates can be used for inhibiting the multiplication of tumor or cancer cells, induce apoptosis in tumor or cancer cells, or for treating cancer in a patient. Representative compounds and/or representative conjugates can be used accordingly in a variety of ways to treat cancer in animals. Conjugate “drug-linker-ligand” may be used to deliver drugs or component “drug” in the tumor or cancer cell. Without pretending to any particular theory, it can be noted that In one embodiment of the invention, the ligand component representative of the conjugate is bound or associated with the antigen of the cancer cells or tumor cells, and this representative conjugate can be absorbed SDA is alevai or cancer cell mediated through receptor endocytosis. The antigen can be attached to the tumor or cancer cell, or it may be an extracellular matrix protein associated with tumor cell or cancer cell. After introducing into the cell one or more specific peptide sequences in the linker component hydrolytically cleaved by one or more proteases tumor or cancer cell that leads to the release of drugs or compounds “drug-linker”. Then the released drug or connection “drug-linker” freely migrates into the cell and induces cytotoxic or cytostatic activity. In an alternative embodiment of the invention, the drug or a component of “drug” is cleaved from a representative conjugate and gets into the environment of a tumor or cancer cell, and then the drug or connection “drug-linker” enters the cell.

In one embodiment of the invention, the ligand component is associated with a tumor or cancer cell.

In another embodiment of the invention, the ligand component binds to the antigen of a tumor or cancer cell, which is located on the surface of this tumor or cancer cell.

In another embodiment, the image is etenia ligand component binds to the antigen of a tumor or cancer cell, which is an extracellular matrix protein associated with tumor cell or cancer cell.

The specificity of the ligand component for a particular tumor or cancer cells may play an important role in determining the type of tumor or cancer cells that best lend themselves to effective destruction. For example, representative conjugates containing ligand BR96, can be used for the treatment of antigen-positive carcinomas including carcinomas of the lung, breast, ovary and pancreas. Representative conjugates containing the ligand, the type of antibodies against CD30 or against CD40, can be used for the treatment of hematologic malignancies.

Other specific types of cancers that can be treated representative conjugates are, but not limited to the cancers listed in table 3.

Table 3

Solid tumors, including, but not limited to:

fibrosarcoma
myxosarcoma
liposarcoma
chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
ehotelier.com
lymphangiosarcoma
lymphangiosarcoma
sinovioma
mesothelioma
Ewing's sarcoma
leiomyosarcoma
rhabdomyosarcoma
colon cancer
cancer of the colon
kidney cancer
pancreatic cancer
bone cancer
breast cancer
ovarian cancer
prostate cancer
cancer of the esophagus
stomach cancer
oral cancer
cancer of the nasal cavity
throat cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
carcinoma of the sweat glands
carcinoma of the sebaceous glands
papillary carcinoma
papillary adenocarcinoma
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
carcinoma of the bile ducts
horiokartsinoma
seminoma
embryonal carcinoma
Wilms tumor
cervical cancer
cancer of body of uterus
testicular cancer
small cell carcinoma of the lung
carcinoma of the bladder
lung cancer
epithelial carcinoma
glioma
multiform glioblastoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
neuroma of the auditory nerve
oligodendroglioma
meningioma
skin cancer
melanoma
neuroblastoma
retinoblastoma

Blood cancer, including, but not limited to:

acute lymphoblastic leukemia “ALL”
acute lymphoblastic b-cell leukemia
acute lymphoblast the th T-cell leukemia
acute myeloblastic leukemia “AML”
acute promyelocytic leukemia, “Ali”
acute monoblastic leukemia
acute erythroleukaemia leukemia
acute megacaryoblastic leukemia
acute myelomonocytic leukemia
acute non-lymphocytic leukemia
acute undifferentiated leukemia
chronic miliitary leukemia “CML”
chronic lymphocytic leukemia “CLL”
leukemia retikulez
multiple myeloma
Acute and chronic leukemias
lymphoblastic
myelogenous
lymphocytic
miliitary
Lymphoma
Hodgkin's disease
not-jackinsky lymphoma
multiple myeloma
macroglobulinemia waldenstrom
the disease is heavy chains
polycythemia Vera

Representative conjugates deliver compounds to tumor or cancer cells that are specific to conjugation, resulting in lower overall toxicity of these compounds. Linker components stabilize representative conjugates in the blood, which, however, split the tumor-specific proteases cells, releasing the drug.

4.8.2. Complex therapy of cancer

Cancer, including, but not limited to IVaS them tumors, metastases, or any other disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by introducing a representative conjugate and/or a representative of the connection.

In other embodiments, the invention describes methods of treating or preventing cancer, comprising the administration to a patient in need, an effective amount of a representative conjugate and chemotherapeutic agents. In one embodiment of the invention, such chemotherapeutic agent is a tool that, as it was discovered, is not effective for the treatment of cancer. In another embodiment of the invention, such chemotherapeutic agent is a tool that, as it was discovered, is an effective treatment for cancer. Representative conjugates can be administered to the patient who also underwent surgery for radical treatment of cancer.

In one embodiment of the invention an additional way cancer treatment is radiation therapy.

In a specific embodiment of the invention representative conjugate is administered concurrently with the chemotherapeutic agent or simultaneously with radiation therapy. In another specific embodiment of the invention the introduction of a chemotherapeutic drug or held what I radiation therapy carried out before or after the introduction of representative conjugates, for example, in one aspect of this introduction is carried out, at least for one hour, five hours, 12 hours, one day, one week, one month before/after the introduction of a representative conjugate, and in other aspects - for/after a few months (for example, three months) before/after the introduction of the conjugate.

Chemotherapeutic agent can be introduced in several sessions. This can be entered one of the chemotherapeutic agents listed in table 4, or a combination thereof. As for radiation therapy, the Protocol for such radiation therapy depends on the type of cancer being treated. Examples of such radiation therapy include, but are not limited to, radiotherapy, and in particular for the treatment of deep tumors can be applied radiation high energy several megavolt (radiation with an energy of more than 1 MV), and for the treatment of skin cancer can be applied to electron radiation and x-rays in a few autovolt. Can also be applied gamma radiation emitted by radioisotopes, such as radioactive isotopes of radium, cobalt and other elements.

In addition, methods of treating cancer using representative compounds and/or representative conjugate can serve as an alternative to chemotherapy or radiation therapy in those cases, to the Yes, such chemotherapy or radiation therapy is or may be too toxic, for example causes unacceptable or unbearable side effects in the individual being treated. The animal in need of treatment, it may be, but not necessarily, subjected to different treatment of cancer, such as surgery, radiation therapy or chemotherapy, depending on which of these therapies, as determined by experts, is acceptable or tolerable.

Representative compounds and/or representative conjugates can also be used inin vivoorex vivothe treatment methods used to treat certain types of cancer, including, but not limited to, leukemias and lymphomas, where such treatment includes the introduction of autologous stem cells. This treatment can be carried out by conducting a multi-stage procedure, in which autologous hematopoietic stem cells of the animal are harvested and purged of all cancer cells, and then the residual population of bone marrow cells of the animal treated by injecting high doses of representative compounds and/or representative conjugate with simultaneous or without radiotherapy with high doses and transplant stem cells again enter the animal. After the restoration of bone marrow function and recovery of the animal spend maintaining Wausau therapy.

4.8.3. Complex therapy of cancer using multiple drug

Below are the methods of treatment of cancer, comprising the administration to a patient in need, an effective amount of a representative conjugate and the other therapeutic agent is an anticancer agent. Suitable anticancer means include, but are not limited to, methotrexate, Taxol, L-asparaginase, mercaptopurine, tioguanin, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosamine, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard gas analogues, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubitsin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel. In one aspect of the invention anticancer means include, but are not limited to, the drugs listed in table 4.

td align="left"> Inhibitors of Ca2+-Asia-Pacific-the basics:
Table 4
Alkylating agents:
Nitrogen mustard gas analogues:cyclophosphamide,
ifosfamide trofosfamide
chlorambucil
melphalan
Nitrosoanatabine:carmustin (BCNU)
lomustin (CCNU)
The alkyl sulphonates:the busulfan
treosulfan
Triazine:dacarbazin
Compounds containing platinum:cisplatin
carboplatin
Plant alkaloids:
Vinylchloride:vincristine
vinblastine
vindesine
vinorelbine
Taxaide:paclitaxel
docetaxel
Inhibitors of DNA topoisomerase
Apoptolidin:etoposide
teniposide
topotecan
9-aminocamptothecin
camptothecin
Kristol
Mitomicina:mitomycin C
Antimetabolites:
Antifolates:
Inhibitors of DHFR:methotrexate
Tr is metrakit
Inhibitors of IMP dehydrogenase:mycofenolate acid
teatterin
ribavirin
EICAR
Inhibitors ribonucleotide-reductase:hydroxyurea
deferoxamine
Analogues of pyrimidine:
Analogues of uracil:5-fluorouracil
floxuridine
doxifluridine
redirected
Analogues of cytosine:cytarabine (Ara-C)
arabinoside cytosine
fludarabine
The purine analogues:mercaptopurine
tioguanin
Hormonal therapeutic agent:
The receptor antagonists:
Antiestrogens:tamoxifen
raloxifene
megestrol
LHRH agonists:goselin
acetate leuprolide
Antiandrogens:flutamide
bikalutamid
Retinoids/deltoids:
Analogues of vitamin D3:EB 1089
ST. 1093
KN 1060
Photodynamic therapeutic agent:verteporfin (BPD-MA)
phthalocyanine
the photosensitizer Pc4
demethoxycurcumin AND (A-2-DMHA)
Cytokines:interferon-α
interferon-γ
the tumor necrosis factor
Other facilities:gemcitabine
walked
reamed
talmid
Inhibitors isoprenaline:lovastatin
Dopaminergic neurotoxins:ion 1-methyl-4-phenylpyridine
Inhibitors of the cell cycle:staurosporin
The aktinomitinov:actinomycin D
dactinomycin
Bleomycin:bleomycin A2
bleomycin B2
peplomycin
The anthracyclines:daunorubicin
doxorubicin (adriamycin)
idarubitsin
epirubicin
pirarubicin
zorubicin
mitoxantrone
The MDR inhibitors:verapamil
thapsigargin

4.8.4. Treatment autoimmune disease

Representative conjugates can be used for preventing or inhibiting the multiplication of cells, which induce autoimmune disease, or for treating autoimmune diseases. Representative conjugates can be used accordingly in a variety of ways of treating autoimmune disease in a patient. Conjugates “drug-linker-ligand” can be used to deliver drugs to the target cell. Without pretending to any particular theory, it can be noted that In one embodiment of the invention, the conjugate drug-linker-ligand” binds to the antigen on the surface of target cells, and such representative conjugate can be absorbed by the cell-target mediated through receptor endocytosis. After introducing into the cell one or more specific peptide sequences in the linker component enzymatically or hydrolytically cleaved to release the drug. Then released the drug migrates freely in the cytosol and induces cytotoxic or cytostatic activity. Alternatively, izaberete the Oia drug is cleaved from a representative conjugate and gets into the external environment of the target cells, after which it enters the cell.

In one embodiment of the invention, the ligand component is associated with an autoimmune antigen. In one aspect of the invention, the antigen present on the surface of cells involved in the induction of autoimmune condition.

In another embodiment of the invention, the ligand component is associated with an autoimmune antigen present on the cell surface.

In one embodiment of the invention the ligand binds to activated lymphocytes that are associated with autoimmune pathological condition.

In another embodiment of the invention representative conjugates prevent or inhibit the proliferation of cells that cause autoimmune antibody associated with a particular autoimmune disease.

Specific types of autoimmune diseases that can be treated representative conjugates, but are not limited to, disorders associated with Th2-lymphocytes (for example, atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, syndrome Omena, systemic sclerosis, and graft versus host”); disorders associated with Th1-lymphocytes (for example, rheumatoid arthritis, multiple sclerosis, psoriasis, rheumatoid Segren, Hashimoto's thyroiditis, graves ' disease, primary bi is irony cirrhosis, Wegener's granulomatosis and tuberculosis); disorders associated with the activated B-lymphocytes (e.g., systemic lupus erythematosus syndrome?, rheumatoid arthritis and diabetes type I); and the disorders listed in Table 5.

Table 5
Chronic active hepatitis
Addison disease
Allergic alveolitis
Allergic reaction
Allergic rhinitis
Syndrome Alport
Anaphylaxis
Ankylosing spondylitis
Antiphospholipid syndrome
Arthritis
Ascaris
Aspergillosis
Atopic Allergy
Atopic dermatitis
Atopic rhinitis
Behcet's disease
Lung disease birdwatchers
Bronchial asthma
Syndrome Kaplan
Cardiomyopathy
Coeliac disease
Chagas disease
Chronic glomerulonephritis
Syndrome Kogan
The disease is associated with the synthesis of cold agglutinins
Congenital infectious rubella
The CREST syndrome
Crohn's disease
The cryoglobulinemia
Cushing's syndrome
Dermatomyositis
Discoid lupus erythematosus
Syndrome Dressler
The syndrome of Eaton-Lambert
Echovirus infection
Encephalomyelitis
Endocrine ophthalmopathy
Infection caused by the Epstein-Barr
The fuse in horses
Erythematous
Evans syndrome
The syndrome still's
Fibromyalgia
Cycle Fuchs
Atrophy of the stomach
Food Allergy
Giant cell arteritis diagnostics
Glomerulonephritis
Syndrome?
Graft-versus-host
Graves disease
Disease Guillain-Barre
Hashimoto's thyroiditis
Hemolytic anemia
Purpura's disease-Selana
Idiopathic atrophy of the adrenal glands
Idiopathic fabric lungs
IgA nephropathy
Inflammatory bowel disease
Insulin-dependent diabetes mellitus
Uvenil the hydrated arthritis
Juvenile diabetes (type I)
The syndrome of Lambert-Eaton
Laminitis
Lichen planus
Lapoinya hepatitis
Common lupus
Lymphopenia
Meniere's disease
Mixed disorder of connective tissue
Multiple sclerosis
Severe myasthenia gravis
Pernicious anemia
Polyglandular syndromes
Presenilny dementia
The primary agammaglobulinemia
Primary biliary of CIRR the C
Psoriasis
Psoriatic arthritis
The phenomenon, Raynaud's disease
Habitual miscarriage
Syndrome, Reiter
Rheumatic fever
Rheumatoid arthritis
Syndrome Santera
Schistosomiasis
Syndrome Schmidt
Scleroderma
Syndrome Shulman
Syndrome Segren
Syndrome stiff man”
Sympathetic ophthalmia
Systemic lupus erythematosus
cheriet, Takayasu's
Temporal Eretria
Thyroiditis
Thrombocytopenia
Thyrotoxicosis
Toxic epidermal necrolysis
Insulin resistance type
Diabetes mellitus type I
Ulcerative colitis
Uveitis
Vitiligo
Macroglobulinemia waldenstrom
Wegener's granulomatosis

4.8.5. Complex therapy of autoimmune diseases with the use of multiple drug

Below are methods of treating autoimmune diseases comprising administration to a patient in need, an effective amount of a representative conjugate and another therapeutic agent,which is a well known tool for the treatment of autoimmune diseases. In one embodiment of the invention such means against autoimmune diseases include, but are not limited to, the tools listed in table 6.

Table 6
Cyclosporine
Cyclosporin a
Mycophenolat mofetil
Siolim
Tackroom
Entanercept
Prednisone
Azathioprine
Cyclophosphamide methotrexate
Prednisone
Aminocaproic acid
Chloroquine
Hydroxychloroquine
Hydrocortisone
Dexamethasone
Chlorambucil
DHEA
Danazol
Bromkriptin
Meloxicam
Infliximab

4.8.6. The treatment of infectious diseases

Representative conjugates can be used for preventing or inhibiting the multiplication of cells, which induce infectious disease or for the treatment of infectious diseases. Representative conjugates can be used accordingly in a variety of ways of treating infectious disease in a patient. Conjugates “drug-linker-ligand” can be used to deliver drugs to the target cell. In one embodiment of the invention, the ligand component is associated with infected cell.

In one embodiment of the invention these conjugates prevent or inhibit the proliferation of cells, inducing a specific infectious disease.

Specific types of infectious dis is evani, which can be treated as a representative conjugates, include, but are not limited to, the diseases listed in table 7.

Table 7
Bacterial diseases:
diphtheria
whooping cough
latent bacteremia
urinary tract infections
gastroenteritis
cellulite
epiglottic
tracheitis
adenoid hypertrophy
zapotocny abscess
impetigo
ectima
pneumonia
endocarditis
septic arthritis
pneumococcal infection
peritonitis
bacteremia
meningitis
acute purulent meningitis
urethritis
cervicitis
proctitis
pharyngitis
salpingitis
epididymitis
gonorrhea
syphilis
listeriosis
anthrax
Nocardia
salmonellosis
typhoid fever
dysentery
conjunctivitis
sinusitis
brucellosis
tularemia
cholera
bubonic plague
tetanus
necrotorous enteritis
the actinomycosis
mixed anaerobic infections
syphilis
recurrent fever
leptospirosis
Lyme disease
the bite fever rat
TB
lymphadenitis
leprosy
chlamydia
pneumonia caused by chlamydia
trachoma
conjunctivitis with inclusions (swimming pool conjunctivitis)

Systemic fungal diseases:

/table>

Rikketsioznye diseases:

histoplasmosis
coccidiodomycosis
blastomycosis
sporotrichosis
cryptococcomas
systemic candidiasis
aspergillosis
mycomics
mycetoma
chronomics
typhoid
Rocky mountain spotted fever
arlais
rickettsiosis caused by bites Eastern tick
smallpox, caused by Rickettsia
Q-fever
Bartonella

Diseases caused by parasites:

malaria
babesias
African sleeping sickness
Chagas disease
leishmaniasis
fever dum-dum
toxoplasmosis
meningoencephalitis
keratitis
entameba
giardias
cryptosporidiosis
isosporous
cyclosporin
microsporidiosis
Ascaris
trichocephalosis
the hookworm
nematodes
eye syndrome “wandering larvae”
trains
dracunculiasis
lymphatic filariasis
Loos
“river blindness
filariasis dogs, call the by nematodes of the family Onchocercidae
schistosomiasis
itching of the bather
paragonimus lungs
paragonimus liver
fasciolosis
fasciolopsis
opisthorchiasis
teniidoz
echinococcosis
alveolar echinococcosis

Viral diseases:

measles
subacute sclerosing panencephalitis
runny nose
mumps
rubella
roseola
fifth disease
chicken pox
infection caused by respiratory syncytial virus
croup
bronchiolitis
infectious mononucleosis
polio
herpetic pharyngitis
foot and mouth disease
bornholmsgat disease
genital herpes
genital warts
aseptic meningitis
myocarditis
pericarditis
gastroenteritis
acquired immunodeficiency syndrome (AIDS)
infection caused by the virus of human immunodeficiency (HIV)
Reye's syndrome.
syndrome Kawasaki
flu
bronchitis
viral outpatient pneumonia
acute infectious respiratory disease
acute pharyngoconjunctival fever
epidemic keratoconjunctivitis
infection caused by the herpes simplex virus 1 (HSV-1)
infection caused by herpes simplex virus 2 (HSV-2)
herpes, Lisi
cytomegalic inclusion disease, inclusiona disease
rabies
p is agressiva multiple leucoencephalopathy
Kuru
malignant hereditary insomnia
disease Creutzfeld-Jakob
disease Gastman-Straussler-Sheinker
tropical spastic prepares
Eastern equine encephalitis
California encephalitis
encephalitis caused by a virus St. Louis
yellow fever
the disease caused by the dengue virus
lymphocytic choriomeningitis
fever Lassa
hemorrhagic fever
pulmonary syndrome caused by Hantavirus
info the tion, caused by the Marburg virus
infections caused by Ebola virus
smallpox

4.8.8. Complex therapy of infectious diseases using multiple drug

Below are methods of treating infectious diseases comprising administration to a patient in need, an effective amount of a representative conjugate and another therapeutic agent that is an anti-infective agent. In one embodiment of the invention, such anti-infective means include, but are not limited to, the tools listed in table 8.

Table 8
β-laktamovogo antibiotics:
penicillin G
penicillin V
cloxacillin
dicloxacillin
methicillin
nafcillin
oxacillin
ampicillin
amoxicillin
bacampicillin
azlotillin
carbenicillin
mezlocillin
piperacillin
the tikarcillin

Aminoglycosides:

amikacin
gentamicin
kanamycin
neomycin
the netilmicin
streptomycin
the tobramycin

Makr the Lida:

azithromycin
clarithromycin
erythromycin
lincomycin
clindamycin

Tetracyclines:

demeclocycline
doxycycline
minocycline
oxytetracycline
tetracycline

Quinolones:

cinoxacin
nalidixic acid

Fluoroquinolones:

ciprofloxacin
enoxacin
greatl xacin
levofloxacin
lomefloxacin
norfloxacin
ofloxacin
sparfloxacin
trovafloxacin

Polypeptides:

bacitracin
colistin
polymyxin

Sulfonamides:

sulfisoxazole
sulfamethoxazole
the sulfadiazine
sulfamethizole
sulfacetamide

Other Antibacterials:

sulfamethazole
chloramphenicol
vancomycin
metronidazole
inupristin
dalfopristin
rifampin
spectinomycin
nitrofurantoin

Antivirals:

Common antiviral agents:

idoxuridine
vidarabine
trifluridine
acyclovir
famciclovir
peniciclovir
valacyclovir
anticyclonic
foscarnet
ribavirin
amantadine
rimantadine
cidofovir
the antisense oligonucleotides
immunoglobulins
interferons

Medicines to treat HIV infections:

tenofovir
emtricitabine
zidovudine
didanosine
zalcitabine
stavudine
lamivudine
nevirapine
delavirdine
shinaver
ritonavir
indinavir
nelfinavir

5. Examples

Example 1-Getting connection AB

Fmoc-val-cit-RAV-HE (14,61 g, a 24.3 mmol, 1.0 EQ., U.S. patent No. 6214345, Firestone et al.) was diluted in DMF (120 ml, 0.2 M) and to this solution was added diethylamine (60 ml). Reaction monitoring was performed using HPLC, and it was found that the reaction was completed after 2 hours. The reaction mixture was concentrated and the resulting residue is precipitated with ethyl acetate (approx. 100 ml), treating with ultrasound for 10 minutes. After this was added ether (200 ml) and the residue was again treated with ultrasound for 5 minutes. The solution was left for 30 minutes without stirring, and then filtered and dried under high vacuum to obtain Val-cit-PAB-OH which was used in the next stage without additional purification. Output: 8,84 g (96%). Conjugate val-cit-PAB-OH (8.0 g, 21 mmol) was diluted in DMF (110 ml) and the resulting solution was treated with MC-OSu (Willner et al. (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 EQ.). The reaction was completed after 2 cha is a, as it was shown by HPLC. The reaction mixture was concentrated and the oil obtained was inundated with ethyl acetate (50 ml). After ultrasonic treatment for 15 minutes was added ether (400 ml) and the mixture was again treated with ultrasound to the complete destruction of all large particles. After this, the solution was filtered and the solid was dried to obtain not quite white solid intermediate compound. Output: 11,63 g (96%); ES-MS m/z 757,9 [M-H].

Fmoc-val-cit-RAV-HE (14,61 g, a 24.3 mmol, 1.0 EQ., U.S. patent No. 6214345, Firestone et al.) was diluted in DMF (120 ml, 0.2 M) and to this solution was added diethylamine (60 ml). Reaction monitoring was performed using HPLC, and it was found that the reaction was completed after 2 hours. The reaction mixture was concentrated and the resulting residue is precipitated with ethyl acetate (approx. 100 ml), treating with ultrasound for 10 minutes. After this was added ether (200 ml) and the residue was again treated with ultrasound for 5 minutes. The solution was left for 30 minutes without stirring, and then filtered and dried under high vacuum to obtain Val-cit-PAB-OH which was used in the next stage without additional purification. Output: 8,84 g (96%). Conjugate val-cit-PAB-OH (8.0 g, 21 mmol) was diluted in DMF (110 ml) and the resulting solution was treated with MC-OSu (Willner et al. (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 EQ.). The reaction was completed after 2 hours, as it was shown by HPLC. The reaction mixture to the centered and the oil obtained was inundated with ethyl acetate (50 ml). After ultrasonic treatment for 15 minutes was added ether (400 ml) and the mixture was again treated with ultrasound to the complete destruction of all large particles. After this, the solution was filtered and the solid was dried to obtain not quite white solid intermediate compound. Output: 11,63 g (96%); ES-MS m/z 757,9 [M-H].

Not quite white solid intermediate compound (8.0 g, 14.0 mmol) was diluted in DMF (120 ml, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28,0 mmol, 2.0 EQ.) and DIEA (3,66 ml, or 21.0 mmol, 1.5 EQ.). The reaction was completed after 1 hour, as it was shown by HPLC. The reaction mixture was concentrated to obtain oil, which was besieging EtOAc, and then triturated with EtOAc (approx. 25 ml). Then the dissolved substance is again precipitated with ether (approx. 200 ml) and triturated for 15 minutes. The solid was filtered and dried under high vacuum, resulting in the received connection AB, which had a purity of 93%, as it was shown by HPLC and used in the next stage without additional purification. Yield: 9.7 g (94%).

Example 2-connection 1

HCl-salt tert-butyl ester of phenylalanine (868 mg, 3 mmol), N-Boc-delaplain (668 mg, 1 EQ.), DEPC (820 μl, 1.5 EQ.) and DIEA (1.2 ml) was diluted in dichloromethane (3 ml). After keeping for 2 hours (h) at room temperature (about 28°C) re Klenow mixture was diluted with dichloromethane (20 ml) and sequentially washed with saturated aqueous (water.) NaHCO 3(2 × 10 ml) and saturated aqueous NaCl (2 × 10 ml). The organic layer was separated and concentrated. The obtained residue resuspendable in ethyl acetate and purified on a flash chromatography in ethyl acetate. The appropriate fractions were combined and concentrated to obtain the dipeptide as a white solid: 684 mg (64%). ES-MS m/z 491,3 [M+H]+.

For the selective removal of the BOC in the presence of tert-butyl methyl ether of the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 ml). Then was added 4M HCl/dioxane (960 μl, 3 EQ.) and the reaction mixture was stirred over night at room temperature. RP-HPLC showed almost complete removal of the protective group BOC with a minimum level of cleavage of tert-butyl methyl ether. The resulting mixture was cooled in an ice bath was added triethylamine (500 μl). After 10 minutes the mixture was removed from the cooling bath, diluted with dichloromethane (20 ml) and sequentially washed with saturated aqueous NaHCO3(2 × 10 ml) and saturated aqueous NaCl (2 × 10 ml). The organic layer was concentrated and received a yellow foam: 287 mg (57%). The intermediate compound was used without further purification.

The Tripeptide Fmoc-Meval-val-dil-O-t-Bu (obtained as described in the application WO 02/088172 entitled “Pentapeptide Compounds and Uses Related Thereto”; 0.73 mmol)was treated with TFA (3 ml) and dichloromethane (3 ml) for 2 hours at room temperature. The mixture is concentrated to dryness, and the residue evaporated with toluene (3 × 20 ml) and dried in vacuum over night. The residue was diluted with dichloromethane (5 ml) and added to the dipeptide with remote protective groups (287 mg, 0.73 mmol)and then was added DIEA (550 μl, 4 EQ.) and DEPC (201 μl, 1.1 EQ.). After incubation for 2 hours at room temperature the reaction mixture was diluted with ethyl acetate (50 ml) and successively washed with 10% aqueous citric acid (2 × 20 ml), saturated aqueous NaHCO3(2 × 10 ml) and saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The obtained residue resuspendable in ethyl acetate and purified on a flash chromatography in ethyl acetate. The appropriate fractions were combined and concentrated to obtain Fmoc-Meval-val-dil-dap-phe-O-t-Bu in the form of a white solid: 533 mg (71%). Rfof 0.4 (EtOAc). ES-MS m/z 1010,6 [M+H]+.

The resulting product (200 mg, 0.2 mmol) was diluted in dichloromethane (3 ml) and diethylamine (1 ml). The reaction mixture was stirred over night at room temperature. Then the solvents were removed and got the oil, which was purified flash chromatography on silica gel in a stepwise gradient of 0-10% Meon in dichloromethane, resulting in the received compound 1 as a white solid: 137 mg (87%). Rf0,3 (10% Meon/CH2Cl2). ES-MS m/z to 788.6 [M+H]+.

Example 3 to Obtain compound 2

Compound 2 was obtained from compound 1 (30 mg, of 0.038 mmol) by treatment with 4M HCl in dioxane (4 ml) for 7 hours at room temperature. The solvent was removed and the residue was dried in vacuum overnight to obtain compound 2 as gigroskopichnomu white solids: 35 mg (120%calculated for the HCl-salt). ES-MS m/z 732,56 [M+H]+.

Example 4 to Obtain compound 3

Fmoc-Meval-val-dil-dap-phe-O-t-Bu (example 2, 50 mg) was treated with 4M HCl in dioxane (4 ml) for 16 hours at room temperature. The solvent was removed and the residue was dried in vacuum overnight to obtain 50 mg of intermediate compounds in the form of gigroskopichnomu white solid.

The white solid intermediate compound (20 mg, 0.02 mmol) was diluted with dichloromethane (1 ml)was added DEPC (5 μl, 0.03 mmol, 1.5 equiv.) and then was added DIEA (11 μl, 0.06 mmol, 3 EQ.) and tert-butylamine (3,2 μl, 0.03 mmol, 1.5 EQ.). After incubation for 2 hours at room temperature the reaction was not completed, as it was shown by RP-HPLC. Then was added DEPC (10 μl) and tert-butylamine (5 ml) and the reaction mixture was stirred for another 4 h and Then the reaction mixture was diluted with dichloromethane (15 ml) and then washed with water (5 ml), 0.1m waters. HCl (10 ml) and saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The remainder of razbam the Yali dichloromethane and purified on a flash chromatography in a step gradient of 0-5% Meon in dichloromethane. The appropriate fractions were combined and concentrated to obtain Fmoc-protected intermediate as a white solid: 7,3 mg (36%). Rf0,75 (10% Meon/CH2Cl2).

Fmoc-protected intermediate compound was diluted with dichloromethane (0.5 ml) and treated with diethylamine (0.5 ml) for 3 hours at room temperature. The reaction mixture was concentrated to dryness. The product was isolated by flash chromatography on silica gel in a stepwise gradient of 0-10% Meon in dichloromethane, resulting in the obtained compound 3 as a white solid: 4 mg (70%). Rf0,2 (10% Meon/CH2Cl2). ES-MS m/z 787 [M+H]+, 809 [M+Na]+.

Example 5 to Obtain compound 4

Boc-L-phenylalanine (265 mg, 1 mmol, 1 EQ.) and onomatology ether of triethylene glycol (164 μl, 1 mmol, 1 EQ.) was diluted with dichloromethane (5 ml). After this was added DCC (412 mg, 2 mmol, 2 equiv.) and then DMAP (10 mg). The reaction mixture was stirred at room temperature overnight. The precipitate was filtered. The solvent was removed in vacuo and the residue was diluted with ethyl acetate, and then purified on a flash chromatography on silica gel in ethyl acetate. The fractions containing the desired product were combined, concentrated and dried in vacuum to obtain white solids: 377 mg (91%). Rfof 0.5 (EtOAc). ES-MS m/z 434 [M+Na]+.

the Removal of the protective group BOC conducted by treatment of the above product in dioxane (10 ml) with a mixture of 4M HCl/dioxane (6 ml) for 6 hours at room temperature. The solvent was removed in vacuum and the residue was dried in vacuum to obtain a white solid.

HCl-salt of phenylalanine-monomethylaniline ether of triethylene glycol (236 mg, 0,458 mmol, 1 EQ.) and N-Boc-delaplain (158 mg, 0.55 mmol, 1.2 EQ.) was diluted with dichloromethane (3 ml). To the mixture was added DEPC (125 μl, 1.5 equiv.) and then DIEA (250 μl, 3 EQ.). After incubation for 2 hours at room temperature the reaction mixture was diluted with ethyl acetate (30 ml) and sequentially washed with saturated aqueous NaHCO3(2 × 10 ml), 10% aqueous citric acid (2 × 10 ml) and saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The obtained residue resuspendable in ethyl acetate and was purified flash chromatography on silica gel in ethyl acetate. The appropriate fractions were combined and concentrated to obtain intermediate compound as a white foam: 131 mg (50%). Rfof 0.25 (EtOAc). ES-MS m/z 581,3 [M+H]+.

The removal of the protective group BOC conducted in dichloromethane (2 ml) and TFA (0.5 ml) at room temperature for 2 h Then the solvent was removed in vacuo and the residue evaporated with toluene (3 × 25 ml), then dried in vacuum to obtain 138 mg TFA-salt of the dipeptide.

Fmoc-Meval-val-dil-OH (example 2, 147 mg, 0.23 mmol, 1 EQ.) and TFA-salt of the dipeptide (138 mg) was diluted in dichloromethane (2 ml). To the mixture was added DEPC (63 μl, 1.5 equiv.) and then DIEA (160 μl, 4 EQ.). After you remove the air traffic management for 2 hours at room temperature the reaction mixture was diluted with dichloromethane (30 ml) and sequentially washed with 10%aqueous citric acid (2 × 20 ml) and saturated aqueous NaCl (20 ml). The organic layer was separated and concentrated. The obtained residue resuspendable in dichloromethane and purified flash chromatography on silica gel in a stepwise gradient of 0-5% Meon in dichloromethane. The appropriate fractions were combined and concentrated to obtain a white foam: 205 mg (81%). Rf0,4 (10% Meon/CH2Cl2). ES-MS m/z 1100,6 [M+H]+, 1122,4 [M+Na]+.

The protective Fmoc group was removed by treatment with diethylamine (2 ml) in dichloromethane (6 ml). After incubation for 6 hours at room temperature the solvent was removed in vacuum and the resulting product was isolated by flash chromatography on silica gel in a stepwise gradient of 0-10% Meon in dichloromethane. The appropriate fractions were combined and concentrated. After evaporation of a mixture of dichloromethane/hexane, 1:1, obtained compound 4 as a white foam: 133 mg (80%). Rf0,15 (10% Meon/CH2Cl2). ES-MS m/z 878,6 [M+H]+.

Example 6 to Obtain compound 5

Fmoc-Meval-val-dil-OH (example 2, 0.50 g, 0.78 mmol) and dap-phe-OMe-HCl (0.3 g, 0.78 mmol, obtained as described G.R. Pettit et al., Anti-Cancer Drug Design 1998, 13, 243-277) was dissolved in CH2Cl2(10 ml)and then added diisopropylethylamine (0,30 ml, 1,71 mmol, 2.2 EQ.). Then was added DEPC (0,20 ml, 1,17, 1.5 EQ.) and the mixture was stirred in an argon atmosphere. The reaction was completed after 1 hour, as it was shown by HPLC. A mixture of koncentrira is whether obtaining oil and was purified by chromatography on SiO 2(on a column of size 300 × 25 mm), elwira 100%EtOAc. The product was isolated as a white foamy solid. Yield: 0.65 g (87%). ES-MS m/z 968,35 [M+H]+, 991,34 [M+Na]+; UV λmax215, 265 nm.

Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene chloride (5 ml) was treated with diethylamine (2 ml) and the mixture was stirred at room temperature for 2 h then the reaction was completed, as it was shown by HPLC, and the reaction mixture was concentrated to obtain an oil, was dissolved in 2 ml DMSO and injectively in preparative HPLC column (column With12-RP, 5 μm, 100A, linear gradient of MeCN in water (containing 0.1% TFA), 40 minutes at 10-100%, and then 20 minutes at 100%, at a flow rate of 25 ml/min). The fractions containing the product is evaporated and getting salt triperoxonane acid as a white powder. Output: 0.126 g (98%). Rf0,28 (100% EtOAc); ES-MS m/z 746,59 [M+H]+, 768,51 [M+Na]+; UV λmax215 nm.

Example 7 to Obtain compound 6

Trifenatate salt of compound 5 (0.11 g, 0.13 mmol), compound AB (0,103 g, 0.14 mmol, 1.1 EQ.) and HOBt (3.4 mg, 26 μmol, 0.2 EQ.) suspended in DMF/pyridine (2 ml/0.5 ml, respectively). Then add diisopropylethylamine (to 22.5 μl, 0.13 mmol, 1.0 EQ.) and the yellow solution was stirred in an argon atmosphere. After 3 hours was added 1.0 EQ. DIEA. After another 24 hours the reaction mixture dobavlyali,5 EQ. activated linker. The reaction was completed after 40 hours. The contents of the vessel evaporated, dissolved in DMSO and was injectively in preparative HPLC column (column With12-RP, 5 μm, 100A, linear gradient of MeCN in water (containing 0.1% TFA), 40 minutes at 10-100%, and then 20 minutes at 100%, at a flow rate of 50 ml/min). The desired fractions evaporated and received the product as a yellow oil. Then was added methylene chloride (approx. 2 ml) and an excess of ether, resulting in the received connection 6 in the form of a white precipitate, which was filtered and dried. Yield: 90 mg (52%). ES-MS m/z 1344,32 [M+H]+, 1366,29 [M+Na]+; UV λmax215, 248 nm.

Example 8 to Obtain compound 7

Compound 4 (133 mg, 0.13 mmol, 1 EQ.), connection AV (123 mg, 0,167 mmol, 1.1 EQ.) and HOBt (4 mg, 0.2 EQ.) was diluted in DMF (1.5 ml). After 2 minutes, was added pyridine (5 ml) and monitored the reaction by using RP-HPLC. It was found that the reaction was completed after 18 hours the Reaction mixture was diluted with dichloromethane (20 ml) and successively washed with 10%aqueous citric acid (2 × 10 ml), water (10 ml) and saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The obtained residue resuspendable in dichloromethane and purified flash chromatography on silica gel in a stepwise gradient of 0-10% Meon in dichloromethane. The appropriate fractions were combined and who has koncentrirebuli to obtain compound 7 as a white foam: 46 mg (21%). Rf0,15 (10% Meon/CH2Cl2). ES-MS m/z 1476,94 [M+H]+.

Example 9 to Obtain tert-butyl ether MC-val-cit-PAB-MMAF 8

Connection 1 (83 mg, 0.11 mmol), compound AB (85 mg, 0.12 mmol, 1.1 EQ.) and HOBt (2.8 mg, 21 μmol, 0.2 EQ.) was dissolved in dry DMF (1.5 ml) and pyridine (0.3 ml) in an argon atmosphere. After 30 hours the reaction was essentially complete, as it was shown by HPLC. Then the mixture was evaporated, dissolved in minimum amount of DMSO and purified preparative HPLC (column12-RP, 5 μm, 100A, linear gradient of MeCN in water (containing 0.1% TFA), 40 minutes at 10-100%, and then 20 minutes at 100%, at a flow rate of 25 ml/min) to give compound 8 as a white solid. Yield: 103 mg (71%). ES-MS m/z 1387,06 [M+H]+, 1409,04 [M+Na]+; UV λmax205, 248 nm.

Example 10 to Obtain the MC-val-cit-PAB-MMAF 9

Compound 8 (45 mg, 32 µmol) suspended in methylene chloride (6 ml)and then was added TFA (3 ml). The resulting solution was kept for 2 hours the Reaction mixture was concentrated in vacuo and was purified preparative HPLC (column12-RP, 5 μm, 100A, linear gradient of MeCN in water (containing 0.1% TFA), 40 minutes at 10-100%, and then 20 minutes at 100%, at a flow rate of 25 ml/min). The desired fractions were concentrated and received maleimidomethyl-valine-citrulline-p-hydroxyethylaminophenol-MMAF (MC-val-it-PAB-MMAF) 9 in the form of not-quite-white solid. Yield: 11 mg (25%). ES-MS m/z 1330,29 [M+H]+, 1352,24 [M+Na]+; UV λmax205, 248 nm.

Example 11 to Obtain tert-butylamide MC-val-cit-PAB-MMAF 10

Connection 3 (217 mg, 0.276 mmol, 1.0 EQ.), connection AV (204 mg, 0.276 mmol, 1.0 EQ.) and HOBt (11 mg, 0,0828 mmol, 0.3 EQ.) was diluted with a mixture of pyridine/DMF (6 ml). To this mixture was added DIEA (0,048 ml) and the mixture was stirred for about 16 hours. Volatile organic compounds evaporated in vacuum. The crude residue was purified using a Chromatotron® (radial thin-layer chromatography with a stepwise gradient 0-5-10% methanol in DHM obtaining tert-butylamide MC-val-cit-PAB-MMAF 10. Yield: 172 mg (45%). ES-MS m/z 1386,33 [M+H]+, 1408,36 [M+Na]+; UV λmax215, 248 nm.

Example 12 to Obtain AS-MS-MMAE by conjugation AS with MC-MMAE

AS, dissolved in 500 mm sodium borate and 500 mm sodium chloride, at pH 8.0, was treated with an excess of 100 mm dithiothreitol (DTT). After incubation at 37°C for 30 minutes, the buffer was replaced by elution of the resin Sephadex G25 and suirable PBS with 1 mm DTPA. The amount of thiol/Ab was assessed by determining the concentration of the recovered antibodies when the optical density of the solution of 280 nm to determine concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determine the concentration at optical density of 412 nm. The recovered antibody dissolve the built in PBS, cooled on ice.

Reagent “drug-linker”, maleimidomethyl-monomethylaniline E, i.e. the MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to chilled recovered antibody AS in PBS. After approximately one hour to extinguish the reaction for kupirovaniya” any unreacted thiol group of the antibody was added excess maleimide. The reaction mixture was concentrated by ultrafiltration centrifuge and AS-MC-MMAE was purified and absoluely by elution G25 resin in PBS, then filtered through 0.2 μm filters under sterile conditions, and frozen for subsequent storage.

Example 13-Getting AS-MC-MMAF by conjugation AS with MC-MMAF

AS-MC-MMAF was prepared by conjugation AS with MC-MMAF in accordance with the procedure described in example 12.

Example 14-Getting AS-MC-val-cit-PAB-MMAE by conjugation AS with MC-val-cit-PAB-MMAE

AS-MC-val-cit-PAB-MMAE was obtained by conjugation AS with MC-val-cit-PAB-MMAE in accordance with the procedure described in example 12.

Example 15 to Obtain AS-MC-val-cit-PAB-MMAF by conjugation AS with MC-val-cit-PAB-MMAF (9)

AS-MC-val-cit-PAB-MMAF was prepared by conjugation AS with MC-val-cit-PAB-MMAF (9) in accordance with the procedure described in example 12.

Example 16 Determination of cytotoxicity of the selected the response

Cytotoxic activity of MMAF and compounds 1-5 can be estimated on the Lewis Y-positive cell lines OVCAR-3 and Lewis Y-positive cell lines breast carcinoma N, carcinoma L2987 lung and carcinoma LS174t colon. To assess the cytotoxicity of compounds 1-5 cells can be seeded at a density of approximately 5-10000 per well in 150 μl of culture medium, and then at the beginning of the analysis, they can be treated with appropriate doses of compounds 1-5 with four repetitions. Tests for cytotoxicity is usually conducted within 96 hours after addition of test compounds. At the end of cultivation in the last 4-6 hours of incubation in each well can be added 50 μl resazurin dye to assess the viability of the cells. Recovery of the dye can be determined using fluorescence spectrometry at a wavelength of excitation and emission wavelength of 535 nm and 590 nm, respectively. To analyze the degree of recovery of resazurin treated cells can be compared with the degree of recovery of resazurin untreated control cells.

1-hour tests for cytotoxicity of the analyzed cells were treated with drug for 1 hour, and then washed; resulting in a cytotoxic effect can be defined p is after incubation for 96 hours.

Example 17 Analysis onin vitrocytotoxicity of selected compounds

Table 10 illustrates the cytotoxic effect SAS-conjugates and compounds 7-10 on CD30+cell line Karpas 299, and analyzed as described in General procedure 1. Data is shown for two separate experiments. It was found that SAS-conjugates and compounds 7-9 have a slightly higher activity than the conjugate SAS-val-cit-MMAE.

Table 10
ConjugateIC50(ng/ml)
SAS-val-cit-MMAE6
SAS-71,0
SAS-815
CAS-90,5
SAS-1020

In other experiments, the conjugate BR96-val-cit-MMAE had at least 250 times more efficient than the free MMAF.

General procedure I-Determination of cytotoxicity. To assess the cytotoxicity representative conjugates 7-10 cells were seeded at a density of approximately 5-10000 per well in 150 μl of culture medium, and then n is the beginning of the analysis they were treated with appropriate doses representative conjugates 7-10 with four repetitions. Tests for cytotoxicity conducted within 96 hours after addition of test compounds. At the end of cultivation, in the last 4-6 hours of incubation, each well can be added 50 μl resazurin dye to assess the viability of the cells. Recovery of the dye was determined by fluorescence spectrometry at a wavelength of excitation and emission wavelength of 535 nm and 590 nm, respectively. To analyze the degree of recovery of resazurin treated cells was compared with the degree of recovery of resazurin untreated control cells.

Example 18 Analysis onin vitrocell proliferation

The efficiency of the ADC can be measured by analysis of cell proliferation in accordance with the following Protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488).

1. An aliquot of 100 μl of cell culture containing about 104cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in the environment, besieged in each well of 96-well plate with opaque walls.

2. Received control wells containing medium not containing cells.

3. In the experimental wells was added ADC and perform incubation for 3-5 days.

4. The tablets were balanced to room temperature for about 30 minutes.

5. To each well was added volume to reage is the CellTiter-Glo, equal volume of cell present culture medium.

6. The contents of the wells were mixed for 2 minutes on an orbital shaker to induce cell lysis.

7. Tablet incubated at room temperature for 10 minutes to stabilize the luminescent signal.

8. Luminescence was recorded and represented in the graphs as RLU=relative luminescence units.

Example 19-Excretion of conjugates and antibodies from the plasma of rats

The pharmacokinetics of excretion of conjugates of the antibody-drug” and total antibodies from plasma was studied on rats Sprague-Dawley (Charles River Laboratories, the body weight of each rat was 250-275 g). Animals were administered a booster dose by injection into the tail vein (IV injection). Approximately 300 μl of whole blood was collected using a cannula through the jugular vein or by caudal puncture in a vessel with lithium/anticoagulant heparin through the following intervals: 0 (prior to dose), after 10 and 30 minutes; 1, 2, 4, 8, 24, and 36 hours; and through 2, 3, 4, 7, 14, 21 and 28 days after administration of the dose. The total titer of antibodies was measured using ELISA using ECD/GxhuFc-HRP. The level of conjugate “antibody-drug” was measured by ELISA using MMAE/MMAF/ECD-Bio/SA-HRP.

Example 20-Excretion of conjugates and antibodies from the plasma of monkeys

Pharmacokinetics elimination of PLA is we conjugates of the antibody-drug” and the total number of antibodies can be tested for abacadabra monkeys. On Fig illustrated two-stage study of the excretion concentrations of plasma after the introduction of the H-MC-vc-MMAE abacadabra monkeys in different doses: 0,5; 1,5; 2,5; and 3.0 mg/kg on days 1 and 21. Concentrations of total antibody and ADC was measured in dependence on time (N=trastuzumab).

Example 21 Determination of tumor volumein vivoin transgenic mice with Explant

Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, NY). For these experiments are suitable animals of many species, but are preferred female FVB mice, because they are most sensitive to the formation of tumors. Male FVB can be used for mating and bred population vasectomysearch mice CD.1 can be used for stimulation of pseudopregnant. Vasectomiesriver mouse can be obtained from any commercially available source. Animals progenitors can be crossed with either FVB mice or mice 129/BL6 x FVB, heterozygous for p53. Mice heterozygous for p53 allele, can be used as a potential animals with increased ability to form tumors. Some tumors F1 come from mixed species. Tumor mice progenitors can be only of type FVB.

The animal is, having tumor (allograft, cultured transgenic mmtv-mice Fo5), can be processed by one or more doses of the ADC by intravenous (i.v.) the injection. Tumor volume can be determined through different periods of time after injection.

Example 22 Synthesis of MC-MMAF through tert-butyl methyl ether

Synthesis 1:

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, of 128.6 mg, 0,163 mmol) suspended in CH2Cl2(0,500 ml). After this was added 6-maleimidomethyl acid (68,9 mg, 0,326 mmol) and 1,3-diisopropylcarbodiimide (0,0505 ml, 0,326 mmol)and then pyridine (0,500 ml). The reaction mixture was left for 1 hour for mixing. HPLC analysis indicated complete depletion of the parent compound 1. Volatile organic compounds evaporated under reduced pressure. The product was isolated using column flash chromatography in a step gradient of 0-5% methanol in CH2Cl2. They received a total of 96 mg of pure MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (yield 60%). ES-MS m/z 981,26 [M+H]+; 1003,47 [M+Na]+; 979,65 [M-H]-.

MC-MeVal-Val-Dil-Dap-Phe-OtBu (compound 12, 74 mg, 0,0754 mmol) suspended in CH2Cl2(2.0 ml) and TFA (1 ml) at room temperature. After 2.5 hours, HPLC analysis indicated complete depletion of the parent compound. Volatile organic compounds evaporated under reduced pressure and the product was isolated with p the power preparative RP-HPLC on a column of Phenomenex C 12Synergi Max-RP 80A (250 × 21,20 mm). Eluent: a linear gradient of 10%-90% MeCN/0.05% of TFA (water.) within 30 minutes, and then socrata a mixture of 90% MeCN/0.05% of TFA (water.) within another 20 minutes. ES-MS m/z 925,33 [M+H]+; 947,30 [M+Na]+; 923,45 [M-H]-.

Example 23a-Synthesis of MC-MMAF (11) through dimethoxybenzoyl ether

Synthesis of 2:

Obtaining Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (Fmoc-Phe-ODMB)

In a three-neck 5-liter round bottom flask was loaded with Fmoc-L-phenylalanine (200 g, 516 mmol, Bachem), 2,4-dimethoxybenzyl alcohol (95,4 g, 567 mmol, Aldrich) and CH2Cl2(2.0 l). To the resulting suspension over 20 minutes in an atmosphere of N2added tert-butylacetyl N,N-dimethylformamide (155 ml, 586 mmol, Fluka), which formed a clear solution. Then the reaction mixture was stirred at room temperature overnight, after which TLC analysis (0,42, heptane/EtOAc=2:1) indicated complete reaction. The reaction mixture was concentrated under reduced pressure and obtained light yellow oil, which was again dissolved in CH2Cl2(200 ml) and was purified on a short column of silica gel (25 cm × 25 cm, CH2Cl2) to obtain a colorless foam (250 g). To the resulting foam was added MeCN (1 l), which completely dissolved the party of the reaction mixture. The mixture is then concentrated to dryness and the ova was dissolved in MeCN (1 l) and the resulting suspension was stirred for 1 hour and filtered, then the filter cake washed with MeCN (2 × 200 ml) and received Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid (113,58 g, 41%, 95.5% of AUC according to HPLC). Data: HPLC.

Obtaining L-phenylalanine-2,4-dimethoxybenzyl ester (Phe-ODMB)

In a 500-ml round bottom flask was loaded with Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ether (26,00 g, to 48.3 mmol), CH2Cl2(150 ml) and diethylamine (75 ml, Acros). The mixture was stirred at room temperature and passing the reaction was monitored by HPLC. After 4 hours the mixture was concentrated (bath temperature <30°C). The remainder resuspendable in CH2Cl2(200 ml) and concentrated. This procedure was repeated one more time. To the residue was added Meon (20 ml), which led to the formation of gel. This residue was diluted with CH2Cl2(200 ml), concentrated and turbid oil was kept under vacuum overnight. The residue is suspended in CH2Cl2(100 ml)and then added toluene (120 ml). The mixture was concentrated and the residue was kept under vacuum overnight. Data: HPLC,1H-NMR.

Obtaining Fmoc-delaplain (Fmoc-Dap)

Vos-delaplain (58,8 g, 0,205 mol) is suspended in 4 BC HCl in 1,4-dioxane (256 ml of 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC analysis indicated the completion of reaction (10% Meon/CH2Cl2), and the mixture kontsentrirovaniya dry. Then downloaded another 50 ml of 1,4-dioxane and the mixture was concentrated to dryness and dried in vacuum over night. The obtained white solid was dissolved in H2O (400 ml) and was transferred into a three-liter three-neck round-bottom flask equipped with a mechanical stirrer and a temperature sensor. Then within 1 minute was added N,N-diisopropylethylamine (214,3 ml of 1.23 mol, Acros), which resulted in a temperature increase of 20.5 28.2°C (internal). The mixture was cooled in an ice bath was added 1,4-dioxane (400 ml). After that, through the addition funnel over 15 minutes was added a solution of Fmoc-OSu (89,90 g, 0,267 mol, Advanced ChemTech) in 1,4-dioxane (400 ml), maintaining the temperature of the reaction mixture below 9°C. the Mixture was left to warm to room temperature and was stirred for 19 hours, after which the mixture was concentrated on a rotary evaporator to obtain aqueous suspensions (390 g). This suspension was diluted with H2O (750 ml) and Et2O (750 ml), which resulted in a copious white precipitate. The layers were separated, maintaining the solids together with the organic layer. The aqueous layer was acidified with concentrated HCl (30 ml) and was extracted with EtOAc (3 × 500 ml). The combined extracts were dried over MgSO4, filtered and concentrated to obtain 59,25 g yellow oil A. Extract Et2O once was extracted with saturated NaHCO3(200 ml), maintaining the firm in the society together with the water layer. The aqueous suspension was acidified with concentrated HCl (50 ml) and was extracted with Et2O (50 ml), maintaining the solids together with the organic layer. The organic layer was filtered and concentrated to obtain 32,33 g yellow oil Century. Two substances in the form of oil (a and b) were combined and purified using flash chromatography on silica gel, elwira CH2Cl2(3.5 l), and then 3% Meon/CH2Cl2(9 l), resulting in a received 68,23 g Fmoc-delaplaine in the form of a white foam (81%, purity of 97.5%, HPLC (AUC)).

Obtaining Fmoc-Dap-Phe-ODMB

Untreated Phe-ODMB (48,3 mmol) suspended in anhydrous DMF (105 ml, Acros) for 5 minutes and then was added Fmoc-Dap(19,80 g, to 48.3 mmol). The mixture was cooled in an ice bath was added TBTU (17,08 g, 53,20 mmol, Matrix Innovations). Then within 3 minutes, the syringe was added N,N-diisopropylethylamine (25,3 ml, 145,0 mmol, Acros). After 1 hour the ice bath was removed and the mixture was left for 30 minutes to heat. Then the mixture was poured into water (1 l) and extracted with ethyl acetate (300 ml). After separation of the layers the aqueous layer was again extracted with ethyl acetate (2 × 150 ml). The combined organic layers were washed with saturated salt solution (150 ml), dried (MgSO4) and was filtered through filter paper) to remove nerastvorim substances (inorganic chemicals and a number of dibenzofuran). After concentration the residue(41 g) adsorbing on silica (41 g) was purified by chromatography (column (22 cm × 8 cm; 65% heptane/EtOAc (2.5 l); 33% heptane/EtOAc (3.8 l), resulting in scores 29.4 g of the product as a white foam (86%, purity 92%, HPLC).

Data: HPLC,1H-NMR, TLC (1:1 EtOAc/heptane, Rf=0,33, vanillic red).

Getting Dap-Phe-ODMB

In a 1-liter round bottom flask was loaded with Fmoc-Dap-Phe-ODMB (27,66 g), CH2Cl2(122 ml) and diethylamine (61 ml, Acros). The solution was stirred at room temperature and passing the reaction was monitored by HPLC. After 7 hours the mixture was concentrated (bath temperature <30°C). The remainder resuspendable in CH2Cl2(300 ml) and concentrated. This procedure was repeated twice. To the residue was added Meon (20 ml) and CH2Cl2(300 ml) and the solution was concentrated. The residue is suspended in CH2Cl2(100 ml) and toluene (400 ml), then concentrated and the residue was kept under vacuum during the night, resulting in a received creamy residue.

Data: HPLC,1H-NMR, MS.

Obtaining Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB

UntreatedDap-Phe-ODMB (39,1 mmol) suspended in anhydrous DMF (135 ml, Acros) for 5 minutes and then was added Fmoc-MeVal-Val-Dil-HE (24,94 g, 39,1 mmol, see connection example 2). The mixture was cooled in an ice bath was added TBTU (13,81 g, 43,0 mmol, Matrix Innovations). Then within 2 minutes, the syringe was added N,N-diisopropylethylamine (20.5 ml, 117,3 mmol, Acros). After 1 hour the ice bath delete the Lee and the mixture was left for 30 minutes to heat. Then the mixture was poured into water (1.5 l) and was diluted with ethyl acetate (480 ml). After standing for 15 minutes the layers were separated and the aqueous layer was extracted with ethyl acetate (300 ml). The combined organic layers were washed with saturated salt solution (200 ml), dried (MgSO4) and was filtered through filter paper) to remove nerastvorim substances (inorganic chemicals and a number of dibenzofuran). After concentration the residue (49 g) is scraped from the flask walls and adsorbing on silica (49 g), and then purified by chromatography (column, 15 cm × 10 cm; 2:1 EtOAc/heptane (3 l); EtOAc (5 l); 250 ml fractions), resulting in a received 31,84 g of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB in the form of a white foam (73%, purity 93%, HPLC (AUC)).

Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0,21, vanillic red).

Getting MeVal-Val-Dil-Dap-Phe-ODMB

In a 1-liter round bottom flask was loaded with Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB (28,66 g), CH2Cl2(80 ml) and diethylamine (40 ml). The solution was stirred at room temperature overnight, and then concentrated under reduced pressure. The residue was adsorbing on silica (30 g)and then purified on a flash chromatography (column, 15 cm × 8 cm; 2% Meon/DHM (2 l), 3% Meon/GHM (1 l), 6% Meon/DHM (4 l); 250 ml fractions), resulting in a received 15,88 g MeVal-Val-Dil-Dap-Phe-ODMB in the form of a white foam (69%, purity 96%, HPLC (AUC)).

Dunn is e: HPLC, TLC (6% Meon/DHM, Rf=0,24, vanillic red).

Getting MC-MeVal-Val-Dil-Dap-Phe-ODMB

In a 50-ml round bottom flask was loaded MeVal-Val-Dil-Dap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4 ml), maleimidomethyl acid (180 mg, 0.85 mmol) and TBTU (300 mg, of 0.93 mmol, Matrix Innovations) at room temperature. Then the syringe was added N,N-diisopropylethylamine (450 μl, 2.57 mmol). After 1.5 hours the mixture was poured into water (50 ml) and was diluted with ethyl acetate (30 ml). To facilitate separation of added NaCl. After separation of the layers the aqueous layer was extracted with ethyl acetate (25 ml). The combined organic layers were dried (MgSO4), filtered and concentrated. The oil obtained (1 g) was purified flash chromatography (100 ml silica; 25% heptane/EtOAc (100 ml), 10% heptane/EtOAc (200 ml), EtOAc (1.5 l)), resulting in the received MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg, 57%, purity 94%; HPLC (AUC)).

Data:1H-NMR, HPLC.

Getting MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF)(11)

In a 50-ml round bottom flask was loaded MC-MeVal-Val-Dil-Dap-Phe-ODMB (compound 13, 428 mg, 0,39 mmol) was dissolved in 2.5% TFA/CH2Cl2(20 ml). After 2 minutes the solution had acquired a pinkish-purple color. Reaction monitoring was performed using HPLC and TLC (6% Meon/DHM, KMnO4-staining). After 40 minutes was added three drops of water and cloudy pinkish-purple mixture was concentrated to obtain mg pink residue. After purification by chromatography (15% IPA/DHM) received 270 mg MC-MMAF (73%, purity 92%, HPLC) as a white solid.

Example 23b-Synthesis of analogue mc-MMAF

MeVal-Val-Dil-Dap-Phe-OtBu (connection 1, 35 mg, 0,044 mmol) suspended in DMF (0,250 ml). After this was added 4-(2,5-dioxo-2,5-dihydropyrrol-1-yl)benzoic acid (11 mg, 0,049 mmol) and HATU (17 mg, 0,044 mmol), and then DIEA (0,031 ml of 0.17 mmol). This reaction mixture was left for 2 hours for mixing. HPLC analysis showed complete depletion of the parent compound 1.

The product was isolated using preparative RP-HPLC on a column with a Phenomenex C12Synergi Max-RP 80A (250 × 21,20 mm). Eluent: a linear gradient of 10%to 80% MeCN/0.05% of TFA (water.) within 8 minutes, then socrata mixture of 80% MeCN/0.05% of TFA (water.) for a further 12 minutes. Was allocated a total of 20 mg of the pure product (14) (0.02 mmol, yield 46%). ES-MS m/z 987,85 [M+H]+; 1019,41 [M+Na]+; 985,54 [M-H]-.

MV-MeVal-Val-Dil-Dap-Phe-OtBu (compound 14, 38 mg, 0,0385 mmol) suspended in CH2Cl2(2.0 ml) and TFA (1 ml). The mixture was stirred for 2 hours, and then volatile organic compounds evaporated under reduced pressure. The product was purified using preparative RP-HPLC on a column with a Phenomenex C12Synergi Max-RP 80A (250 × 21,20 mm). Eluent: a linear gradient of 10%to 80% MeCN/0.05% of TFA (water.) within 8 minutes, and then socrata a mixture of 90% MeCN/0.05% of TFA (water.) for a further 12 minutes. It was kind of the Leno just 14.4 mg of MC-MMAF-product (0.015 mmol, yield 40%). ES-MS m/z 930,96 [M+H]+, 952,98 [M+Na]+; 929,37 [M-H]-.

Example 23C-Receiving MC-MeVal-Cit-PAB-MMAF (16)

To a suspension of Fmoc-MeVal-OH (3.03 g, to 8.57 mmol) and N,N'-diskrimineerimata (3,29 g, 12,86 mmol) in CH2Cl2(80 ml) at room temperature was added DIEA (4,48 ml, 25,71 mmol). This reaction mixture was left for 3 hours stirring, and then poured into a separating funnel and the organic mixture was extracted with 0.1m HCl (water.). The crude organic residue was concentrated under reduced pressure and the product was isolated by column flash chromatography on silica gel using a linear gradient of 20-100% ethyl acetate/hexane. They received a total of 2.18 g of pure Fmoc-MeVal-OSu (4,80 mmol, yield 56%).

To a suspension of Fmoc-MeVal-OSu (2,18 g, 4,84 mmol) in DME (13 ml) and THF (6.5 ml) at room temperature was added a solution of L-citrulline of 0.85 g, 4,84 mmol) and NaHCO3(0,41 g, 4,84 mmol) in H2O (13 ml). The suspension was left for 16 hours at room temperature for mixing, and then was extracted with a mixture of tert-BuOH/CHCl3/H2O and acidified using 1M HCl to a pH of 2.3. The organic phase was separated, dried and concentrated under reduced pressure. The residue is triturated with diethyl ether and received a 2.01 g of Fmoc-MeVal-Cit-COOH, which was used without further purification.

The crude Fmoc-MeVal-Cit-COOH suspended in 2:1 CH2l 2/Meon (100 ml) and to this suspension was added n-aminobenzoyl alcohol (0.97 g, 7.9 mmol) and EEDQ (1,95 g, 7.9 mmol). This suspension was left to 125 hours for mixing, and then the volatile organic substances were removed under reduced pressure and the residue was purified column flash chromatography on silica gel using 10% Meon/CH2Cl2. The result has been pure Fmoc-MeVal-Cit-RAV-HE (0.55 g, 0,896 mmol, yield of 18.5%), ES-MS m/z 616,48 [M+H]+.

To a suspension of Fmoc-MeVal-Cit-RAV-HE (0.55 g, 0,896 mmol) in CH2Cl2(40 ml) was added STRATOSPHERESTM(related piperazine-resin) (>5 mmol/g, 150 mg). After stirring at room temperature for 16 hours the mixture was filtered through celite (pre-washed Meon) and concentrated under reduced pressure. The residue is triturated with diethyl ether and hexane. The obtained solid substance, MeVal-Cit-RAV-HE, suspended in CH2Cl2(20 ml), and then to the suspension was added MC-OSu (0.28 g, 0,896 mmol), DIEA (0.17 ml, 0,99 mmol) and DMF (15 ml). This suspension was stirred for 16 hours, but GVHD analysis of the reaction mixture indicated incomplete reaction, and therefore the suspension was concentrated under reduced pressure to a volume of 6 ml, and then added a 10% solution of NaHCO3(water) and the suspension was stirred for another 16 hours. The solvent was removed under reduced pressure and the residue was purified colon is manual flash chromatography on silica gel in a gradient of 0-10% Meon/CH 2Cl2in the result that was obtained 42 mg (0,072 mmol, yield 8%) MC-MeVal-Cit-RAV-HE.

To a suspension MC-MeVal-Cit-RAV-HE (2.37 g, 4.04 mmol) and bis(nitrophenyl)carbonate (2,59 g, charged 8.52 mmol) in CH2Cl2(10 ml) was added DIEA (of 1.06 ml, the 6.06 mmol). This suspension was stirred for 5.5 h, then concentrated under reduced pressure and was purified by trituration with diethyl ether. MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0,196 mmol) suspended in a solution of pyridine/DMF (1:5) (3 ml) and to the suspension was added HOBt (5 mg, 0,039 mmol), DIEA (0.17 ml, 0,978 mmol) and MMAF (compound 2, 150 mg, 0,205 mmol). This reaction mixture was stirred for 16 hours at room temperature, and then was purified preparative RP-HPLC (× 3) on a column with a Phenomenex C12Synergi Max-RP 80A (250 × 21,20 mm). Eluent: a linear gradient of 10%-90% MeCN/0.05% of TFA (water.) within 30 minutes, then socrata a mixture of 90% MeCN/0.05% of TFA (water.) within another 20 minutes. MC-MeVal-Cit-PAB-MMAF (16) was obtained as a yellowish solid (24.5 mg, 0,0182, the output of 0.45%). ES-MS m/z 1344,95 [M+H]+; 1366,94 [M+Na]+.

Example 23d-Receiving Succinimidyl suberin-Val-Cit-PAB-MMAF (17)

Compound I (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, or 0.57 mmol, 1.5 EQ.) suspended in anhydrous pyridine, then added 5 ml HOBt (10 mg, 0,076 mmol, 0.2 EQ.) and DIEA (199 μl, to 1.14 mmol, 3 EQ.). The reaction mixture was treated with ultrasound for 10 minutes, and then stirred p. and room temperature over night. The pyridine was removed under reduced pressure and the residue resuspendable in CH2Cl2. The mixture was separated on a flash chromatography on silica gel in a stepwise gradient Meon, from 0 to 10%, in CH2Cl2. The fractions containing the product were combined, concentrated, dried in vacuum over night and received 317 mg (yield 59%) of Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m/z 1415,8 [M+H]+.

Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20% TFA/CH2Cl2(10 ml) for 2 hours. The mixture was diluted with CH2Cl2(50 ml). The organic layer was sequentially washed with water (2 × 30 ml) and saturated salt solution (1 × 30 ml). The organic phase was concentrated and loaded on a layer of silica gel in 10% Meon/CH2Cl2. The product was suirable 30% Meon/CH2Cl2. After drying in vacuum over night received Fmoc-Val-Cit-PAB-MMAF in the form of a white solid, 38 mg, yield 40%. ES-MS m/z 1357,7 [M-H]-.

Fmoc-Val-Cit-PAB-MMAF, 67 mg, suspended in CH2Cl2(2 ml), diethylamine (2 ml) and DMF (2 ml). The mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure. The residue is evaporated with pyridine (2 ml)and then with toluene (2 × 5 ml) and dried in vacuum. Val-Cit-PAB-MMAF was received in the form of a brown oil and used without further purification.

All Val-Cit-PAB-MMAF, obtained from 67 mg of Fmoc-Val-Cit-PAB-MMAF, suspended in pyridine (2 ml) and add the Yali to a solution of disuccinimidyl (74 mg, 0.2 mmol, 4 equiv.) in pyridine (1 ml). The reaction mixture was stirred at room temperature. After 3 hours was added ether (20 ml). The precipitate was collected and washed with additional ether. Reddish solid is suspended in 30% Meon/CH2Cl2and filtered through a layer of silica gel using as eluent 30% Meon/CH2Cl2. Compound 17 was obtained as a white solid (20 mg, yield 29%). ES-MS m/z 1388,5 [M-H]-.

Example 24-In vivothe effectiveness of mcMMAF-conjugates of the antibody-drug”

Efficiency SAS-mcMMAF in ALCL-xenografts Karpas-299. To assessin vivothe effectiveness of the conjugate SAS-mcMMAF, containing, on average, 4 molecules of the drug to the antibody (SAS-mcF4), humanALCLcells Karpas-299 subcutaneously implanted in immunodeficient mice SV-17 SCID (5 × 106cells per mouse). Tumor volume was calculated according to the formula (0,5 × L × W2), where two bi-directional measurements one of L and W is longer and the other shorter. When the average tumor volume of the investigated animals reached approximately 100 mm3(within 48 to 162), the mice were divided into 3 groups (5 mice per group), and these mice either were not subjected to processing or into the tail vein did a single intravenous injection of 1 mg/kg or 2 mg/kg CAS-mcF4 (figure 1). Tumor neobrabotannim mice grew rapidly, and 7 days after start of the experiment, their average amount was reached >1000 mm3. In contrast, all SAS-mcF4-treated mice was observed rapid decrease in tumor size, 3 of 5 mice group that received a dose of 1 mg/kg, and 5 of 5 mice group that received a dose of 2 mg/kg, there was complete antitumor response. One of the mice with absolute twicemonthly in the group that received a dose of 2 mg/kg, was observed tumor recurrence after about 4 weeks after beginning therapy, but the rest of the mice of this group, i.e. in 4 out of 5 mice, were not found to have any detectable tumors, and 3 mice with absolute twicemonthly in the group that received a dose of 1 mg/kg, tumors were not observed 10 weeks after initiation of therapy.

EfficiencycBR96-mcMMAF in NSCLC-xenografts L2987.cBR96 is a chimeric antibody that recognizes the antigen LeY. To assessin vivothe effectiveness of the conjugate cBR96-mcMMAF, containing 4 molecules of the drug to the antibody (cBR96-mcF4), Nude (naked) mice implanted fragments of tumor non-small cell lung cancer (NSCLC) L2987. When the average tumor volume reached approximately 100 mm3mice were divided into 3 groups: one group that was not subjected to processing, and 2 groups of treatment. For therapy, as shown nafiga, mice every 4 days, was introduced cBR96-mcF4, at a dose of 3 mg/kg or 10 mg/kg/injection, total of 4 injections (q4d×4). As shown in fig.3b, mice, every 4 days, was introduced cBR96-mF4 or nesvanulica control conjugate SAS-mcF4 at a dose of 10 mg/kg/injection, total of 4 injections (q4d×4). As shown in figa and 3b, BR96-mcF4 provided significant slowing of tumor growth compared to control.

Figure 2 illustrates thein vivoanalysis on the effectiveness of one dose SAS-mcMMAF in subcutaneous L540CY. These studies were performed on 4 mice untreated group and 10 mice in each group processing.

Example 25-In vitrothe efficiency of MC-MMAF-conjugates of the antibody-drug”

The activity of conjugates “SAS-antibody-drug” against CD30+cell lines. On figa and 16b presents curves dose-response, constructed according to a representative experiment in which cell culture Karpas-299 (both anaplastic lymphoma) and L428 (jackinsky lymphoma) were incubated with serially diluted SAS-mcMMAF (figa) or SAS-vcMMAF (fig.4b) within 96 hours. Cultures were labeled with 50 μm of resazurin [10-oxide of 7-hydroxy-3H-phenoxazin-3-it] for 4 hours and measured the fluorescence intensity. These data were transformed using the program GraphPad Prism version 4.00 in accordance with the procedure of constructing a 4-parameter kr is the howl of the dose-response. The value of the IC50was defined as the concentration at which cell growth was reduced by 50% compared with untreated control culture. Each concentration was tested with four repetitions.

The activity of conjugates “cBR96the antibody-drug” againstLeY+cell lines. On figa and 5b presents curves dose-response, constructed according to a representative experiment in which cell culture N (breast carcinoma) and L2987 (non-small cell lung carcinoma) were incubated with serially diluted cBR96-mcMMAF (figa) or cBR96-vcMMAF (fig.5b) within 96 hours. Cultures were labeled with 50 μm of resazurin for 4 hours and measured the fluorescence intensity. These data were transformed using the program GraphPad Prism version 4.00 in accordance with the procedure of constructing a 4-parameter curve dose-response. The value of the IC50was defined as the concentration at which cell growth was reduced by 50% compared with untreated control culture. Each concentration was tested with four repetitions.

The activity of conjugates of the antibodyc1F6-drug” against CD70+cell lines of renal carcinoma. On figa and 6b presents Kryvyi “dose-response, constructed according to a representative experiment in which culture cells Caki-1 and 786-O incubi is ovali with serially diluted c1F6-mcMMAF (figa) or c1F6-vcMMAF (fig.6b) within 96 hours. Cultures were labeled with 50 μm of resazurin for 4 hours and measured the fluorescence intensity. These data were transformed using the program GraphPad Prism version 4.00 in accordance with the procedure of constructing a 4-parameter curve dose-response. The value of the IC50was defined as the concentration at which cell growth was reduced by 50% compared with untreated control culture. Each concentration was tested with four repetitions.

Example 26-Cleaning trastuzumab

The contents of one vessel, comprising 440 mg of the antibody Herceptin® (huMAb4D5-8, rhuMAb HER2, U.S. patent No. 5821337), was dissolved in 50 ml of MES buffer (25 mm MES, 50 mm NaCl, pH 5,6) and loaded on a cation exchange column (sepharose S, 15 cm × 1.7 cm), which is then balanced in the same buffer. Then the column was washed with the same buffer (5 column volumes). Trastuzumab was suirable by adjusting the NaCl concentration of the buffer up to 200 mm. The fractions containing the antibody were pooled, diluted to 10 mg/ml were dialyzed in a buffer containing 50 mm potassium phosphate, 500 mm NaCl, 2 mm EDTA, pH 6.5.

Example 27 to Obtain conjugate trastuzumab-MC-MMAE by conjugation of trastuzumab with MS-MAE

Trastuzumab, dissolved in 500 mm sodium borate and 500 mm sodium chloride at pH 8.0, was treated with an excess of 100 mm dithiothreitol (DTT). After incubation at 37º within 30 minutes, the buffer was replaced by the ay elution of the resin Sephadex G25, and then suirable PBS with 1 mm DTPA. The amount of thiol/Ab was assessed by determining the concentration of the recovered antibodies when the optical density of the solution of 280 nm and the concentration of the thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determine the concentration at optical density of 412 nm. The recovered antibody dissolved in PBS, cooled on ice.

Reagent “drug-linker”, maleimidomethyl-monomethylaniline E (MMAE), i.e., MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to chilled recovered antibody trastuzumab in PBS. After approximately one hour to extinguish the reaction for kupirovaniya” any unreacted thiol group of the antibody was added excess maleimide. The reaction mixture was concentrated by ultrafiltration centrifuge and trastuzumab-MC-MMAE was purified and absoluely by elution G25 resin in PBS, and then filtered through 0.2 μm filters under sterile conditions, and frozen for subsequent storage.

Example 28 to Obtain the conjugate trastuzumab-MC-MMAF by conjugation of trastuzumab with MC-MMAF

Conjugate trastuzumab-MC-MMAF was prepared by conjugation of trastuzumab with MC-MMAF in accordance with the procedure of example 27.

Example 29 to Obtain the conjugate trastuzumab-MC-val-cit-RAV-MMAE by conjuga the Finance of trastuzumab with MC-val-cit-RAV-MAE

Conjugate trastuzumab-MC-val-cit-RAV-MMAE was obtained by conjugation of trastuzumab with MC-val-cit-RAV-MAE in accordance with the procedure of example 27.

Example 30 to Obtain the conjugate trastuzumab-MC-val-cit-RAV-MMAF by conjugation of trastuzumab with MC-val-cit-RAV-MMAF9

Conjugate trastuzumab-MC-val-cit-RAV-MMAF was prepared by conjugation of trastuzumab with MC-val-cit-RAV-MMAF9in accordance with the procedure of example 27.

Example 31-Toxicity for rats

Profile acute toxicity of the free drug and the ADC was evaluated in young rats, Sprague-Dawley (weight 75-125 grams each, Charles River Laboratories (Hollister, CA). Animals were injected injection on day 1 and then on day 0, day 3 and day 5 performed a full chemical and hematological tests, and on the 5th day performed a full autopsy. All animals were measured levels of enzymes in the liver, and for three arbitrarily taken animals of each group were performed routine histological analysis of the following tissues: chest, liver, kidney, thymus, spleen, colon and small intestine. The experimental groups were the following groups:

For conjugates of trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-RAV-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-RAV-MMAF value µg MMAF/m2was calculated using weight (Mw) MMAF equal 731,5, and mass (Mw) of Herceptin, equal 14516.

The surface area was calculated according to the following formula: [{(weight in grams to the extent 0,667) × 11,8}/10000] (Guidance for Industry and Reviewers, 2002).

Dose solution was injected intravenously into the tail vein in a single loading dose on day 1 of the study at a dose of 10 ml/kg Before the introduction of dose on day 1 of the study and after each day of its introduction was measured body weight of the animals. Whole blood was collected in EDTA-containing tubes for hematological analysis. Whole blood was collected in tubes-separators serum for clinical biochemical analyses. Blood samples were taken before administration of the dose on study day -4, day 3 and day 5. At necropsy, whole blood was also collected in tubes containing sodium heparin, and plasma was frozen at -70°C for further analysis. At necropsy, were taken and placed in neutral buffered formalin tissue following organs: liver, kidneys, heart, thymus, spleen, brain, chest and sections of the gastrointestinal tract, including the stomach and small and large intestine. Also conducted research in the chest, small intestine, large intestine, liver, thymus, spleen and kidneys.

The levels of serum enzymes of the liver in each time period was within, compared with their levels in normal female rats, Sprague-Dawley (5 and 95 percentiles). The number of leukocytes and trombi the comrade in each time period was within, compared with those in normal female rats, Sprague-Dawley (5 and 95 percentiles).

Studies of high doses was performed on normal rats Sprague-Dawley for the following groups:

Group 1:Media
Group 2:trastuzumab-MC-MMAF, 52,24 mg/kg, 4210 mg/m2
Group 3:trastuzumab-MC-MMAF, 68,25 mg/kg 5500 mg/m2
Group 4:trastuzumab-MC-MMAF, of 86.00 mg/kg, 6930 g/m2

In 11 animals was routine histological analysis of the tissue. These animals participated in the research phase to the limits of an acute dose-dependent toxicity immunoconjugate trastuzumab-MC-MMAF. Animals were monitored for 12 days after administration of the dose.

Example 32-Toxicity/safety for abacadabra monkeys

Three groups of four abacadabra monkeysMacaca fascicularic(2 males and 2 females)that were not previously in uchastvovashih experiments investigated the effect of conjugates of trastuzumab-MC-vc-RAV-MMAE and trastuzumab-MC-vc-RAV-MMAF. Intravenous held on the 1st and the 22nd day of research.

Group
SampleDose
Media1
1M/1F
Day 1
Day 22
H-MC-vc-RAV-MAE2
2M/2F
180 g/m2(0.5 mg/kg) on day 1
1100 g/m2(3.0 mg/kg) on day 22
H-MC-vc-RAV-MAE3
2M/2F
550 mg/m2(1.5 mg/kg) on day 8
550 mg/m2(1.5 mg/kg) on day 29
H-MC-vc-RAV-MAE4
2M/2F
880 mg/m2(2.5 mg/kg) on day 15
880 mg/m2(2.5 mg/kg) on day 36

SampleGroupDose
Media1
1M/1F
Day 1
Day 22
H-MC-vc-RAV-MMAF2
2M/2F
180 g/m2(0.5 mg/kg) on day 1
1100 g/m2(3.0 mg/kg) on day 22
H-MC-vc-RAV-MMAF3
2M/2F
550 mg/m2(1.5 mg/kg) on day 1
550 mg/m2(1.5 mg/kg) on day 22
H-MC-vc-RAV-MMAF4
2M/2F
880 mg/m2(2.5 mg/kg) on day 1
880 mg/m2(2.5 mg/kg) on day 22

H=trastuzumab

The dose was expressed as the surface area of the animal's body, in order to match the doses administered to the animals of other species, because the dose expressed in mg/m2not depends on the type of animal, and therefore doses for different animals can be compared. Drugs ADC contained PBS, 5.4 mm of sodium phosphate, 4.2 mm potassium phosphate, 140 mm sodium chloride, pH 6.5.

For hematological analysis blood was taken after 5 minutes, 6 hours, 10 hours and after 1, 3, 5, 7, 14 and 21 days after administration of each dose. The number of erythrocytes (RBC) and platelets (PLT) were measured by the light scattering method. The number of leukocytes (WBC) were measured by the method using peroxidase/basophils. The number of reticulocytes were measured by the light scattering method using a cationic dye. The number of cells was measured with the apparatus of the Advia 120. The level of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) was measured in units/l using UV/NADH, methodology, developed by the International Federation of clinical chemistry (IFCC) and carried out on the apparatus Olympus AU400, and the total titer of antibodies was measured using ELISA using ECD/GxhuFc-HRP. The level of Ab-conjugate was measured using ELISA tests-MMAE/MMAF/ECD-Bio/SA-HRP.

Example 3-Production, characterization and humanization of monoclonal antibodies 4D5 against ErbB2

Mouse monoclonal antibody 4D5, which specifically binds to the extracellular domain of ErbB2, was produced as described in Fendly et al. (1990) Cancer Research 50:1550-1558. Briefly, cells NIH-T/HER2-3400(expressing approximately 1 × 105the ErbB2 molecules/cell)produced as described in Hudziak et al. Proc. Natl. Acad. Sci. (USA) 84:7159-7163 (1987)), was collected using a phosphate buffered saline (PBS)containing 25 mm EDTA and used for immunization of BALB/c mice. These mice I.P. Pavlova. introduced injections of 107cells in 0.5 ml PBS at 0-, 2-, 5 - and 7-th week. Mice with anticorodal, which contained as immunoprecipitate32P-labeled ErbB2, at 9 and 13 weeks were injected I.P. Pavlova. injection membrane extract ErbB2, purified using agglutinin wheat germ (WGA)- sepharose. And then were injected i.v. injection of 0.1 ml of ErbB2 and splenocytes were subjected to fusion with mouse myeloma line H-Ad. Supernatant hybridoma was skanirovali binding with ErbB2 using ELISA and radioimmunoprecipitation.

Mapping and characterization of epitopes

The epitope of ErbB2 associated with monoclonal antibody 4D5, characterized by analyzing competitive binding (Fendly et al.,Cancer Research50:1550-1558 (1990)). The study of cross-blocking spent is by direct measurement of fluorescence on intact cells using skaniruyushchego device PANDEX TMto quantify fluorescence. Monoclonal antibody conjugatively with fluoresceinisothiocyanate (FITZ) in accordance with well known procedures (Wofsy et al. Selected Methods in Cellular Immunology, p.287, Mishel & Schiigi (eds.) San Francisco: W.J. Freeman Co. (1980)). Confluently monolayers of cells NIH-T/HER2-3400was trypsinization, once washed and resuspendable with a 1.75 × 106cells/ml in cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN3. In order to avoid clogging of the membranes tablets PANDEXTMwas added to a final concentration of 1% latex particles (IDC, Portland, OR). In wells PANDEXTMwas added to the cell suspension, 20 μl, and 20 μl of purified monoclonal antibodies (100 µg/ml 0.1 µg/ml) and the mixture incubated on ice for 30 minutes. To each well was added a pre-defined breeding FITZ-labeled monoclonal antibody in 20 μl, were incubated for 30 minutes, washed, and the fluorescence intensity was quantitatively determined by PANDEXTM. It was believed that monoclonal antibodies have a common epitope if each antibody blocks the binding of another antibody by 50% or more compared with irrelevant control monoclonal antibody. In this experiment, monoclonal antibody 4D5 was attributed to the epitope I (amino acid residues from about 529 to 625, including the remains of Valeto the aqueous domain of ErbB2).

The tumor-inhibiting properties of monoclonal antibody 4D5 was assessed in cell lines of breast cancer, SK-BR-3 (see Hudziak et al. (1989) Molec. Cell. Biol. 9(3):1165-1172). Briefly, cells SK-BR-3 were separated with the use of 0.25% (vol./about.) trypsin and suspended in complete medium at a density of 4 × 105cells / ml. Aliquots of 100 μl (4 × 104cells) were seeded in 96-well plates to microdesmidae, then the cells were left for adhesion, after which was added 100 μl of the same medium or medium containing monoclonal antibody (final concentration 5 µg/ml). After 72 hours the tablets twice washed with PBS (pH 7.5), stained with crystal violet (0.5% in methanol) and analyzed for the relative cell proliferation, as described by Sugarman et al., (1985) Science 230:943-945. Monoclonal antibody 4D5 inhibited relative cell proliferation of SK-BR-3 of about 56%.

Monoclonal antibody 4D5 was also evaluated for its ability to inhibit HRG-stimulated tyrosine phosphorylation of proteins withMrabout 180000, in whole lysates of MCF7 cells (Lewis et al. (1996) Cancer Research 56:1457-1465). It was reported that MCF7 cells Express all known ErbB receptors, but at relatively low levels. Because ErbB2, ErbB3 and ErbB4 have almost the same molecular weight, when carrying out a Western blot analysis of cell lysates impossible to determine which is the first of these proteins fosfauriliruetsa on tyrosine. However, these cells are ideal for analyses on HRG-stimulated phosphorylation on tyrosine as used in analytical conditions, in the absence of exogenously added HRG, these cells produce low or redetection levels of phosphorylated by cirasino proteins withMrabout 180000.

The MCF7 cells were sown in 24-hole plates and each well was added monoclonal antibodies against ErbB2, and then incubation was performed for 30 minutes at room temperature, and then to each well was added rHRGβ1177-244to a final concentration of 0.2 nm, and the incubation was continued for 8 minutes. Then from each well was carefully sucked out of the environment and the reaction was stopped by adding 100 μl of the LTO-sample buffer (5% LTOs, 25 mm DTT, and 25 mm Tris-HCl, pH 6.8). Each sample (25 µl) were subjected to electrophoresis in 4-12% gradient gel (Novex), and then electrophoresis was transferred to the membrane from polyvinylidenedifluoride. Immunoblot antibodies against phosphotyrosine (4G10, UBI used in a concentration of 1 μg/ml) showed and the intensity of the predominant reactive band atMr=180000 quantitatively evaluated using reflection densitometry, as described in the literature (Holmes et al. Science, 256:1205-1210 (1992); Sliwkowski et al. J. Biol. Chem., 269(20):14661-14665 (1994)).

Monoclonal antibody 4D5 was found significant and generowanie generate HRG-induced signal phosphorylation of tyrosine at Mr180000. In the absence of HRG was not any stimulation of tyrosine phosphorylation of proteins by massMrabout 180000. In addition, this antibody did not engage in cross-react with EGFR (Fendly et al.,Cancer Research50:1550-1558 (1990)), ErbB3 or ErbB4. Monoclonal antibody 4D5 had the ability to block HRG-stimulation of tyrosine phosphorylation by 50%.

The tumor-inhibiting properties of monoclonal antibody 4D5 was evaluated on cells MDA-MB-175 and SK-BR-3 in the presence or in the absence of exogenous rHRGβ1 (see Schaefer et al. Oncogene 15:1385-1394 (1997)). The levels of ErbB2 in cells MDA-MB-175 were 4-6 times higher than the levels found in normal epithelial breast cells, and the receptor ErbB2-ErbB4 cells MDA-MB-175 is constitutive fosfauriliruet on tyrosine. Monoclonal antibody 4D5 able to inhibit cell proliferation of MDA-MB-175 in the presence and in the absence of exogenous HRG. The inhibition of proliferation of cells under the action of 4D5 depends on the level of ErbB2 expression (Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). The maximum detectable inhibition of cells SK-BR-3 was 66%. However, a greater effect can be achieved with the use of exogenous HRG.

Mouse monoclonal antibody 4D5 was humanitarno using the strategy of “gene conversion mutagenesis”as described in U.S. patent No. 5821337, which in all its floor the OTE is introduced into the present description by reference. Gumanitarnoe monoclonal antibody 4D5 used in the following experiments, was marked huMAb4D5-8. This antibody is of the IgG1 isotype.

Cited works

Scope of the present invention is not limited to specific variants of its implementation, described in the examples which are presented merely to illustrate several aspects of the present invention, and its scope includes any functionally equivalent variants. Indeed, in addition to the modifications that have been illustrated and described in this application, the present invention may be made and various other modifications, obvious to the experts, and such modifications are also included in the scope of the attached claims.

All work cited here in its entirety and for all purposes of practical application are entered into the present description by reference as if each individual publication, patent or patent application were specifically and individually entered in its entirety and for all purposes of practical application by reference.

1. The compound having the formula DF:

or pharmaceutically acceptable salt of the compound DF,
where independently each position:
R2selected from the group comprising H and-C1-C8alkyl;
R3selected from g is uppy, including the-N and-C1-C8alkyl;
R4selected from the group comprising H and-C1-C8alkyl;
R5selected from the group comprising-H and methyl;
R6selected from the group comprising H and-C1-C8alkyl;
R7selected from the group comprising H and-C1-C8alkyl;
R8represents-O-(C1-C8alkyl);
R9selected from the group comprising H and-C1-C8alkyl;
R10represents a C6-C10aryl;
Z represents O or NH;
R11selected from the group comprising-H, -C1-C20alkyl, C6-C10aryl and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents-C2-C8-alkyl;
R14represents-H or-C1-C8alkyl.

2. The compound according to claim 1, having the formula:

or its pharmaceutically acceptable salt.

3. The compound or its pharmaceutically acceptable salt according to any one of claims 1 to 2 in isolated and purified form.

4. Conjugate having the formula:
L-(LU-DF)P
or its pharmaceutically acceptable salt;
where L represents a ligand component;
the linker component (LU) has the formula:
-Aa-Ww-Yy-,
where-A - is an extension component,
but ili 1,
each W independently represents an amino acid component,
w is an integer from 0 to 12,
-Y - is a spacer elements component
y is 0, 1 or 2; and p is from 1 to about 20;
DFhas the formula:

or its pharmaceutically acceptable salt,
where the wavy line in the DFspecify the site of covalent binding ligand component L is selected from the group comprising a protein, polypeptide and peptide through a linker component LU;
independently at each position:
R2selected from the group comprising H and C1-C8alkyl;
R3selected from the group comprising H and C1-C8alkyl;
R4selected from the group comprising H and C1-C8alkyl;
R5selected from the group consisting of H and methyl;
R6selected from H and C1-C8of alkyl;
R7selected from the group comprising H and C1-C8alkyl;
R8represents O-(C1-C8alkyl);
R9selected from the group comprising H and C1-C8alkyl;
R10represents a C6-C10aryl;
Z represents O or NH;
R11selected from the group including H, C1-C20alkyl, C6-C10aryl and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents a C2/sub> -C8-alkyl;
R14represents N or C1-C8alkyl.

5. Conjugate having the formula:
LU-DF
or its pharmaceutically acceptable salt,
where-LU - is a linker component;
where the linker component (LU) contains;
-Aa-Ww-Yy-,
where-A - is an extension component,
and is 0 or 1,
each W independently represents an amino acid component,
w is an integer from 0 to 12,
-Y - is a spacer elements component, and
y is 0, 1 or 2;
DFhas the formula:

where the wavy line in the DFspecify the site of covalent binding of the linker component of the LU, and
independently at each position:
R2selected from the group comprising H and C1-C8alkyl;
R3selected from the group comprising H and C1-C8alkyl;
R4selected from the group comprising H and C1-C8alkyl;
R5selected from the group consisting of H and methyl;
R6selected from H and C1-C8of alkyl;
R7selected from the group comprising H and C1-C8alkyl;
R8represents O-(C1-C8alkyl);
R9selected from the group comprising H and C1-C8alkyl;
R10represents a C6-C10aryl;
Z represents O or NH;
R11/sup> selected from the group including H, C1-C20alkyl, C6-C10aryl and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents a C2-C8-alkyl;
R14represents N or C1-C8alkyl.

6. The conjugate according to claim 4, where the conjugate is a conjugate of the antibody-drug", having the formula Ia':

or its pharmaceutically acceptable salt,
where Ab represents the antibody
-Aa-Ww-Yy- is a linker component,
But is an extension component,
and is 0 or 1,
each W independently represents an amino acid component,
w is an integer from 0 to 12,
Y is a spacer elements component
y is 0, 1 or 2, and
p is in the range from 1 to about 20.

7. Conjugate the antibody-drug" according to claim 6 where each of a, w and y, or a is 1 and each of w and y is 0
or its pharmaceutically acceptable salt.

8. Conjugate the antibody-drug" according to claim 6 having the formula:

or its pharmaceutically acceptable salt,
where L is an antibody and R17represents a C1-C10alkylene-, -C3-C8carbocycle-, -O-(C1-C8alkyl)-, -who rylan-, -C1-C10alkalinuria-, -aralen-C1-C10alkylene-, -C1-C10alkylene-(C3-C8-carbocycle)-, -(C3-C8carbocycle)-C1-C10alkylene-, -C3-C8heterocycle-, -C1-C10alkylene-(C3-C8heterocycle)-, -(C3-C8heterocycle)-C1-C10alkylene-, -(CH2CH2O)r- and - (CH2CH2O)r-CH2-; and r is an integer from 1 to 10.

9. Conjugate the antibody-drug" 6 or its pharmaceutically acceptable salt, in which the antibody is attached to the linker component or connection DFthrough a cysteine residue of the antibody.

10. Conjugate the antibody-drug" according to claim 9 or its pharmaceutically acceptable salt, in which p is equal to from 2 to 5.

11. Conjugate the antibody-drug" according to claim 9, having the formula:

or its pharmaceutically acceptable salt,
where L is an antibody and R17represents a C1-C10alkylene-, -C3-C8carbocycle-, -O-(C1-C8alkyl)-, -aralen-, -C1-C10alkalinuria-, -aralen-C1-C10alkylene-, -C1-C10alkylene-(C3-C8-carbocycle)-, -(C3-C8carbocycle)-C1-C10alkylene-, -C3-C8 heterocycle-, -C1-C10alkylene-(C3-C8heterocycle)-, -(C3-C8heterocycle)-C1-C10alkylene-, -(CH2CH2O)r- and -(CH2CH2O)r-CH2-; and r is an integer from 1 to 10.

12. Conjugate the antibody-drug" of claim 8 having the formula:

or its pharmaceutically acceptable salt.

13. Conjugate the antibody-drug" according to claim 11 having the formula:

or its pharmaceutically acceptable salt,
where each of w and y is equal to 0.

14. Conjugate the antibody-drug" 6 or its pharmaceutically acceptable salt, where w is an integer from 2 to 12.

15. Conjugate the antibody-drug" 14 or its pharmaceutically acceptable salt, where w is 2.

16. Conjugate the antibody-drug" 15 or its pharmaceutically acceptable salt, where Wwrepresents-valine-citrulline-, -phenylalanine-lysine - or-N-methylvaline-citrulline-.

17. Conjugate the antibody-drug" under item 8, or its pharmaceutically acceptable salt, where Wwrepresents a 5-aminovaleric acid, homophenylalanine lysine, tetraethylammonium lysine, cyclohexylamin lysine, isonipecotic acid lysine, beta is Lanin lysine, glycine series valine glutamine or isonicotinoyl acid.

18. Conjugate the antibody-drug" under article 16, having the formula:

or its pharmaceutically acceptable salt.

19. Conjugate the antibody-drug" under article 16, having the formula:

or its pharmaceutically acceptable salt.

20. Conjugate the antibody-drug" under article 16, having the formula:

or its pharmaceutically acceptable salt.

21. Conjugate the antibody-drug" on any of PP-8 and 12-20, where DFhas the formula:

or its pharmaceutically acceptable salt.

22. Conjugate the antibody-drug" 6 or its pharmaceutically acceptable salt, where the antibody is selected from the group consisting of monoclonal antibodies;
especifismo antibodies, chimeric antibodies, gumanitarnogo antibodies, diately and antibody fragment.

23. Conjugate the antibody-drug" according to claim 6 having the formula:

or

or its pharmaceutically acceptable salt,
where Ab is an antibody, Val is a valine and Cit is citrulline.

24. Conjugate the antibody-drug" item 23 and the and its pharmaceutically acceptable salt, where the antibody binds to CD30, CD20, CD33, or antigen Lewis y

25. Pharmaceutical composition for treating cancer containing an effective amount of a conjugate according to any one of claims 4 and 6-24 or its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent, carrier or filler.

26. The conjugate according to any one of claims 4 and 6-24 or its pharmaceutically acceptable salts in the manufacture of a medicinal product for the treatment of cancer.

27. Use p where the cancer is a lymphoma.

28. Use p where the cancer is a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colon, thyroid, pancreatic, prostate or bladder.

29. A method of treating cancer, introducing a number of conjugate "antibody-drug" according to any one of claims 4 and 6-24 or its pharmaceutically acceptable salt, where the specified number is an effective treatment for cancer, the antibody conjugate "antibody-drug" binds to the antigen expressed by a cancer cell.

30. Use p where the cancer is a cancer of the breast.

31. Use p where the cancer is a cancer of the kidneys.

32. Conjugate or pharmaceutically acceptable salt according to any one of the p.4-24 in isolated and purified form.

33. Conjugate the antibody-drug"containing the antibody is covalently attached to one or more molecules of the drug, where the conjugate antibody-drug" has the formula Ic:

or its pharmaceutically acceptable salt,
where Ab is an antibody that binds to one or several associated with tumor antigens (1)-(35):
(1) BMPR1B (the receptor protein of bone morphogenesis type IB);
(2) E16 (LAT1, SLC7A5);
(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate);
(4) 0772P (CA125, MUC16);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);
(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 family soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II);
(7) Sema 5b (FLJ10372, KIAA1445; Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5);
(8) PSCA nlg (2700050C12Rik, C530008016Rik, RIKEN cDNA S, cDNA gene RIKEN S);
(9) ETBR (endothelin receptor type b);
(10) MSG783 (RNF124, hypothetical protein FLJ20315);
(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony TRANS is membrany epithelial antigen of prostate 2, testimony transmembrane protein of the prostate);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, derived from teratocarcinoma);
(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/receptor of the virus of Epstein-Barr) or Hs.73792);
(15) CD79b (IGb (beta protein associated with immunoglobulin), V);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C);
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20Rα;
(21) Brevican;
(22) Ephb2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R;
(27) CD22;
(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha protein);
(29) CXCR5 (receptor 1 lymphoma, Burkitt's lymphoma);
(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes);
(31) RG (ion channel 5, an opening purinergic ligand receptor RH);
(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2);
(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I);
(34) FCRH1 (Fc receptor-like protein: 1); and
(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily, 2),
But is an extension component,
and is 0 or 1,
each W independently represents, own the th amino acid component,
w is an integer from 0 to 12,
Y is a spacer elements component
y is 0, 1 or 2,
p is in the range from 1 to about 20, and
D is a molecule drugs selected from the group consisting of formulas DEand DF:

where the wavy line in the DEand DFindicates the sites of covalent joining A, W or Y, and independently at each position:
R2selected from the group comprising H and C1-C8alkyl;
R3selected from the group comprising H and C1-C8alkyl;
R4selected from the group comprising H and C1-C8alkyl;
R5selected from the group consisting of H and methyl;
R6selected from the group comprising H and C1-C8alkyl;
R7selected from the group comprising H and C1-C8alkyl;
R8independently selected from the group including H, HE, C1-C8alkyl and O-(C1-C8alkyl);
R9selected from the group comprising H and C1-C8alkyl;
R10represents a C6-C10aryl;
Z represents O or NH;
R11selected from the group including H, C1-C20alkyl, C6-C10aryl, and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents a C2-C8-alkyl;
R14the present is the focus of a N or C 1-C8alkyl; and
R18represents-C(R8)2-C(R8)2-C6-C10aryl.

34. Conjugate the antibody-drug" p, in which D has the formula DE:

or its pharmaceutically acceptable salt.

35. Conjugate the antibody-drug" p, in which D has the formula DF:

or its pharmaceutically acceptable salt.

36. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody is attached to a molecule drugs through a cysteine residue of the antibody.

37. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where p is from 1 to 4.

38. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where p is from 2 to 8.

39. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where p is from 2 to 5.

40. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt.

41. Conjugate the antibody-drug" p, where each of w and y is 0, having formula:

or f is rmaceuticals acceptable salt.

42. Conjugate the antibody-drug" paragraph 41 or its pharmaceutically acceptable salt, where D has the formula DE.

43. Conjugate the antibody-drug" under § 42, in which the formula DEhas the formula:

or its pharmaceutically acceptable salt.

44. Conjugate the antibody-drug" paragraph 41 or its pharmaceutically acceptable salt, where D has the formula DF.

45. Conjugate the antibody-drug" item 44, in which the formula DFhas the formula:

or its pharmaceutically acceptable salt.

46. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt.

47. Conjugate the antibody-drug" p.46, having the formula:

or its pharmaceutically acceptable salt.

48. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt.

49. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where D has the formula DE.

50. Conjugate the antibody-drug" § 49, in which the formula DEhas the formula:
or its pharmaceutically acceptable salt.

51. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, in which D has the formula DF.

52. Conjugate the antibody-drug" under § 51, in which formula DFhas the formula:

or its pharmaceutically acceptable salt.

53. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where w is an integer from 2 to 12.

54. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where w is 2.

55. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where Wwrepresents-valine-citrulline-.

56. Conjugate the antibody-drug" p, in which D is selected from the group consisting of the formulas:

and

or its pharmaceutically acceptable salt.

57. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody specifically binds to the HER2 receptor.

58. Conjugate the antibody-drug" § 57 or its pharmaceutically acceptable salt, which specifically binds to the extracellular domain of the HER2 receptor and inhibits R. the art of tumor cells, which sverkhekspressiya receptor HER2.

59. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody is selected from the group consisting of monoclonal antibodies, especifismo antibodies, chimeric antibodies, gumanitarnogo antibodies and fragments of antibodies.

60. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody fragment is a Fab fragment.

61. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where humanitariannet antibody selected from the group consisting of huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

62. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody is a huMAb4D5-8 (trastuzumab).

63. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt,
where Val is a valine and Cit is citrulline.

64. Conjugate the antibody-drug" p or its pharmaceutically acceptable salt, where the antibody is a huMAb4D5-8 (trastuzumab).

65. Pharmaceutical composition for treating cancer containing an effective amount conjuga is as "antibody-drug" on any of PP-64 or its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent, the carrier or filler.

66. The way of lysis or inhibition of proliferation of tumor or cancer cells, including the treatment of tumor or cancer cells by the number of conjugate "antibody-drug" on any of PP-64, where the specified number is effective for lysis or inhibition of proliferation of tumor or cancer cells.

67. A method of treating cancer, comprising introducing quantity conjugate the antibody-drug" on any of PP-64, where the specified amount is effective to treat cancer.

68. The method according to p where the cancer is selected from the group consisting of breast cancer, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colon, thyroid, pancreatic, prostate and bladder.

69. The method according to p where the cancer is a lymphoma.

70. The method according to p where lymphoma is nahodkinskuju lymphoma.

71. Method of inhibiting cell proliferation, including:
treatment of mammalian cells in cell culture medium conjugate the antibody-drug" on any of PP-64 or its pharmaceutically acceptable salt and
measurement of cytotoxic activity of the conjugate the antibody-drug or its pharmaceutically acceptable salt.

72. the manual treatment of cancer, including the introduction of an effective amount of the composition of the conjugate the antibody-drug" on any of PP-64 or its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent, carrier or filler.

73. The method according to item 72, where the conjugate antibody-drug or its pharmaceutically acceptable salt specifically binds to a receptor encoded by the genome EbrB2.

74. The method according to item 72, in which the number of conjugate "antibody-drug or its pharmaceutically acceptable salt, administered to the patient, is in the range from about 0.1 to about 10 mg/kg of body weight of the patient.

75. The method according to item 72, where the conjugate antibody-drug or its pharmaceutically acceptable salt is introduced at intervals of about three weeks.

76. The method according to item 72, where the specified conjugate antibody-drug or its pharmaceutically acceptable salt is prepared together with a pharmaceutically acceptable parenteral carrier, and is administered parenterally.

77. The method according to item 72, wherein said conjugate the antibody-drug or its pharmaceutically acceptable salt is prepared in the form of an injectable form of the drug.

78. The method according to item 72, wherein said conjugate the antibody-drug or its pharmaceutically p is yemlemuyu salt is injected.

79. The method according to item 72, further including the introduction of a second antibody that binds to a tumor-associated antigen selected from (1)to(35):
(1) BMPR1B (the receptor protein of bone morphogenesis type IB);
(2) E16 (LAT1, SLC7A5);
(3) STEAP1 (testimony transmembrane epithelial antigen of the prostate);
(4) R (SA, MUC16);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);
(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 family soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transferring protein 3b type II);
(7) Sema 5b (FLJ 10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, SEMA domain, samedomain repetitions of thrombospondin (type 1 and is similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin) 5B);
(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA S, cDNA gene RIKEN S);
(9) ETBR (endothelin receptor type b);
(10) MSG783 (RNF124, hypothetical protein FLJ20315);
(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, 1 gene associated with prostate cancer protein 1 associated with prostate cancer, testimony transmembrane epithelial antigen of prostate 2, testimony transmembrane protein of the prostate);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of the subfamily M);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor, etc is coming from teratocarcinoma);
(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/receptor of the virus of Epstein-Barr) or Hs.73792);
(15) CD79b (IGb (beta protein associated with immunoglobulin), V);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (fosfatazy anchor protein 1A containing SH2 domain), SPAP1B, SPAP1C);
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20Rα;
(21) Brevican;
(22) Ephb2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R;
(27) CD22;
(28) CD79a (CD79A, CD79α, associated with immunoglobulin alpha protein);
(29) CXCR5 (receptor 1 lymphoma, Burkitt's lymphoma);
(30) HLA-DOB (beta subunit of MHC molecules class II (Ia antigen)that binds peptides and presents them to CD4+T-lymphocytes);
(31) RG (ion channel 5, an opening purinergic ligand receptor RH);
(32) CD72 (antigen CD72 line b-cell differentiation, Lyb-2);
(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein belonging to the family of proteins with leucine rich repeats (LRR), type I);
(34) FCRH1 (Fc receptor-like protein 1); and
(35) IRTA2 (associated with translocation of the receptor of the immunoglobulin superfamily, 2).

80. Method of inhibiting the growth of tumor cells, which sverkhekspressiya tumor-associated antigen, providing for the introduction of conjugate "antibody-drug" on any of PP-64 or its pharmaceutically acceptable salt, which specifically binds to the tumor-associated antigen, and chemotherapeutic the agent who, where each of the specified conjugate the antibody-drug or its pharmaceutically acceptable salt and chemotherapeutic agent are introduced in amounts effective to inhibit the growth of tumor cells.

81. The method according to item 80, where the specified conjugate antibody-drug or its pharmaceutically acceptable salt according tumor cells sensitivity to a given chemotherapeutic agent.

82. The method of treatment of a person susceptible to the disease, or a person who was diagnosed with a disease characterized by overexpression of ErbB2 receptor, where the method includes introducing an effective amount of a combination of conjugate "antibody-drug", or its pharmaceutically acceptable salt according to any one of p-64 and chemotherapeutic agent.

83. Analysis for the detection of cancer cells, including:
treatment of the cells with the conjugate antibody-drug or its pharmaceutically acceptable salt according to any one of p-64 and
determination of the degree of binding of the conjugate to the antibody-drug or its pharmaceutically acceptable salt cells.

84. Analysis p where the cells are tumor cells of the breast.

85. Analysis p, where the degree of binding is determined by measuring the levels of nucleic key is lots encoding the tumor-associated antigen, through fluorescent in situ hybridization (FISH).

86. Analysis p, where the degree of binding determined by immunohistochemical analysis (IHC).

87. Product contains:
conjugate the antibody-drug or its pharmaceutically acceptable salt according to any one of p-64;
container; and
the liner is attached to the packaging, or label indicating that the conjugate antibody-drug or its pharmaceutically acceptable salt can be used to treat cancer.

88. Product by p, where the specified liner in the package or label indicates that the conjugate antibody-drug or its pharmaceutically acceptable salt can be used to treat cancer characterized by overexpression of ErbB2 receptor.

89. Product by p where the cancer is a cancer of the breast.

90. A method of treating cancer in a mammal, where the cancer is characterized by overexpression of ErbB2 receptor (HER2) and is unresponsive or poorly responsive to treatment with an antibody against rb2, and where the method involves the administration to a mammal a therapeutically effective amount of the conjugate the antibody-drug" on any of PP-64 or its pharmaceutically acceptable salt.

91. The conjugate the antibody-Lek is rstone tool" on any of PP-64 or its pharmaceutically acceptable salts in the manufacture of a medicine for lysis or inhibition of proliferation of tumor cells or cancer cells.

92. The conjugate the antibody-drug" on any of PP-64 or its pharmaceutically acceptable salts in the manufacture of medicinal products for the treatment of hyperproliferative disorders.

93. Use p where hyperproliferative violation is a cancer selected from the group consisting of breast cancer, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colon, thyroid, pancreatic, prostate and bladder.

94. Use p where the cancer is a cancer of the breast Jeezy.

95. Use p where the cancer is a lymphoma.

96. The compound having the formula:

where R2selected from the group comprising-H and C1-C8alkyl;
R3selected from the group comprising-H and C1-C8alkyl;
R4selected from the group comprising-H and C1-C8alkyl;
where R5selected from the group consisting of H and methyl;
R6selected from the group comprising-H and C1-C8alkyl;
R7selected from the group comprising-H and C1-C8alkyl;
R8represents O-(C1-C8alkyl);
R9selected from the group comprising-H and C1-C8alkyl;
R10represents a C6-C10aryl;
R11selected from the group comprising-H, C1-C20alkyl, C6-C10aryl and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents-C2-C8-alkyl;
R14represents-H or C1-C8alkyl;
or its pharmaceutically acceptable salt.

97. The compound having the formula:

where independently each position:
R2selected from the group comprising H and-C1-C8alkyl;
R3selected from the group comprising H and-C1-C8alkyl;
R4selected from the group comprising-N =C1-C8alkyl;
where R5selected from the group consisting of H and methyl;
R6selected from the group comprising H and-C1-C8alkyl;
R7selected from the group comprising H and-C1-C8alkyl;
R8represents-O-(C1-C8alkyl);
R9selected from the group comprising H and-C1-C8alkyl;
R10represents a C6-C10aryl;
or its pharmaceutically acceptable salt.

98. The compound having the formula:

where R2selected from the group comprising-H and C1-C8alkyl;
R3selected from the group comprising-H and C1-C8alkyl;
R4selected from the group comprising-H and C1 -C8alkyl;
where R5selected from the group comprising-H and methyl;
R6selected from the group comprising H and-C1-C8alkyl;
R7selected from the group comprising H and-C1-C8alkyl;
R8represents-O-(C1-C8alkyl);
R9selected from the group comprising H and-C1-C8alkyl;
R10represents a C6-C10aryl;
Z represents-O-;
R11selected from the group comprising-H, C1-C20alkyl, C6-C10aryl and -(R13O)m-R14;
m is an integer from 1 to 1000;
R13represents-C2-C8-alkyl;
R14represents-H or C1-C8alkyl;
or its pharmaceutically acceptable salt.

99. The compound according to any one of PP-98 or its pharmaceutically acceptable salt in isolated and purified form.

100. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt, where Val is a valine, and Cit is citrulline.

101. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt.

102. Conjugate the antibody-drug" p having the formula:

or its pharmaceutically acceptable salt.

103. The method according to p where the cancer is a cancer selected from the group comprising breast cancer, ovarian, kidney, prostate and liver.

104. The method according to p where the cancer is a hematopoietic cancer.

105. The method according to p, where hematopoietic cancer is nahodkinskuju lymphoma.

106. Conjugate the antibody-drug" on item 23, where the antibody is associated with CD70.

107. The compound according to any one of claims 1 and 96-98 or its pharmaceutically acceptable salt, where each R2and R6represents methyl, and R9represents-N.

108. The conjugate according to any one of claims 4 to 20, and 22 or its pharmaceutically acceptable salt, where each R2and R6represents methyl, and R9represents-N.

109. The compound according to any one of claims 1 and 96-98 or its pharmaceutically acceptable salt, where each R2and R6represents methyl, R5represents-H, R7represents sec-butyl, in each case, R8represents-och3and R9represents-N.

110. The conjugate according to any one of claims 4 to 20, 22 and 24, or its pharmaceutically acceptable salt, where each R2and R6represents methyl, R5represents-H, R7represents sec-butyl, in each case, R8 represents-och 3and R9represents-N.

111. Conjugate the antibody-drug" according to claim 6 having the formula:

or its pharmaceutically acceptable salt, where Ab represents the antibody, Val represents valine, and Cit is citrulline.

112. Conjugate the antibody-drug" according to claim 6 having the formula:

or its pharmaceutically acceptable salt, where Ab represents the antibody, Val represents valine, and Cit is citrulline.

113. Conjugate the antibody-drug" according to claim 9 or its pharmaceutically acceptable salt, where p is from 1 to 4.

114. Conjugate the antibody-drug" according to claim 9 or its pharmaceutically acceptable salt, where p is from 2 to 8.

115. The conjugate according to any one of p-42, 44, 46-49, 51, 53-55, 57-62 and 64 or its pharmaceutically acceptable salt, where R2and R6each represents methyl, and R9represents-N.

116. The conjugate according to any one of p-42, 44, 46-49, 51, 53-55, 57-62 and 64 or its pharmaceutically acceptable salt.

117. The compound according to any one of claims 1, 96 and 98, or its pharmaceutically acceptable salt, where R11selected from the group including H, C1-C20alkyl and (R13O)m-R14.

118. The conjugate according to any one of claims 4 to 20, 22 and 24, or its pharmaceutically acceptable with the ü, where R11selected from the group including H, C1-C20alkyl and (R13O)m-R14.



 

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30 tbl, 14 ex

FIELD: medicine, pharmaceutics.

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27 cl, 19 dwg, 8 tbl, 18 ex

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Ox40l antibodies // 2423383

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19 cl, 7 tbl, 23 ex

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26 cl, 26 dwg, 9 ex

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29 cl, 15 dwg, 6 tbl, 9 ex

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60 cl, 18 dwg, 15 tbl, 8 ex

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10 cl, 1 tbl, 2 dwg, 1 ex

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