RU2448117C2 - Monomethylvaline compounds capable of forming conjugates with ligands - Google Patents

Abstract

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

Description

Links to previous applications

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

1. The prior art

The present invention relates to a drug compound, and in particular to drug-linker-ligand conjugates, drug-linker compounds and to drug-ligand conjugates, to compositions comprising the above compounds and conjugates, and to methods for them applications for treating cancer, an autoimmune disease or an infectious disease. The present invention also relates to antibody-drug conjugates, to compositions comprising these conjugates, and to methods of their use for treating cancer, autoimmune disease and infectious disease. The present invention also relates to methods of using the compounds comprising antibody-drug conjugates for in vitro , in situ and in vivo diagnosis or treatment of mammalian cells or for the treatment of pathological conditions.

2. The prior art

For many years, intensive studies have been carried out to improve the delivery of drugs and other agents to cells, tissues and target tumors in order to achieve maximum efficiency with minimal toxicity. Although many attempts have been made to develop effective methods for delivering biologically active molecules to cells, both in vivo and in vitro , none of them have given satisfactory results. Optimizing the conjugation of a drug with its intracellular target, as well as minimizing the intracellular redistribution of the drug, for example, into neighboring cells, often presents certain difficulties or is ineffective.

Most of the modern means parenterally administered to the patient are not targeted, which leads to their systemic delivery to cells and tissues of the body in which the presence of these agents is not required and is often undesirable. As a result, these drugs cause undesirable side effects, which often leads to a limitation of the doses of the drug (e.g., chemotherapeutic (anti-cancer) agent, cytotoxic agent, enzyme inhibitor and antiviral or antimicrobial agents) intended for administration. Although oral administration of drugs, in comparison with parenteral administration, is considered a convenient and economical route of administration, however, in this case, the problem of non-specific toxic effects of the drug, after it enters the circulatory system, on normal cells remains relevant. Other problems arising from oral administration are related to the bioavailability of the administered drug and its retention in the intestine, which leads to an additional effect of the drug on the intestine, and therefore to an increased risk of toxic effects on the intestine. Accordingly, the main objective of the present invention is to develop methods for the specific delivery of agents to cells and tissues. The advantages of this treatment are the ability to prevent the general physiological effects associated with the unwanted delivery of these agents to other cells and tissues, such as uninfected cells. Intracellular delivery can be achieved by methods that provide for the accumulation or presence of active agents, i.e. active metabolites, within the cells.

Monoclonal antibody therapy has been developed for the targeted treatment of patients with cancer, immunological and angiogenic disorders.

Use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, for example, drugs for killing or suppressing 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; US Pat. No. 4,975,278), theoretically allows targeted drug delivery to tumors and their accumulation within cells, while systemic administration of these unconjugated drugs can lead to the production of toxicity levels that are unacceptable for normal cells and also insufficient for tumor cells that need to be destroyed (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 maximum effectiveness is combined with minimal toxicity. It has been reported that both polyclonal and monoclonal antibodies can be used in this strategy (Rowland et al., 1986, Cancer Immunol. Immunother. 21: 183-87). The drugs used in these methods are daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., 1986, see above). The toxins used in antibody-toxin conjugates are bacterial toxins such as diphtheria toxin, plant toxins such as ricin, and small toxin molecules 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), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58: 2928; Hinman et al. (1993) Cancer Res. 53: 3336-3342). Toxins can exert their cytotoxic and cytostatic effects in accordance with mechanisms including binding to tubulin, DNA binding or inhibition of topoisomerase (Meyer DL & Senter PD “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, when conjugated to large antibodies or receptor protein ligands, tend to lose activity or decrease activity.

Zevalin® (ibritumab tiuksetan, Biogen / Idec) is an antibody-radioisotope conjugate consisting of a mouse monoclonal antibody IgG1-kappa directed against the CD20 antigen present on the surface of normal and malignant B-lymphocytes and ¹¹¹ radioactive isotopes In or ⁹⁰ Y bound to the thiourea-linker chelating complex (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27 (7): 766-77; Wiseman et al. (2002) Blood 99 (12 ): 4336-42; Witzig et al. (2002) J. Clin. Oncol. 20 (10): 2453-63; Witzig et al. (2002) J. Clin. Oncol. 20 (15): 3262-69) . Although zevalin has activity against non-Hodgkin’s B-cell lymphoma (NHL), its administration leads to severe and chronic cytopenia in most patients. In 2000, permission was granted for the use of Mylotarg® (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), that is, an antibody-drug conjugate consisting of an anti-human CD33 antibody bound to calicheamicin for the treatment of acute myeloid leukemia by injections of the indicated drug (Drugs of the Future (2000) 25 (7): 686; U.S. Patent Nos. 4,970,198; 5,079,233; 5,585,989; 5,606,040; 5693762, 5,739,116, 5,767,285, 577,3001). Cantuzumab mertansin (Immunogen, Inc.), an antibody-drug conjugate consisting of an anti-human C242 antibody linked via a disulfide linker SPP to a maytansinoid drug, DM1, was used in phase II trials for the treatment of cancers expressing CanAg, such as cancers of the colon, pancreas, stomach, etc. MLN-2704 (Millenium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate consisting of a monoclonal antibody against a membrane-bound prostate antigen (PSMA) attached to a maytansinoid drug, DM1, is under investigation possible use for the treatment of prostate tumors. This maytansinoid drug, DM1, was conjugated to the TA.1 mouse monoclonal antibody via a non-disulfide linker, SMCC (Chari et al. (1992) Cancer Research 52: 127-131). It was reported that said conjugate was 200 times less effective than the corresponding conjugate linked via a disulfide linker. In this work, the SMCC linker is considered “non-splittable”.

Several short-chain peptide compounds have been isolated from the marine mollusk Dolabella auricularia and found to have 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). Analogs of these compounds were also obtained, and it was found that some of them have biological activity (for a review, see Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277). So, for example, auristatin E (US patent No. 5635483) is a synthetic analogue of the natural marine product dolastatin 10, i.e. an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anticancer drug vincristine (GR Pettit (1997) Prog. Chem. Org. Nat. Prod. 70: 1-79). Dolastatin 10, auristatin PE and auristatin E are linear peptides having four amino acids, three of which are unique to compounds of the dolastatin class, and the C-terminal amide.

Auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), i.e. synthetic analogues of dolastatin were conjugated: (i) with chimeric monoclonal antibodies cBR96 (specific for Lewis Y antigen on carcinomas); (ii) with CAC10, which is specific for CD30 present in hematologic 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; published US application 2004/0018194); (iii) with antibodies against CD20, such as rituxan® (WO 04/032828), used to treat CD20-expressing cancers and immune disorders; (iv) with the 2H9 anti-EphB2 antibody and the anti-IL-8 antibody used to treat colon cancer (Mao et al. (2004) Cancer Research 64 (3): 781-788)); (v) with an anti-E-selectin antibody (Bhaskar et al. (2003) Cancer Res. 63: 6387-6394); and (vi) with other anti-CD30 antibodies (WO 03/043583).

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

Contrary to in vitro data obtained in clinical studies of compounds of the class of dolostatins and their analogues, the doses necessary to achieve a therapeutic effect produce significant general toxicity, which reduces their effectiveness. In accordance with this, the need to develop derivatives of dolastatin / auristatin with significantly lower toxicity but valuable therapeutic activity remains relevant. Thus, the present invention seeks to eliminate these and other limitations and problems that still remain unresolved.

ErbB family tyrosine kinase receptors are important mediators of cell growth, differentiation and survival. This receptor family includes four different members, including the epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185 ^neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). An anti-ErbB2 antibody panel was characterized using an SCBR3 human breast tumor cell line (Hudziak et al. (1989) Mol. Cell. Biol. 9 (3): 1165-1172. Maximum inhibition was achieved using an antibody called 4D5, which 56% inhibited cell proliferation. In this assay, other antibodies in this antibody panel reduced cell proliferation to a lesser extent.In addition, it was found that the 4D5 antibody sensitizes ErbB2 overexpressing breast tumor cell lines producing cytotoxicity effects of TNF-α (US Pat. No. 5,677,171). Anti-ErbB2 antibodies discussed by Hudziak et al. have been further characterized by 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. Cell. 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 antibodies having various properties are described by 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 US patent No. 5783186 and Klapper et al. (1997) Oncogene 14: 2099-2109.

Homology screening allowed the identification of two other members of the ErbB receptor family, namely ErbB3 (US patent No. 5183884; US 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 receptors showed increased expression, at least in some breast cancer cell lines.

Herceptin® (trastuzumab) is a recombinant DNA “humanized” monoclonal antibody that, in a cell analysis, selectively and with high affinity (Kd = 5 nM) binds to the extracellular domain of the human epidermal growth factor-2 receptor protein, HER2 (ErbB2) (patent US No. 5821337; US patent No. 6054297; US patent No. 6407213; US patent No. 6639055; Coussens L. et al. (1985) Science 230: 1132-9; Slamon DJ et al. (1989) Science 244: 707-12) . Trastuzumab is an IgG1-kappa antibody that contains the framework regions of a human antibody and the hypervariable regions (complementarity determining regions) of a murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen and thereby inhibits the growth of cancer cells. Since trastuzumab is a humanized antibody, this minimizes any NAMA response in patients. The humanized anti-HER2 antibody was produced in a suspension culture of mammalian cells (Chinese hamster ovary cells, CHO). The HER2 proto-oncogen (or c-erbB2) encodes a 185 kDa transmembrane receptor protein whose structure is akin to the epidermal growth factor receptor structure. Overexpression of the HER2 protein is observed in 25% -30% of primary breast tumors and can be determined by immunohistochemical evaluation of fixed tumor sections (Press MF et al. (1993) Cancer Res. 53: 4960-70). In vitro and animal analyzes have shown that trastuzumab inhibits the proliferation of human tumor cells that overexpress HER2 (Hudziak RM et al. (1989) Mol. Cell. Biol. 9: 1165-72; Lewis GD et al. (1993 ) Cancer Immunol. Immunother; 37: 255-63; Baselga J. et al. (1998) Cancer Res. 58: 2825-2831). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling TE et al. (1996) [abstract]. Proc. Annual. Meeting Am. Assoc. Cancer Res; 37: 471; Pegram MD et al. (1997) [abstract] Proc. Am. Assoc. Cancer Res; 38: 602). In vitro , trastuzumab-mediated ADCC has been shown to be observed predominantly on HER2 overexpressing cancer cells compared to cancer cells that do not exhibit overexpression of HER2. Herceptin® is the only drug indicated for the treatment of patients suffering from breast cancer with metastases that overexpress HER2 protein, and for patients undergoing one or more chemotherapy courses for cancer with metastases. Herceptin® in combination with paclitaxel is indicated for the treatment of patients suffering from breast cancer with metastases that overexpress HER2 protein, and for patients who have not undergone chemotherapy for cancer with metastases. Herceptin® is clinically active in patients with ErbB2 overexpressing cancer metastatic breast tumors, where these patients underwent an intensive course of treatment before anticancer therapy (Baselga et al. (1996) J. Clin. Oncol. 14: 737-744).

A murine monoclonal anti-HER2 antibody inhibits the growth of breast cancer cell lines, in which there is overexpression of HER2 at the levels of 2+ and 3+ (1-2 × 10 ⁶ HER2 receptors per cell), but this antibody did not have activity against cells, which expressed low levels of HER2 (Lewis et al. (1993) Cancer Immunol. Immunother. 37: 255-263). Given these observations, the 4D5 antibody was humanized (huMab4D5-8, rhuMab HER2, US patent No. 5821337; Carter et al. (1992) Proc. Natl. Acad. Sci., USA, 89: 4285-4289) and tested in patients with breast cancers that overexpress HER2 but still progress after standard chemotherapy (Cobleigh et al. (1999) J. Clin. Oncol. 17: 2639-2648).

Although receiving herceptin is an outstanding scientific achievement in treating patients with ErbB2 overexpressing breast cancers that have previously undergone intensive anticancer therapy, some patients in this group did not find any response or showed only a slight response to treatment with herceptin.

Therefore, the need remains to develop other methods of HER2-directed anticancer therapy of patients with HER2-overexpressing tumors or with other diseases associated with HER2 expression that are not or are difficult to treat with herceptin.

Any work cited in this application should not be construed as prototypes of 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 - (A _a --W _w --Y _y -D) _P (Ia)

or their pharmaceutically acceptable salts or solvates,

where: L is a ligand component;

-A _a -W _w -Y _y - represents a linker component (LU), where in the specified linker component:

-A- is an extension component;

equal to 0 or 1,

each of -W- independently represents an amino acid component,

w is an integer from 0 to 12,

-Y- means spacer component

y is 0, 1 or 2;

p ranges from 1 to about 20; and

—D is a “drug” component having the formulas D _E and D _F ;

where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH, where n is an integer from 0 to 6; and

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle).

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

or their pharmaceutically acceptable salts or solvates,

where: R ² selected from hydrogen and C ₁ -C ₈ alkyl;

R ^{3 is} selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle), where R ^{5 is} selected from H and methyl, or R ⁴ and R ⁵ taken together with the carbon atom to which they are attached form a ring of the formula - ( CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle, and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ^{10 is} selected from an aryl group or a C ₃ -C ₈ heterocycle;

Z is —O—, —S—, —NH— or —NR ¹² , where R ¹² is C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH, and

n is an integer from 0 to 6.

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

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

In another aspect, the present invention relates to pharmaceutical compositions comprising an effective amount of a drug-linker compound and a pharmaceutically acceptable carrier or excipient.

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

In another aspect, the present invention relates to methods for lysing or inhibiting the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods for lysing or inhibiting the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate containing a drug component cleaved from said drug drug conjugate ligand agent. "

In yet another aspect, the present invention relates to methods for treating cancer, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

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

In another aspect, the present invention relates to methods for treating cancer, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate containing a drug component that is cleaved from said drug-ligand conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting replication of a cell that expresses an autoimmune antibody, wherein said method comprises administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods for lysing or inhibiting replication of a cell that expresses an autoimmune antibody, wherein said method comprises administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting the replication of a cell that expresses an autoimmune antibody, wherein said method comprises administering to a patient in need thereof an effective amount of a drug-ligand conjugate containing the drug component cleaved from the specified drug-ligand conjugate.

In yet another aspect, the present invention relates to methods for treating an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

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

In another aspect, the present invention relates to methods for treating an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate comprising a drug component that is cleaved from said drug-ligand conjugate.

In yet another aspect, the present invention relates to methods for treating an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

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

In another aspect, the present invention relates to methods for treating an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate comprising a drug component cleavable from said drug-ligand conjugate.

In yet another aspect, the present invention relates to methods for lysing the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

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

In another aspect, the present invention relates to methods for preventing the proliferation of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate comprising a drug component cleavable from said drug-conjugate ligand. "

In yet another aspect, the present invention relates to methods for the prevention of cancer, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods for the prevention of cancer, comprising administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for preventing cancer, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate comprising a drug component that is cleaved from said drug-ligand conjugate.

In yet another aspect, the present invention relates to methods for preventing the proliferation of cells expressing an autoimmune antibody, wherein said methods comprise administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods for preventing the proliferation of cells expressing an autoimmune antibody, wherein said methods comprise administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for preventing the proliferation of cells expressing an autoimmune antibody, wherein said methods comprise administering to a patient in need thereof an effective amount of a drug-ligand conjugate containing the drug component cleavable from said conjugate “ ligand drug. "

In yet another aspect, the present invention relates to methods for preventing an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods for the prevention of an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for the prevention of an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate comprising a drug component that is cleaved from said drug-ligand conjugate.

In yet another aspect, the present invention relates to methods of preventing an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-linker compound.

In another aspect, the present invention relates to methods of preventing an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

In another aspect, the present invention relates to methods for preventing an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-ligand conjugate containing a drug component that is cleaved from said drug-ligand conjugate.

In another aspect, the present invention relates to a drug compound that can be used as an intermediate for the synthesis of a drug-linker compound containing a drug component cleaved from said drug-ligand conjugate.

In another aspect, the present invention relates to a drug-linker compound, which can be used as an intermediate for the synthesis of a drug-linker-ligand conjugate.

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

Ab - (A _a --W _w --Y _y --D) _P (Ia ')

or their pharmaceutically acceptable salts or solvates,

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

A is an extension component;

equal to 0 or 1,

each of -W- independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer component

y is 0, 1 or 2,

p ranges from 1 to about 20; and

D is a “drug” component selected from compounds of formulas D _E and D _F ;

where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH, and

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

n is an integer from 0 to 6.

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

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

(2) E16 (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (six-domain transmembrane prostate epithelial antigen, Genbank reg. No. NM_012449);

(4) 0772P (CA125, MUC16, Genbank reg. No. AF361486);

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

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, member 2 of the family of 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, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorine) 5B, Genbank Reg. No. AB040878);

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

(9) ETBR (type B endothelin receptor, 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, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein of the prostate Genbank Reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of 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 / Epstein-Barr virus receptor), or Hs. 73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), B29, Genbank reg. No. NM_000626);

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

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

(18) NCA (Genbank Reg. No. M18728);

(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, immunoglobulin-bound alpha protein, B-cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with IgM molecules on the cell surface, transmitting a signal involved in the differentiation of B- cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (Burkitt’s lymphoma receptor 1, a G-protein bound receptor activated by chemokine CXCL13, provides lymphocyte migration and humoral defense, and also plays a role in HIV-2 infection and, probably, in the development of AIDS, lymphomas, myelomas and leukemia, Genbank reg. No. NP_001707.1);

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

(31) P2X5 (the -5 ion channel opened by the P2X purinergic receptor ligand; the extracellular ATP opening channel can participate in synaptic transmission and neurogenesis, and its deficiency can play a role in the pathophysiology of idiopathic bladder dysfunction, Genbank reg. No. NP_002552.2);

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

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

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

(35) IRTA2 (an immunoglobulin 2 superfamily receptor associated with translocation, a putative immunoreceptor that possibly plays a role in the development of B cells and lymphogenesis; moreover, in some malignant B cells, gene regulation is disturbed by translocation, Genbank reg. No. NP_112571.1).

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

In yet another aspect, the present invention relates to compositions comprising an effective amount of a drug-antibody conjugate, wherein said drug (part) is cleaved from said drug-antibody conjugate, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the present invention relates to methods for lysing or inhibiting the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-linker-antibody conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting the growth of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component that is cleaved from said drug drug conjugate agent is an antibody. "

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

In another aspect, the present invention relates to methods for treating cancer, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate comprising a drug component that is cleaved from said drug-antibody conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting replication of a cell that expresses an autoimmune antibody, wherein said method comprises administering to a patient in need thereof an effective amount of a drug-linker-antibody conjugate.

In another aspect, the present invention relates to methods for lysing or inhibiting the replication of a cell that expresses an autoimmune antibody, wherein said method comprises administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component that is cleaved from the specified drug-antibody conjugate.

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

In yet another aspect, the present invention relates to methods for treating an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate comprising a drug component that is cleaved from said drug-antibody conjugate.

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

In another aspect, the present invention relates to methods for treating an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate comprising a drug component that is cleaved from said drug-antibody conjugate.

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

In another aspect, the present invention relates to methods for preventing the proliferation of tumor cells or cancer cells, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component that is cleaved from said drug-drug conjugate antibody".

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

In yet another aspect, the present invention relates to methods for preventing cancer, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component that is cleaved from said drug-antibody conjugate.

In another aspect, the present invention relates to methods for preventing the proliferation of cells expressing an autoimmune antibody, wherein said methods comprise administering to a patient in need thereof an effective amount of a drug-linker-antibody conjugate.

In another aspect, the present invention relates to methods for preventing the proliferation of cells expressing an autoimmune antibody, wherein said methods comprise administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component cleaved from said conjugate “ drug antibody. "

In yet another aspect, the present invention relates to methods for the prevention of an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-linker-antibody conjugate.

In another aspect, the present invention relates to methods for the prevention of an autoimmune disease, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate containing a drug component that is cleaved from said drug-antibody conjugate.

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

In another aspect, the present invention relates to methods for preventing an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug-antibody conjugate comprising a drug component that is cleaved from said drug-antibody conjugate.

In another aspect, the present invention relates to a drug compound, which can be used as an intermediate for the synthesis of a drug-linker compound containing a drug component cleaved from said drug-antibody conjugate.

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

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

Ab - (A _a --W _w --Y _y --D) _P (Ic)

or their pharmaceutically acceptable salts or solvates,

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

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

(2) E16 (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (six-domain transmembrane prostate epithelial antigen, Genbank reg. No. NM_012449);

(4) 0772P (CA125, MUC16, Genbank reg. No. AF361486);

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

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 of the family of 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, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorine) 5B, Genbank Reg. No. AB040878);

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

(9) ETBR (type B endothelin receptor, 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, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein of the prostate Genbank Reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of 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 / Epstein-Barr virus receptor), or Hs. 73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), B29, Genbank reg. No. NM_000626);

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

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

(18) NCA (Genbank Reg. No. M18728);

(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, immunoglobulin-bound alpha protein, B-cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with IgM molecules on the cell surface, transmitting a signal involved in the differentiation of B- cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (Burkitt’s lymphoma receptor 1, a G-protein bound receptor activated by chemokine CXCL13, provides lymphocyte migration and humoral defense, and also plays a role in HIV-2 infection and, probably, in the development of AIDS, lymphomas, myelomas and leukemia, Genbank reg. No. NP_001707.1);

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

(31) P2X5 (the -5 ion channel opened by the P2X purinergic receptor ligand; the extracellular ATP opening channel can participate in synaptic transmission and neurogenesis, and its deficiency can play a role in the pathophysiology of idiopathic bladder dysfunction, Genbank reg. No. NP_002552.2);

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

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

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

(35) IRTA2 (an immunoglobulin 2 superfamily receptor associated with translocation, a putative immunoreceptor that possibly plays a role in the development of B cells and lymphogenesis; moreover, in some malignant B cells, gene regulation is disturbed by translocation, Genbank reg. No. NP_112571.1).

A is an extension component;

equal to 0 or 1,

each of -W- independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer component

y is 0, 1 or 2;

p ranges from 1 to about 20; and

D is a drug selected from compounds of formulas D _E and D _F ;

where: the wavy line in the formulas D _E and D _F means a covalent binding site with A, W or Y and where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H and C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH, and

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

n is an integer from 0 to 6.

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

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

In another aspect, the present invention relates to an article comprising a compound in the form of an antibody-drug conjugate according to the invention; a container and package insert or label on the package indicating that the compound can be used to treat a cancer characterized by overexpression of the ErbB2 receptor.

In another aspect, the present invention relates to a method for treating cancer in a mammal, wherein said cancer is overexpressed by an ErbB2 receptor and does not respond or is poorly treated with an anti-ErbB2 antibody, and wherein said method comprises administering to said mammal a therapeutically effective amount of an antibody-drug conjugate according to the invention.

In another aspect of the present invention, a significant amount of said drug molecule is not cleaved from the antibody until said antibody-drug conjugate compound penetrates into a cell having on its surface a receptor that is specific for a given antibody present in said an antibody-drug conjugate, and said drug molecule is cleaved from said antibody only when the antibody-drug conjugate your ”will penetrate into the indicated cell.

In another aspect of the present invention, the bioavailability of the antibody-drug conjugate compound or intracellular metabolite of the compound in a mammal is better than the bioavailability of the drug compound containing the drug molecule present in the antibody-drug conjugate or better than the bioavailability of an analogue of a compound not containing a drug molecule.

In another aspect of the present invention, said drug molecule is a molecule that undergoes intracellular cleavage from an antibody that is a component of a given compound or an intracellular metabolite of that compound in a mammal.

In another aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of an antibody-drug conjugate compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, carrier or excipient. The 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 for lysing or inhibiting the proliferation of tumor or cancer cells, comprising treating the tumor or cancer cells with a specific amount of an antibody-drug conjugate compound of the invention or a pharmaceutically acceptable salt or solvate thereof that is effective for lysis or inhibiting the proliferation of said tumor or cancer cells.

In another aspect, the present invention relates to a method for inhibiting cell proliferation, comprising treating mammalian cells present in a cell culture medium with an antibody-drug conjugate compound of the invention, wherein said antibody-drug conjugate compound penetrates the cell, and then the drug is cleaved from the specified antibody-drug conjugate compound, resulting in inhibition of the proliferator cell cells.

In another aspect, the present invention relates to a method for treating cancer, comprising administering to a patient a composition comprising an antibody-drug conjugate compound and a pharmaceutically acceptable diluent, carrier or excipient.

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

(a) treating the cells with an antibody-drug conjugate compound of the invention and

(b) determining the degree of binding of the antibody-drug conjugate compound to said cells.

For a better understanding of the present invention, the following is a detailed description of examples of its implementation, illustrated in the attached graphic material and diagrams. The discussion below is merely descriptive, illustrative, and representative and should not be construed as limiting the scope of the appended claims.

A brief description of the graphic material

Figure 1 illustrates an effective in vivo assay by administering a single dose of cAC10-mcMMAF into subcutaneous Karpas-299 ALCL xenografts.

Figure 2 illustrates an effective in vivo assay by administering a single dose of cAC10-mcMMAF into subcutaneous L540cy. This study was performed on 4 mice of the untreated group and 10 mice in each treatment group.

Figures 3a and 3b illustrate in vivo the efficacy of cBR96-mcMMAF in subcutaneous L2987. The shaded triangles in FIG. 3a and the arrows in FIG. 3b indicate days of therapy.

Figures 4a and 4b illustrate the in vitro activity of the conjugates “antibody cAC10 drug" directed against CD30 ⁺ cell lines.

Figures 5a and 5b illustrate in vitro activity of the cBR96 antibody drug conjugates directed against Le ^{y +} cell lines.

FIGS. 6a and 6b illustrate in vitro activity of the “antibody c1F6 drug” conjugates directed against CD70 ⁺ cell lines of renal carcinoma.

Figure 7 illustrates an in vitro analysis of cell proliferation using SK-BR-3 cells treated with antibody-drug conjugates (ADC): - ● - trastuzumab-MS-vc-RAB-MMAF, 3.8 MMAF / Ab, -o-trastuzumab-MS-MMAF, 4.1 MMAF / Ab, and -Δ-trastuzumab-MS-MMAF, 4.8 MMAF / Ab, measured in relative fluorescence units (RLU) with respect to ADC concentration (μg / ml). H = trastuzumab, where H is attached via cysteine [cys].

On Fig illustrates an in vitro analysis of cell proliferation using BT-474 cells treated with ADC: - ● - trastuzumab-MS-vc-RAB-MMAF, 3.8 MMAF / Ab, -o-trastuzumab-MS-MMAF, 4.1 MMAF / Ab, and -Δ- trastuzumab-MS-MMAF, 4.8 MMAF / Ab.

Figure 9 illustrates an in vitro analysis of cell proliferation using MCF-7 cells treated with ADC: - ● - trastuzumab-MS-vc-RAB-MMAF, 3.8 MMAF / Ab, -o-trastuzumab-MS- (N -Me) -vc-PAB-MMAF, 3.9 MMAF / Ab, and -Δ- trastuzumab-MS-MMAF, 4.1 MMAF / Ab.

Figure 10 illustrates an in vitro analysis of cell proliferation using MDA-MB-468 cells treated with ADC: - ● - trastuzumab-MS-vc-RAB-MMAE, 4.1 MMAE / Ab, -o- trastuzumab-MS- vc-PAB-MMAE, 3.3 MMAE / Ab, and -Δ- trastuzumab-MS-vc-PAB-MMAF, 3.7 MMAF / Ab.

11 illustrates a study on the removal of plasma concentrations after administration of H-MS-vc-PAB-MMAF-TEG and H-MS-vc-PAB-MMAF to Sprague-Dawley rats: the administered dose was 2 mg ADC per kg of rat body weight . Concentrations of total antibodies and ADC were measured versus time (H = trastuzumab).

On Fig illustrates a study on the removal of concentrations from plasma after the introduction of H-MS-vc-MMAE dog-like monkeys in various doses: 0.5; 1.5; 2.5; 3.0 mg / kg on days 1 and 21. Concentrations of total antibodies and ADC were measured as a function of time (H = trastuzumab).

On Fig illustrated the change in the average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which on day 0 was administered: carrier, trastuzumab-MS-vc-RAV-MMAE (1250 μg / m ² ) and trastuzumab-MS-vc-RAB-MMAF (555 μg / m ² ) (H = trastuzumab).

On Fig illustrated the change in average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which were administered on day 0: 10 mg / kg (660 μg / m ² ) trastuzumab-MS- MMAE and 1250 μg / m ² trastuzumab-MS-vc-RAV-MMAE.

On Fig illustrated the change in the average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which on day 0 was administered: the carrier and 650 μg / m ² trastuzumab-MS-MMAF.

On Fig illustrates the change in the average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which were administered on day 0: vehicle and 350 μg / m ^{2 of} four trastuzumab-MS-MMAF conjugates, where the ratio of MMAF / trastuzumab (H) was 2, 4, 5.9 and 6.

On Fig illustrates the change in average body weight, with the magnitude of the error, for groups of animals (rats) (mean ± cf. from.) After the introduction of the carrier, trastuzumab-MS-val-cit-MMAF, trastuzumab-MS (Me) -val-cit-RAV-MMAF, trastuzumab-MS-MMAF and trastuzumab-MS-val-cit-RAV-MMAF.

On Fig illustrates the change in average body weight for groups of animals (rats) (mean ± cf. from.) After the introduction of 9.94 mg / kg H-MS-vc-MMAF, 24.90 mg / kg H-MS -vc-MMAF, 10.69 mg / kg H-MS (Me) -vc-RAV-MMAF, 26.78 mg / kg H-MS (Me) -vc-RAV-MMAF, 10.17 mg / kg N -MS-MMAF, 25.50 mg / kg H-MS-MMAF and 21.85 mg / kg H-MS-vc-RAB-MMAF. H = trastuzumab. In each conjugate, the MS linker is linked via cysteine trastuzumab.

On Fig illustrates the change in average body weight, with the magnitude of the error, for groups of Sprague-Dawley rats (mean ± cf. from.) After the administration of trastuzumab (H) -MS-MMAF in doses of 2105, 3158 and 4210 μg / m ² . In each conjugate, the MS linker is linked via cysteine trastuzumab.

4. Detailed Description of Representative Embodiments of the Invention

4.1. Definitions and abbreviations

Unless otherwise specified, the terms and expressions used here have the following meanings.

The term “trademarks” as used by applicants independently means a trademarked finished product, a generic drug, and the active ingredient (s) of a product protected by a trademark.

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

As used herein, the term “antibody” also means a full-sized immunoglobulin molecule or an immunologically active part of a full-sized immunoglobulin molecule, that is, a molecule containing an antigen-binding site that immunospecifically binds to the antigen of the target or part of interest, where the target targets are, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulins described herein may be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass. Immunoglobulins can come from molecules of any kind. However, in one aspect of the invention, the immunoglobulin is a human, mouse or rabbit immunoglobulin. In another aspect of the invention, said antibodies are polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv-, Fab-, F (ab ') -, F (ab') ₂ fragments, fragments produced by the library of expressed Fab fragments, anti-idiotypic (anti-Id) antibodies, CDRs and epitope-binding fragments of any of the above antibodies that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens.

As used herein, the term “monoclonal antibody” means an antibody obtained from a population of substantially homogeneous antibodies, that is, individual antibodies within a given population that are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic determinant. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against one 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 antibody obtained essentially from a homogeneous population of antibodies, but this term does not mean that this antibody must be produced by any specific method. Thus, for example, the monoclonal antibodies of the invention can be obtained using the hybridoma technology first described by Kohler et al. (1975) Nature 256: 495, or by recombinant DNA methods (see US patent No. 4816567). "Monoclonal antibodies" can 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.

Monoclonal antibodies used herein include, in particular, “chimeric” antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding antibody sequences originating from a particular species or belonging to a particular class or subclass of antibodies, and the rest (s) i) is identical (s) or homologous (s) to the corresponding sequences of antibodies derived from another species or belonging to another class or subclass of antibodies, and also include fragments of such antibodies, provided that on they exhibit the desired biological activity (U.S. Patent № 4,816,567 and Morrison et al (1984) Proc Natl Acad Sci, USA, 81:..... 6851-6855).

Various methods have been used to produce monoclonal antibodies (MAb). In hybridoma technology applied to cloned cell lines that produce the same type of antibody, cells of various types are used, including mice, hamsters, rats, and humans. Another method for producing MAb involves the use of genetic engineering techniques, including recombinant DNA methods. Monoclonal antibodies obtained by such methods are, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines regions encoded by DNA and originating from more than one species. Thus, for example, a chimeric antibody can be derived from the variable region of a murine antibody and the constant region of a human antibody. A humanized antibody is derived primarily from a human antibody, even if it contains regions of a non-human antibody. Like a chimeric antibody, a humanized antibody may contain a fully human constant region. However, unlike a chimeric antibody, its variable region can partially come from a human antibody. Non-human synthetic parts of a humanized antibody are often derived from murine antibody CDRs. In any case, these regions play a decisive role in the recognition by the antibody of a specific antigen and in its binding to this antigen.

As already noted, murine antibodies can be used. Although murine antibodies can be used in diagnostics and in short courses of therapy, their administration to humans over a long period of time leads to an increased risk of producing an undesirable immunogenic response in humans. This response is to produce a human anti-mouse antibody (NAMA), which is produced when the human immune system recognizes the mouse antibody as foreign and attacks it. The NAMA response can cause toxic shock or even death.

Chimeric and humanized antibodies can reduce the likelihood of a NAMA response by minimizing the non-human parts of the introduced antibodies. In addition, chimeric and humanized antibodies have the advantage that they activate secondary immune responses in humans, such as antibody-dependent cellular cytotoxicity.

“Antibody fragments” comprise a portion of an intact antibody, and preferably an antigen binding or variable region thereof. Examples of antibody fragments are Fab, Fab ', F (ab') _2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules and multispecific antibodies derived from antibody fragment (s).

An “intact” antibody is an antibody containing an antigen binding variable region, as well as a constant domain of the light chain (CL) and constant domains of the heavy chain, CH1, CH2 and CH3. The constant domains can be constant domains of a native sequence (for example, constant domains of a human native sequence) or their amino acid sequence variants.

An intact antibody may have one or more “effector” functions, which means biological activities attributed to the Fc region (Fc region of a native sequence or Fc region of an amino acid sequence variant) of an antibody. Examples of antibody effector functions are C1q binding; complement dependent cytotoxicity; binding to the Fc receptor; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; inhibition of cell surface receptors (e.g., B cell receptor; BCR) and the like.

Intact antibodies, depending on the amino acid sequence of the constant domain of their heavy chains, can be assigned to various “classes”. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of these classes can be divided into “subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of immunoglobulins of various classes are well known.

As used herein, the terms “ErbB2” and “HER2” are used interchangeably to mean the human HER2 protein described, for example, by Semba et al. Proc. Natl. Acad. Sci., USA, 82: 6497-6501 (1985) and Yamamoto et al. (1986) Nature, 319: 230-234 (Genbank reg. No. X03363). The term “erbB2” means a gene encoding human ErbB2, and “neu” means a gene encoding rat p185 ^neu . Preferred ErbB2 is human ErbB2 with a 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.

“ErbB ligand” means a polypeptide that binds to and / or activates an ErbB receptor. The ErbB ligand may be a human ErbB ligand having a native 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 schwanoma 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)); betacellululin (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, betacellululin, HB-EGF and epiregulin. ErbB ligands that bind to ErbB3 are heregulins. ErbB ligands capable of binding to ErbB4 are betacelluline, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4, and heregulins. The ErbB ligand may also be a synthetic ErbB ligand. Such a synthetic ligand may be specific for a particular ErbB receptor, or it may recognize specific ErbB receptor complexes. An example of a synthetic ligand is the synthetic heregulin / chimera “EGF-biregulin” (see, for example, Jones et al. (1999) FEBS Letters, 447: 227-231, which is incorporated herein by reference).

“Heregulin” (HRG) means a polypeptide encoded by the product of the heregulin gene described in US patent No. 5641869 or Marchionni et al., Nature, 362: 312-318 (1993). Examples of Heregulins are Heregulin-α, Heregulin-β1, Heregulin-β2 and Heregulin-β3 (Holmes et al. Science, 256: 1205-1210 (1992) and US Patent No. 5641869); differentiation factor neu (NDF) (Peles et al. Cell 69: 205-216 (1992)); acetylcholine receptor inducing factor (ARIA) (Falls et al. (1993) Cell 72: 801-815); glial cell growth factors (GGF) (Marchionni et al., Nature 362: 312-318 (1993)); a factor derived from afferent and motor neurons (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 amino acid sequence variants of a native HRG polypeptide, such as a fragment of an EGF-like domain (e.g., HRGβ1177-244).

An “ErbB hetero-oligomer” is a non-covalently linked oligomer comprising at least two different ErbB receptors. An “ErbB dimer” is a non-covalently linked oligomer comprising at least two different ErbB receptors. Such complexes can be formed if a cell expressing two or more ErbB receptors is exposed to an ErbB ligand. ErbB oligomers, such as ErbB dimers, can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described, for example, in Sliwkowski et al. J. Biol. Chem., 269 (20): 14661-14665 (1994). Examples of such ErbB hetero-oligomers are EGFR-ErbB2 (also called HER1 / HER2), ErbB2-ErbB3 (HER2 / HER3) and ErbB3-ErbB4 (HER3 / HER4) complexes. In addition, an ErbB hetero-oligomer may contain two or more ErbB2 receptors combined with other ErbB receptors, such as ErbB3, ErbB4 or EGFR (ErbB1). Other heterologous proteins, such as the cytokine receptor subunit (e.g. gp130), may also be part of such a hetero-oligomer.

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

The term “amino acid sequence variant” means polypeptides having amino acid sequences that differ to some extent from native polypeptide sequences. Typically, amino acid sequence variants are at least about 70% homologous to at least one native ligand receptor binding domain or at least one native receptor ligand binding domain, such as a tumor associated antigen, and preferably they can be at least about 80%, and more preferably at least about 90%, are homologous to the domains that bind to such a receptor or to a 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 an amino acid sequence variant that are identical after aligning sequences and including spaces in them, if necessary to achieve the maximum percentage of sequence identity. Methods and computer programs for such alignment are well known in the art. One such computer program is “Align 2,” developed by Genentech, Inc. and filed with the user documentation with the Copyright Office, Washington, DC 20559, December 10, 1991.

“Antibody-dependent cell cytotoxicity” and “ADCC” mean a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer cells (NK), neutrophils and macrophages) recognize a bound antibody on the target cell, which leads to the lysis of the target cell. Primary cells mediating ADCC, i.e., NK cells, express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression data on hematopoietic cells are summarized in Table 3 on page 464 of Ravetch & Kinet (1991) Annu. Rev. Immunol. 9: 457-92. In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as the assay described in U.S. Patent Nos. 5,500,362 or 5,821,337. Suitable effector cells for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer cells ( NK). Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo , for example, in a mouse model, such as the model described by 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 an antibody. Preferred FcR is human sequence native FcR. In addition, a preferred FcR is FcR, which binds to an IgG antibody (gamma receptor), and receptors of subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors are FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains a tyrosine-based immunoreceptor motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains in its cytoplasmic domain an inhibitory tyrosine-based immunoreceptor motif (ITIM) (see review by M. Daeron, Annu. Rev. Immunol., 15: 203-234 (1997)). FcRs are described by Ravetch & Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods, 4: 25-34 (1994) and de Haas et al., J. Lab. Clin. Med., 126: 330-41 (1995). As used herein, the term “FcR” also covers other FcRs, including FcRs, which will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transmission 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 lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (for example, an antibody) that forms a complex with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996).

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

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for binding to an antigen. Such a hypervariable region typically contains amino acid residues derived from a “hypervariable region” or “CDR” (complementarity-determining region) (for example, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable the light chain domain 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 the “hypervariable loop ”(For example, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the 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 residues of the “framework region” or “FR” are residues of the variable domain that are not residues of the hypervariable region defined herein.

As a result of the hydrolysis of antibodies by papain, two identical antigen-binding fragments are formed, called “Fab” fragments, each of which has one antigen-binding site, and one remaining “Fc” fragment, the name of which indicates its ability to crystallize easily. Pepsin treatment leads to the formation of an F (ab ') ₂ fragment that has two antigen-binding sites and is capable of cross-linking with the antigen.

“Fv” is a minimal antibody fragment containing a full-sized antigen-recognizing site and an antigen-binding site. This region consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected to each other by a non-covalent bond. This region has a configuration in which three hypervariable regions of each variable domain interact with each other in such a way that they form an antigen-binding site on the surface of the VH-VL dimer. In total, six hypervariable regions confer antigen binding specificity to the antibody. However, even one variable domain (or half of the Fv, containing only three hypervariable regions specific for the antigen) has the ability to recognize the antigen and bind to it, albeit with less affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments in that they have several additional residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines originating from the hinge region of the antibody. Used here, the designation Fab'-SH means Fab ', in which the cysteine (e) residue (s) of the constant domains has (s) at least one free thiol group. F (ab ') ₂ fragments of an antibody are inherently produced as pairs of Fab'fragments between which cysteines of the hinge region are located. Other chemical bonds connecting antibody fragments are also known.

The “light chains" of antibodies originating from vertebrates of any kind can be assigned to one of two different types of chains, called kappa (k) and lambda (λ), based on the amino acid sequences of their constant domains.

A “single chain Fv fragment” or “scFv fragment” of an antibody includes the VH and VL domains of an antibody, wherein said domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which provides the formation of scFv with the structure necessary for binding to the antigen. A description of scFv can be found at Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg & Moore eds., Springer-Verlag, New York, p. 269-315 (1994).

The term “diabodies” means small fragments of antibodies with two antigen-binding sites, where these fragments include the variable domain of the heavy chain (VH) connected to the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). If the linker is too short to form a pair between two domains on the same chain, then the domains are forced to mate with the complementary domains of the other chain and form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404097; in WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci., USA, 90: 6444-6448.

“Humanized forms” of non-human antibodies (eg, rodent antibodies) are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (a recipient antibody) in which residues derived from the hypervariable region of a given recipient antibody are replaced by residues derived from a hypervariable region of a non-human antibody (donor antibody), such as a mouse antibody, a rat antibody , a rabbit or non-human primate antibody, wherein said antibodies have the desired specificity, affinity and binding ability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or in the donor antibody. These modifications are introduced to improve the properties of the antibodies. In general terms, a humanized antibody can comprise essentially all or at least one, and usually two, variable domains in which all or almost all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin and all or almost all of FR are FR with a human immunoglobulin sequence. A humanized antibody also contains, but not necessarily, at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin region. For 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.

Humanized anti-ErbB2 antibodies are huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN® Table 3, US Pat. which is fully incorporated into the present description by reference; and humanized antibodies 520C9 (WO 93/21319) and 2C4, described below.

An “isolated” antibody is an antibody that has been identified and isolated and / or purified from components of its natural environment. The polluting components of its natural environment are materials that adversely affect the diagnostic or therapeutic efficacy of the antibody, and such components may be enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments of the invention, said antibody can be purified (1) by more than 95% by weight of the antibody, as can be determined by the Lauri method, and more preferably by more than 99% by weight of the antibody, (2) to a degree sufficient for administration, at least 15 residues at the N-terminal or internal part of the amino acid sequence, using a sequencer equipped with a centrifuge vessel, or (3) until homogeneous by SDS-PAGE electrophoresis under reducing or non-reducing conditions with staining Coomassie blue or preferably silver. The term “isolated antibody” includes an in situ antibody in recombinant cells if at least one naturally occurring component of the antibody is missing. However, usually isolated antibody can be obtained in at least one purification step.

An antibody that “binds” to an antigen of interest is capable of binding to said antigen with an affinity that is sufficient for the antibody to be used for delivery to a cell expressing the antigen.

An antibody that “induces apoptosis” is an antibody that induces programmed cell death, as determined by binding to annexin V, DNA fragmentation, cell shrinkage, expansion of the endoplasmic reticulum, cell fragmentation and / or formation of membrane vesicles (called apoptotic bodies) ) Such a cell is a tumor cell, for example, a cell in a breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, or bladder. Various methods exist for evaluating cellular events associated with apoptosis. So, for example, translocation of phosphatidylserine (PS) can be determined by binding to annexin; DNA fragmentation can be assessed by the formation of a DNA ladder; and nucleus / chromatin condensation together with DNA fragmentation can be estimated by any increase in hypodiploid cells.

The term “disorder or disorder” means any condition whose symptoms can be alleviated as a result of treatment according to the invention. Such disorders or disorders are chronic and acute disorders or diseases, including pathological conditions that may lead to the development in the mammal of the disorder or disorder in question. Non-limiting examples of disorders treated in accordance with the present invention are benign and malignant tumors; leukemia and lymphoid malignancies, and in particular, cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid gland, pancreas, prostate or bladder; disorders associated with damage to nerve cells, glial cells, astrocytes, hypothalamic cells and other glandular cells, macrophages, epithelial cells, stromal cells and blastocellial cells, and inflammatory, angiogenic and immunological disorders or disorders.

The term “therapeutically effective amount” means an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, such a therapeutically effective amount of a drug can reduce the number of cancer cells and the size of tumor cells; inhibit (i.e. slow down to some extent, and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e. slow down to some extent, and preferably stop) the development of tumor metastases; inhibit, to some extent, tumor growth; and / or relieve, to some extent, one or more of the symptoms associated with cancer. The drug, to some extent, can prevent the growth of cancer cells and / or destroy cancer cells, and it can be cytostatic and / or cytotoxic. The effectiveness of anti-cancer therapy can be assessed, for example, by determining the time before the onset of disease progression (TTR) and / or determining the response rate (RR).

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

The term “intracellular metabolite” means a compound resulting from intracellular metabolic processes or reactions to which an antibody-drug conjugate (ADC) is subjected. Such metabolic processes or reactions may be an enzymatic reaction, such as proteolytic cleavage of a peptide linker ADC or hydrolysis of a functional group, such as a hydrazone group, an ester group or an amide group. Intracellular metabolites are, but are not limited to, antibodies and free drugs that undergo intracellular cleavage upon delivery to the cell, diffusion in the cell, absorption by the cell, or transport to the cell.

The terms “intracellular cleaved” and “intracellular cleavage” refer to an intracellular metabolic process or reaction that undergoes a drug-ligand conjugate, a drug-linker-ligand conjugate, an antibody-drug conjugate (ADC), or etc., resulting in a covalent bond, such as a linker, between the drug component (D) and the antibody (Ab) being cleaved, resulting in cleavage of the free drug from the antibody within the cell. Thus, molecules cleaved from the drug-ligand conjugate, drug-linker-ligand conjugate or ADC are intracellular metabolites.

The term “bioavailability” means systemic bioavailability (ie, blood / plasma levels) for a given amount of a drug administered to a patient. “Bioavailability” is an absolute term that defines such parameters as the time of action (speed) and the total amount (level) of a drug that is released from the administered dosage form and enters the general bloodstream.

The term “cytotoxic activity” means the cytolytic, cytostatic or antiproliferative effect of a compound such as an antibody-drug conjugate or its intracellular metabolite. Cytotoxic activity can be expressed as an IC ₅₀ value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.

The terms “cancer” and “cancerous” mean or describe the physiological state in mammals, which is usually characterized by a 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 specific examples of such cancers are squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma and squamous cell carcinoma, abdominal cancer, hepatocellular cancer, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon cancer, cancer bodochnoy colon carcinoma, endometrial or uterine carcinoma, salivary gland, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

An “ErbB2 expressing cancer tumor” is a tumor on the surface of the cells of which certain levels of ErbB2 are produced sufficient for the anti-ErbB2 antibody to bind to ErbB2 and provide a therapeutic anti-cancer effect.

Cancer, “characterized by excessive activation” of the ErbB2 receptor, is cancer in which 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 result from overexpression of the ErbB2 receptor and / or the presence of levels exceeding the normal level of the ErbB2 ligand available for activation of the ErbB2 receptor in cancer cells. Such excessive activation can lead to the appearance of malignant cancer cells and / or can be caused by cancer cells. In some embodiments of the invention, a diagnostic or prognostic analysis of cancer can be performed to determine if amplification and / or overexpression of the ErbB2 receptor occurs, resulting in such excessive activation of the ErbB2 receptor. Alternatively or additionally, a diagnostic or prognostic analysis of the cancer may be performed to determine whether amplification and / or overexpression of the ErbB2 ligand in the cancer tumor that causes excessive activation of the receptor occurs. In a subpopulation of such cancers, excessive activation of this receptor may be due to the autocrine mechanism of stimulation.

A cancer tumor that “overexpresses” an ErbB2 receptor is a cancer tumor that, on the surface of its cells, has 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 the ErbB2 receptor can be determined in a diagnostic or prognostic analysis by detecting elevated levels of ErbB2 protein present on the cell surface (for example, by immunohistochemical analysis; IHC). Alternatively or additionally, levels of ErbB2-coding nucleic acid in a cell can be measured, for example, by in situ fluorescence hybridization (FISH; see WO 98/45479), Southern blotting, or polymerase chain reaction (PCR), such as real-time quantitative PCR time (RT-PCR). Overexpression of the ErbB2 ligand can be determined in a diagnostic analysis by measuring the levels of the ligand (or nucleic acid encoding that ligand) in a patient, for example, by biopsy of a tumor or by various diagnostic assays, such as IHC, FISH, Southern blotting, PCR, or in vivo assays described above. Overexpression studies of the ErbB2 receptor can also be carried out by measuring the level of a “desiccant” antigen (eg, the extracellular domain of ErbB2) in a biological fluid such as serum (see, for example, US Patent No. 4933294; WO 91/05264; US Patent No. 5401638 and Sias et al. (1990) J. Immunol. Methods, 132: 73-80). In addition to the above analyzes, various other in vivo assays are known to practitioners. For example, the cells in the patient’s body can be treated with an antibody that is optionally labeled with a detectable label, for example, a radioactive isotope, and the binding of the antibody to the patient’s cells can be evaluated, for example, by external scanning for radioactivity or by analyzing a biopsy from a patient treated with antibody.

Tumors overexpressing HER2 are evaluated on an immunohistochemical scale corresponding to the number of copies of the expressed HER2 molecules per cell, and such an assessment can be carried out using biochemical analysis, where points on this scale mean: 0 = 0-10000 copies / cell; 1 + = at least about 200,000 copies / cell; 2 + = at least about 500,000 copies / cell; 3 + = approximately 1-2 x 10 ⁶ copies / cell. Overexpression of HER2 at the 3+ level, which leads to ligand-independent activation of tyrosine kinase (Hudziak et al. (1987) Proc. Natl. Acad. Sci., USA, 84: 7159-7163), is observed in approximately 30% of patients with breast cancer gland, and in these patients there is a decrease in the duration of the relapse-free period and life expectancy in general (Slamon et al. (1989) Science, 244: 707-712; Slamon et al. (1987) Science, 235: 177-182).

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

As used herein, the term “cytotoxic agent” means a substance that inhibits or prevents the functioning of cells and / or causes destruction of cells. This term includes radioactive isotopes (for example, ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ⁶⁰ C and radioactive isotopes Lu), chemotherapeutic agents and toxins, such as small toxin molecules or enzymatically active toxins originating 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.

A “chemotherapeutic agent” is a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents are alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA ^TM ); acetogenins (in particular, bullatacin and bullatacinon); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMTOSAR®), acetyl camptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callistatin; SS-1065 (including its adoselisin, carzelisin and biselezin synthetic analogues); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen analogues of mustard gas, such as chlorambucil, chlorofazine, chlorophosphamide, extramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterol, prednimustine, trophosphamide, uracil analog of mustard gas; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; bisphosphonates such as clodronate; antibiotics such as enediin antibiotics (e.g. calicheamicin, and in particular calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines, such as Annamycin, AD 32, Alcarubicin, Daunorubicin, Dexrazoxane, DX-52-1, Epirubicin, GPX-100, Idarubicin, KRN5500, Menogaryl, Dinemycin, including Dinemycin A, Esperamycin, Neocarzine-Statin Chromophore and Related Enzymerinotinines , cactinomycin, autramycin, azaserin, bleomycin, actinomycin, carabicin, carmine mitsin, carzinophilin, chromomycins, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morfolinodoksorubitsin, tsianomorfolinodoksorubitsin, 2-pyrroline-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, martsellomitsin, mitomycins such such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; folic acid analogs such as denopterin, pteropterin and tremetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine and phloxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan and testolactone; antiadrenergic agents such as aminoglutethimide, mitotan and trilostane; folic acid deficiency supplement such as folinic acid (leucovorin); Aceglaton; antitumor folate antagonists such as ALIMTA®, pemetrexed LY231514, dihydrofolate reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-1 and capecitabine inhibitors synthases, glycinamide ribonucleotide formyl transferase inhibitors such as raltitrexed (TOMUDEX ^RM , TDX); dihydropyrimidine dehydrogenase inhibitors such as eniliuracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatraxate; dopofamine; demesolcin; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and anzamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyrarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenauzonic acid; triazichone; 2,2 ', 2 ”trichlorotriethylamine; trichotecenes (in particular, T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids and taxanes, for example, paxlitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ^TM , a cremophor-free nanoparticle-based paclitaxel preparation based on albumin (American Pharmaceutical Partners, Schaumberg, Illinois) and doxetaxel TAXOTERE® (Rhone -Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogues or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloids; vinorelbine (NAVELBINE®); novantron; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMF); retinoids such as retinoic acid; pharmaceutically acceptable salts, oxides or derivatives of all of the above compounds, as well as combinations of two or more of the above compounds, such as CHOP, where this abbreviation means a combination of therapeutic agents, such as cyclophosphamide, doxorubicin, vincristine and prednisone, and FOLFOX, where this abbreviation means a combination therapeutic agents such as oxaliplatin (ELOXATIN ^™ ), 5-FU and leucovorin.

This term also includes anti-hormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifen , keoxifene, LY117018, onapriston and toremifene FARESTON®; aromatase inhibitors that inhibit the aromatase enzyme and regulate the production of estrogen in the adrenal cortex, such as 4 (5) -imidazoles, aminoglutetimide, megestrol acetate MEGASE®, exemestane AROMASIN®, formestanin, fadrozole, Vorozol RIVISOR®, letrozole® REXISAR® EXEXEMEMEMEMAR® ; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (a nucleoside cytosine analog of 1,3-dioxolane); antisense oligonucleotides, and in particular oligonucleotides that inhibit gene expression of signaling pathways involved in the proliferation of unwanted cells, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; rIL-2 PROLEUKIN®; LURTOTECAN® topoisomerase 1 inhibitor; rmRH ABARELIX® and pharmaceutically acceptable salts, acids or derivatives of all of the above compounds.

As used herein, the term “EGFR drug” means a therapeutic agent that binds to EGFR and optionally inhibits EGFR activation. Examples of such agents are antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR are MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see US patent No. 4943533, Mendelsohn et al.) And variants thereof, such as the chimeric antibody 225 (C225 or cetuximab; ERBITUX®) and the reconstructed human antibody 225 (H225) (see WO 96/40210, Imclone Systems Inc.); antibodies that bind to mutant EGFR type II (US patent No. 5212290); humanized and chimeric antibodies that bind to EGFR, as described in US patent No. 5891996; and human antibodies that bind to EGFR, such as ABX-EGF (see WO 98/50433, Abgenix). An anti-EGFR antibody can be conjugated to a cytotoxic agent, thereby forming an immunoconjugate (see, for example, EP 659439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR are ZD1839 or Gefitinib (IRESSA ^TM , Astra Zeneca), erlotinib-HCl (CP-358774, TARCEVA ^TM ; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen).

A “tyrosine kinase inhibitor" is a molecule that to some extent inhibits the tyrosine kinase activity of tyrosine kinase, such as the ErbB2 receptor. Examples of such inhibitors are the “EGFR drugs” described in the previous paragraph, as well as quinazolines such as PD 153035, 4- (3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines such as CGP 59326, CGP 6026 and pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d] pyrimidines, curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino) phthalamide), tyrphostins containing nitrothiophene groups; PD-0183805 (Warner-Lambert); antisense molecules (for example, molecules that bind to an ErbB-coding nucleic acid); quinoxalines (US patent No. 5804396); trifostins (US patent No. 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); imatinib mesylate (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 of patents and patent applications: in US 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 “anti-angiogenic agent” means a compound that blocks the development of blood vessels or, to some extent, inhibits such development. Such an anti-angiogenic factor may be, for example, a small molecule or antibody that binds to a growth factor or to a growth factor receptor involved in stimulating angiogenesis. In one embodiment of the invention, said anti-angiogenic factor is an antibody that binds to vascular 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 other cells as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and standard polypeptide hormones. In addition to the above definitions, the term “cytokines” also includes growth hormones, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); hepatocyte growth factor, fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Mueller inhibitory factor; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I or -II; erythropoietin (EPO); factors inducing osteogenesis; interferons, such as interferon-α, -β and -γ; colony stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); and granulocytic 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; tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term “cytokine” includes proteins derived from natural sources or from recombinant cell culture, and biologically active equivalents of cytokines with a native sequence.

As used herein, the term “prodrug” means a precursor or derivative of a pharmaceutically active substance that, compared to the parent drug, is less cytotoxic to tumor cells and is capable of enzymatically or hydrolytically activating or transforming into a 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 of the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified drugs, glycosylated prodrugs, β-lactam-containing prodrugs; prodrugs optionally containing substituted phenoxyacetamide, or prodrugs optionally containing substituted phenylacetamide; 5-fluorocytosine and other 5-fluoruridine prodrugs that can be converted into a more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized to provide a prodrug form for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids, phospholipids and / or surfactants that can be used for drug delivery (eg, including anti-CD30, anti-CD40, anti-CD70 antibodies or anti-CD antibodies Lewis-Y and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are usually arranged so that they form a bilayer similar to the lipid bilayer in biological membranes. The term “package insert” as used herein means instructions that are usually included in the intended sale of the package of therapeutic products and which contain information regarding indications, method of use, dose, route of administration, contraindications and / or warnings regarding the use of such therapeutic products.

An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one impurity nucleic acid molecule with which it is usually associated in a natural source of nucleic acid encoding an antibody. An isolated nucleic acid molecule, in its form or structure, differs from a molecule found in nature. Therefore, the isolated nucleic acid molecules are different from the nucleic acid molecules contained in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that typically express an antibody, where, for example, said nucleic acid molecule is on the chromosome at a position different from its position in natural cells.

The term “regulatory sequences” means DNA sequences necessary for the expression of a functionally linked coding sequence in a particular host organism. Regulatory sequences suitable for prokaryotes are, for example, a promoter, optionally, an operator sequence and a ribosome binding site. In eukaryotic cells, promoters, polyadenylation signals, and enhancers are known to be used.

A nucleic acid is “functionally linked” if it is in functional relationship with another nucleic acid sequence. So, for example, DNA for a pre-sequence or secretory leader sequence is operably linked to DNA encoding a polypeptide if it is expressed in the form of a precursor protein that participates in the secretion of the polypeptide; either a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if the location of this site facilitates translation. In general terms, the term “operably linked” means that the DNA sequences are adjacent, and in the case of a secretory leader sequence, they are adjacent and are in the same reading frame. However, enhancers do not have to be adjacent to the attachment sequence. Accession can be carried out by ligation at suitable restriction sites. If such sites are not available, then synthetic oligonucleotide adapters or linkers may be used in accordance with standard practice.

As used herein, the terms “cell”, “cell line” and “cell culture” are used interchangeably and all include offspring of these cells. So, for example, the words “transformants” and “transformed cells” include primary cells and cultures derived from them without indicating the number of transfers. It should be noted that all offspring cannot be exactly identical in DNA composition, which is due to artificially introduced or spontaneous mutations. These terms also include mutant progeny that have the same function or biological activity that was established by screening for the original transformed cell. A more precise definition of these terms will be apparent from the context of the following description.

As used herein, the term “autoimmune disease” means diseases or disorders that occur in an individual's own tissues and are directed against these tissues, or diseases that accompany them or their symptoms, or conditions that develop 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, psoriasis arthritis and ankylosing spondylitis), psoriasis, dermatitis, including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria; polymyositis / dermatomyositis, toxic epidermal necrosis, systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (IBD) (Crohn’s disease, ulcerative colitis) and IBD with accompanying gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis and / or episiscleritis; respiratory distress syndrome, including adult respiratory distress syndrome (RDSV); meningitis, IgE-mediated diseases, such as anaphylactic and allergic rhinitis, encephalitis, such as Rasmussen encephalitis; uveitis; colitis, such as histologically diagnosed colitis and collagenous colitis; glomerulonephritis (GN), such as membrane GN, idiopathic membrane GN, membrane proliferative GN (MPHN), including type I and type II glomerulonephritis and rapidly progressing GN; allergic conditions, eczema, asthma, conditions associated with T cell infiltration and chronic inflammatory reactions; atherosclerosis; autoimmune myocarditis; conditions associated with impaired leukocyte adhesion; systemic lupus erythematosus (SLE), such as cutaneous SLE, lupus (including nephritis, cerebritis, lupus erythematosus, non-renal lupus, discoid lupus, alopecia); juvenile diabetes mellitus; multiple sclerosis (PC), such as spinal ophthalmic PC; allergic encephalomyelitis; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; tuberculosis; sarcoidosis; granulomatosis, including Wegener's granulomatosis, agranulocytosis, vasculitis (including large-vessel vasculitis (including polymyalgia rheumatism and giant cell arteritis (Takayasu arthritis)), middle-vessel vasculitis (including Kawazaki disease and polyarteritis nodosa), CNS vasculitis, and ANCA-associated vasculitis, vasculitis or Cherg-Strauss syndrome (CSS)); aplastic anemia; Coombs positive anemia; Diamond-Blackfan anemia; immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA); pernicious anemia; true red blood cell aplasia (IEA); diseases associated with factor VIII deficiency; hemophilia A; autoimmune neutropenia; pancytopenia; leukopenia; diseases associated with leukocyte diapedesis; inflammatory disorders of the central nervous system; multiple organ failure syndrome; severe myasthenia gravis; diseases mediated by the antigen-antibody complex; glomerular basement membrane disease catalyzed by the antibody-antigen reaction; syndrome associated with the production of antibodies against phospholipids; allergic neuritis; Behcet's disease; Castleman's syndrome; Goodpasture syndrome; Lambert-Eaton myasthenic syndrome; Raynaud's syndrome; Sjögren's syndrome; Stevens-Johnson syndrome; solid organ transplant rejection (including reactions to the preliminary administration of high titers of test angigenic reactive antibodies, to IgA deposition in tissues, and to kidney transplant, liver transplant, small bowel transplant, heart transplant, etc.); graft versus host reaction (GVHD); bullous pemphigoid; pemphigus (including pemphigus vulgaris, leaf pemphigus and pemphigoid mucous membranes resembling pemphigus); autoimmune polyendocrinopathies; Reiter's disease; syndrome "stiff person"; immunocomplex nephritis; IgM polyneuropathy or IgM-mediated neuropathy; idiopathic thrombocytopenic purpura (ITP); thrombotic thrombocytopenic purpura (TTP); thrombocytopenia (developing, for example, in patients with myocardial infarction), including autoimmune thrombocytopenia; autoimmune disease of the testicles and ovary, including autoimmune orchitis and oophoritis; primary hypothyroidism; autoimmune endocrine diseases, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis; idiopathic hypothyroidism; Addison's disease; Graves disease; autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes); type I diabetes, also called insulin-dependent diabetes mellitus (IDDM), including IDDM in children and Sheehan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchitis obliterans (non-transmitted), in contrast to NSIP; Guillain-Barré syndrome; Berger's disease (IgA nephropathy), primary biliary cirrhosis; intestinal sprue (celiac enteropathy); intractable spru with extensive foci of herpetiform dermatitis; cryoglobulinemia; amylotrophic lateral sclerosis (ABS; Louis Gerig's disease); coronary heart disease; autoimmune disease of the inner ear (HAZA); autoimmune alopecia; dancing eye syndrome (LNG); polychondritis, such as intractable polychondritis; pulmonary alveolar proteinosis; amyloidosis; giant cell hepatitis; scleritis; monoclonal gammopathy of unknown / unknown etiology (MGUS); peripheral neuropathy, paraneoplastic syndrome; “Canalopathies” such as epilepsy, migraine, arrhythmia, muscle disorders, deafness, blindness, periodic paralysis, CNS “canalopathies”, autism, inflammatory myopathy, and focal segmental glomerulosclerosis (OSGS).

“Alkyl” is a C ₁ -C ₁₈ hydrocarbon containing a normal, secondary, tertiary carbon atom or a carbon atom in the ring. Examples are methyl (Me, —CH ₃ ), ethyl (Et, —CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, —CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr , isopropyl, CH ₂ (CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, isobutyl, - CH ₂ CH (CH ₃ ) ₂ ), 2-butyl (s-Bu, sec-butyl, CH (CH ₃ ) CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, tert-butyl, C (CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH (CH ₃ ) CH ₂ CH ₂ CH ₃ ), 3-pentyl ( -CH (CH ₂ (CH ₃ ) ₂ ), 2-methyl-2-butyl (-C (CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH (CH ₃ ) CH (CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH (CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH (CH ₃ ) CH ₂ CH ₃ ), 1- hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH _3), 2-n Force (-CH (CH ₃₎ CH ₂ CH ₂ CH ₂ CH _3), 3-hexyl (-CH (CH ₂ CH ₃₎ (CH ₂ CH ₂ CH _3)), 2-methyl-2-pentyl (-C (CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH (CH ₃ ) CH (CH ₃ ) CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH (CH ₃ ) CH ₂ CH (CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C (CH ₃ ) (CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH (CH ₂ CH ₃ ) CH (CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C (CH ₃ ) ₂ CH (CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH (CH ₃ ) C (CH ₃ ) ₃ ).

“Alkenyl” is a C ₂ -C ₁₈ hydrocarbon containing a normal, secondary, tertiary carbon atom or a carbon atom in the ring with at least one unsaturated bond, i.e. carbon-carbon bond and sp ² double bond. Examples are, but are not limited to, ethylene or vinyl (—CH = CH ₂ ), allyl (—CH ₂ CH = CH ₂ ), cyclopentenyl (—C ₅ H ₇ ), and 5-hexenyl (—CH ₂ CH ₂ CH ₂₎ CH ₂ CH = CH ₂ ).

“Alkynyl” is a C ₂ -C ₁₈ hydrocarbon containing a normal, secondary, tertiary carbon atom or a carbon atom on the ring with at least one unsaturated bond, i.e. with carbon-carbon bond and sp- triple bond. Examples are, but are not limited to, acetylene (—CH≡CH) and propargyl (—CH ₂ CH≡CH).

“Alkylene” is a saturated, branched, single-chain or cyclic hydrocarbon radical consisting of 1-18 carbon atoms and having two monovalent radical centers formed by the removal of two hydrogen atoms from two identical or different carbon atoms of the starting alkane. Typical alkylene radicals include, but are not limited to, methylene (—CH ₂ -), 1,2-ethyl (—CH ₂ CH ₂ -), 1,3-propyl (—CH ₂ CH ₂ CH ₂ -), 1, 4-butyl (-CH ₂ CH ₂ CH ₂ CH ₂ -), etc.

“Alkenylene” is an unsaturated, branched, single-chain or cyclic hydrocarbon radical consisting of 2-18 carbon atoms and having two monovalent radical centers formed by the removal of two hydrogen atoms from two identical or different carbon atoms of the starting alkane. Typical alkenylene radicals are, but are not limited to, 1,2-ethylene (—CH = CH—).

“Alkynylene” is an unsaturated, branched, single-chain or cyclic hydrocarbon radical consisting of 2-18 carbon atoms and having two monovalent radical centers formed as a result of the removal of two hydrogen atoms from two identical or different carbon atoms of the starting alkyne. Typical alkynylene radicals include, but are not limited to, acetylene (-C≡C-), propargyl (-CH ₂ C≡C-) and 4-pentynyl (-CH ₂ CH ₂ CH ₂ C≡CH-).

“Aryl” is a monovalent aromatic hydrocarbon radical, consisting of 6-20 carbon atoms and resulting from the removal of one hydrogen atom from one carbon atom of the original aromatic system. Some aryl groups in representative structures are designated as “Ar." Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” is an acyclic alkyl radical in which one of the hydrogen atoms bonded to the carbon atom, usually the terminal carbon atom or the sp ³ carbon atom, is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylene-1-yl, naphthobenzyl, 2- naphthophenylethan-1-yl and the like. The arylalkyl group contains from 6 to 20 carbon atoms; for example, the alkyl part, including the alkanyl, alkenyl or alkynyl parts of the arylalkyl group, has 1-6 carbon atoms, and the aryl part has 5-14 carbon atoms.

“Heteroarylalkyl” is an acyclic alkyl radical in which one of the hydrogen atoms bonded to the carbon atom, usually the terminal carbon atom or the sp ³ carbon atom, is replaced by a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkyl group has from 6 to 20 carbon atoms; for example, the alkyl part, including the alkanyl, alkenyl or alkynyl parts of the heteroarylalkyl group, has 1-6 carbon atoms, and the heteroaryl part has 5-14 carbon atoms and 1-3 heteroatoms selected from N, O, P and S. Heteroaryl part of the heteroarylalkyl group may be a monocycle having 3 to 7 members on the ring (2-6 carbon atoms), or a bicycle having 7 to 10 members on the ring (4-9 carbon atoms and 1-3 heteroatoms selected from N , O, P and S), for example a 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 substituted by a substituent. Typical substituents are, but are not limited to, -X-, -R, -O ^- , -OR, -SR, -S ^- , -NR ₂ , -NR ₃ , = NR, -CX ₃ , -CN, -OCN , -SCN, -N = С = О, -NCS, -NO, -NO ₂ , = N ₂ , -N ₃ , NC (= О) R, С (= О) R, -С (= О) NR ₂ , -SO ₃ ^- , -SO ₃ H, -S (= О) ₂ R, -OS (= О) ₂ OR, -S (= O) ₂ NR, -S (= О) R, -OP ( = O) (OR) ₂ , -P (= O) (OR) ₂ , -PO ₃ ^- , -PO ₃ H ₂ , -C (= O) R, -C (= O) X, -C (= S) R, —CO ₂ R, —CO ₂ ^- , —C (= S) OR, —C (= O) SR, —C (= S) SR, —C (= O) NR ₂ , —C ( = S) NR ₂ , -C (= NR) NR ₂ , where each X independently represents halogen: F, Cl, Br or I, and each R independently represents H, C ₂ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₄ heterocycle, protective group or prodrug group. The alkylene, alkenylene and alkynylene groups described above may also be substituted in a similar manner.

“Heteroaryl” and “heterocycle” are a cyclic system in which one or more atoms on the ring are heteroatoms, for example, nitrogen, oxygen and sulfur atoms. The 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 from 3 to 7 members on the ring (2-6 carbon atoms and 1-3 heteroatoms, selected from N, O, P and S), or a 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 a bicyclo [4, 5] -, [5,5] -, [5,6] - or [6,6] -system.

Heterocycles are described by Paquette, Leo A .; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), and in particular in chapters 1, 3, 4, 6, 7, and 9; in “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), and in particular in volumes 13, 14, 16, 19 and 28; and in J. Am. Chem. Soc. (1960) 82: 5566.

Examples of heterocycles are, for example, but are not limited to, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur-oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, iminylazole, indidiol, thiindiol, indidiol, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetra oisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, nafteridinil, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aN-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthroinyl, phenazinyl, phenothiazinyl, furazanil, enoksazinil, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, and benzoxazolin izatinoil.

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

Non-limiting examples of nitrogen bonded heterocycles are heterocycles bonded at position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 2-pyrazoline -pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole; at position 2 of isoindole or isoindoline, at position 4 of morpholine and at position 9 of carbazole or β-carboline. The most typical nitrogen-bound heterocycles are 1-aziridyl, 1-azetidyl, 2-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

A “carbocycle” is a saturated or unsaturated monocyclic ring having 3-7 carbon atoms, or a bicyclic ring having 7-12 carbon atoms. Monocyclic carbocycles have 3-6 carbon atoms on the ring, and usually 5 or 6 carbon atoms on the ring. Bicyclic carbocycles have 7-12 carbon atoms on the ring, for example, in the form of a bicyclo [4,5] -, [5,5] -, - [5,6] - or [6,6] system, or 9 or 10 carbon atoms on the ring, in the form of a 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 “bond” mean a chemical group containing a covalent bond or chain of atoms that covalently binds an antibody to a drug molecule. In various embodiments of the invention, the linker is designated LU. The linker is a divalent radical such as alkyldiyl, aryldiyl, heteroaryldiyl, groups such as - (CR ₂ ) _n O (CR ₂ ) _n -, repeating units of alkyloxy (e.g. polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethylene amino, Jeffamine ^™ ), and dibasic esters and amides, including succinate, succinamide, diglycolate, malonate and caproamide.

The term “chiral” refers to molecules that do not overlap with its mirror-like counterpart, and the term “achiral” refers to molecules that overlap with its mirror-like counterpart.

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

The term “diastereomer” means a stereoisomer with one or more chiral centers, the molecules of which are not mirror images of each other. Diastereomers have various physical properties, for example, melting points, boiling points, spectral properties and reactivity. Mixtures of diastereomers can be separated by high resolution analytical methods such as electrophoresis and chromatography.

The term “enantiomers” means two stereoisomers of a compound whose mirror images are not compatible with each other.

Definitions and notation of the stereochemical terms used herein are broadly provided by SP 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, that is, they are able to rotate the plane of plane-polarized light. In the description of an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule relative to its chiral center (s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by this compound, and (-) or l means that this compound is levorotatory. A connection prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical, except that they are mirror images of each other. A specific stereoisomer may also be called an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can be formed if there is no stereoselectivity or stereospecificity in the chemical reaction or in the chemical process. The terms “racemic mixture” and “racemate” mean an equimolar mixture of two enantiomeric molecules that do not have optical activity.

Examples of a “patient” include, but are not limited to, a 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 human.

The term “aryl” means a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a heterocyclic aromatic group may be unsubstituted or may be substituted by one or more groups, including, but not limited to, —C ₁ -C ₈ alkyl, —O- (C ₁ -C ₈ alkyl), aryl, —C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ') ₂ , -NHC (O) R ', -S (O) ₂ R', -S (O) R ', -OH, halogen, N ₃ , -NH ₂ -, -NH (R'), -N (R ' ) ₂ and —CN, where each of R ′ is independently selected from H, —C ₁ -C ₈ alkyl and aryl.

As used herein, the term “C ₁ -C ₈ alkyl” means a straight or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “C ₁ -C ₈ alkyl” 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 C ₁ -C ₈ alkyls are, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl; unsaturated C ₁ -C ₈ alkyls include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 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-pentynyl, 2-pentinine, 3-methyl-1- butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpe tyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl and isooctyl. C ₁ -C ₈ alkyl group may be unsubstituted or may be substituted by one or more groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O- (C ₁ -C ₈ alkyl), -aryl, - C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ') ₂ , -NHC (O) R ', -SO ₃ R', -S (O) ₂ R ', -S (O) R', -OH, halogen, N ₃ , -NH ₂ -, -NH (R ' ), —N (R ′) ₂ and —CN, where each R ′ is independently selected from H, —C ₁ -C ₈ alkyl and aryl.

The term “C ₃ -C ₈ carbocycle” means a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C ₃ -C ₈ carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, cycloheptyl, -1,3-cycloheptadienyl, - 1,3,5-cycloheptatrienyl, cyclooctyl and cyclooctadienyl. C ₃ -C ₈ carbocyclic group may be unsubstituted or may be substituted by one or more groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O- (C ₁ -C ₈ alkyl) -, aryl, - C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ') ₂ , -NHC (O) R ', -S (O) ₂ R', -S (O) R ', -OH, halogen, N ₃ , -NH ₂ -, NH (R'), -N (R ' ) ₂ and —CN, where each of R ′ is independently selected from H, —C ₁ -C ₈ alkyl and aryl.

The term “C ₃ -C ₈ carbocyclo” means a C ₃ -C ₈ carbocyclic group as defined above, wherein one of the hydrogen atoms of the carbocyclic group is replaced by a bond.

The term “C ₁ -C ₁₀ alkylene” means a direct saturated hydrocarbon group of the formula - (CH ₂ ) _1-10 -. Examples of C ₁ -C ₁₀ alkylene are methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decalene.

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

where the phenyl group may be unsubstituted or may be substituted by one to four groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O- (C ₁ -C ₈ alkyl) -, aryl, -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ') ₂ , -NHC ( O) R ′, —S (O) ₂ R ′, —S (O) R ′, —OH, halogen, N ₃ , —NH ₂ -, —NH (R ′), —N (R ′) _2, and —CN, where each of R ′ is independently selected from H, —C ₁ -C ₈ alkyl and aryl.

The term “C ₃ -C ₈ heterocycle” means an aromatic or non-aromatic C ₃ -C ₈ carbocycle in which one to four carbon atoms on the ring are independently substituted by a heteroatom selected from the group consisting of O, S and N. Representative examples C ₃ - C ₈ heterocycles include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl pyridinyl pyridinyl pyridinyl isoxazolyl and tet azolyl. The C ₃ -C ₈ heterocycle may be unsubstituted, or it may be substituted with 1-7 groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O- (C ₁ -C ₈ alkyl) -, -aryl , -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ' ) ₂ , -NHC (O) R ', -S (O) ₂ R', -S (O) R ', -OH, halogen, N ₃ , -NH ₂ -, -NH (R'), -N (R ') ₂ and -CN, where each of R' is independently selected from H, -C ₁ -C ₈ alkyl and aryl.

The term “C ₃ -C ₈ heterocyclo” means a C ₃ -C ₈ heterocyclic group as defined above, wherein one of the hydrogen atoms of the heterocyclic group is replaced by a bond. C ₃ -C ₈ heterocyclo may be unsubstituted or may be substituted with 1-6 groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O- (C ₁ -C ₈ alkyl) -, -aryl, - C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH ₂ , -C (O) NHR', -C (O) N (R ') ₂ , -NHC (O) R ', -S (O) ₂ R', -S (O) R ', -OH, halogen, N ₃ , -NH ₂ -, -NH (R'), -N (R ') ₂ and -CN, where each R' is independently selected from H, -C ₁ -C ₈ alkyl and aryl.

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

The term “representative conjugate” means a drug-ligand conjugate in which a drug is cleaved from said drug-ligand conjugate or a drug-linker-ligand conjugate.

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

Examples of “hydroxyl protecting groups” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, tert-butylmethyl ether acetic acid ester, substituted acetic acid esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

The term “leaving group” means a functional group that may be substituted with another functional group. Such leaving groups are well known in the art, and examples of these groups include, but are not limited to, halide (e.g. chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethyl sulfonyl (triflate) and trifluoromethyl sulfonate.

The term “pharmaceutically acceptable salt”, as used herein, means pharmaceutically acceptable organic or inorganic salts of a representative compound or representative conjugate. These representative compounds and representative conjugates contain at least one amino group, and this amino group can form the corresponding acid addition salts. Examples of such salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sugar, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e. 1,1'-methylene bis- ( 2-hydroxy-3-naphthoate)). The term “pharmaceutically acceptable salt” may include another molecule, such as an acetate ion, a succinate ion or another counterion. Such a counterion can be any organic or inorganic molecule that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If many charged atoms are part of a pharmaceutically acceptable salt, then such a salt can have many counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

The terms “pharmaceutically acceptable solvate” or “solvate” mean a 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 that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The abbreviations used in the present description have the following meanings: AE means auristatin E; Boc means N- (tert-butoxycarbonyl), cit means citrulline, dap means dolaproin, DCC means 1,3-dicyclohexylcarbodiimide, DCM (DCM) means dichloromethane, DEA means diethylamine, DEAD means diethyl azodicarboxylate, DEPC means diethylphosphorylcyanidodicarboxydisoxydisopropyl aminobenzoate, DIAD means N, N-diisopropylethylamine, dil means dolaisoleucine, DMAP means 4-dimethylaminopyridine, DME means ethylene glycol dimethyl ether (or 1,2-dimethoxyethane), DMF (DMF) means N, N-dimethylformamide, DMSO (DMSO) means dimethyl sulfoxide, doe means dol phenin, dov means N, N-dimethylvaline, DNTB means 5,5'-dithiobis (2-nitrobenzoic acid), DTPA means diethylene triaminopentaacetic acid, DTT means dithiothreitol, EDCI means 1- (3-dimethylaminopropyl) -3-ethyl-ethylbenethylbenzene means 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS means electrospray mass spectrometry, EtOAc means ethyl acetate, Fmoc means N- (9-fluorenylmethoxycarbonyl), gly means glycine, HATU means hexafluorophosphate O- (7- azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium, HOBt means 1-hydroxybenzotriazole, HPLC (VE X) means high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH ₃ CN) is acetonitrile, MeOH is methanol, Mtr is 4-anizildifenilmetil (or 4-methoxytrityl), nor (holes) is (1S, 2R ) - (+) - norephedrine, RAB 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-maleimidocaproyl, phe means L-phenylalanine, PyBrop means bromo-tris-pyrrolidinophosphonium hexafluorophosphate, SEC means exc flash chromatography, Su means succinimide, TBTU means tetrafluoroborate O-benzotriazol-1-yl-N, N, N, N-tetramethyluronium, TFA means trifluoroacetic acid, TLC (TLC) means thin layer chromatography, UV (UV) means ultraviolet radiation, and val means valine.

The abbreviations used in the present description for linkers have the following meanings: Val-Cit means valine-citrulline, a dipeptide site in a protease cleavable linker; RAV means p-aminobenzylcarbamoyl; (Me) vc means N-methyl-valine-citrulline, where the linker peptide bond has been modified to prevent the cleavage of this linker by cathepsin B; MS (PEG) ₆ —OH means maleimidocaproyl polyethylene glycol; SPP is N-succinimidyl-4- (2-pyridylthio) pentanoate, and SMCC is N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate.

The terms “treat” or “treatment”, if they do not have any other meaning in the context of the description, mean therapeutic treatment and prophylactic or preventive measures that are aimed at preventing or slowing down (weakening) an individual of an undesirable physiological change or disorder, such as the development or progression of cancer. In accordance with the present invention, beneficial or desirable clinical effects include, but are not limited to, alleviating symptoms, reducing the severity of the disease, stable (i.e. not worsening) condition, delaying or slowing the progression of the disease, reducing or temporarily relieving the symptoms of the disease, and remission (partial or complete), regardless of whether these symptoms are detectable or undetectable. The term “treatment” may also mean an increase in life expectancy compared to life expectancy if untreated. The term “individuals in need of treatment” refers to individuals already suffering from a given condition or disorder, as well as to individuals who are predisposed to a given condition or disorder, or to individuals who need to be prevented from a given condition or disorder.

As for cancer, the term “treatment” includes any or all of the following effects, such as preventing the growth of tumor cells, cancer cells, or tumors; preventing the replication of tumor cells or cancer cells, reducing the total tumor mass or reducing the number of cancer cells, and alleviating one or more symptoms associated with the disease.

For an autoimmune disease, the term “treatment” includes any or all of the following effects, such as preventing the replication of cells associated with an autoimmune disease, including, but not limited to, cells producing an autoimmune antibody; a decrease in the titer of autoimmune antibodies and a weakening of one or more symptoms of an autoimmune disease.

For an infectious disease, the term “treatment” includes any or all of the following effects, such as preventing the growth, reproduction or replication of pathogens that cause the infectious disease and alleviating one or more symptoms of the infectious disease.

The abbreviations used in the present description for cytotoxic drugs have the following meanings: MMAE means monomethyl-auristatin E (molar mass (MW 718); MMAF means N-methylvaline-valine-dolaisoleucine-dolaproin-phenylalanine (MW 731.5); MMAF-DMEAEA means MMAF with DMAEA (dimethylaminoethylamine) 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 amide to the C-terminus of MMAF; AEVB means valerylbenzylhydrazone auristatin and E linked by an acid labile linker to the C-terminus of AE (MW 732), and AFP is p-phenylenediamine monoamide to the C-terminal of phenylalanine of auristatin F (MW 732).

4.2. Compounds of the Invention

4.2.1. The compound of formula ( Ia )

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

L - (A _a --W _w --Y _y -D) _P (Ia)

or their pharmaceutically acceptable salts or solvates,

where: L is a ligand component;

-A _a -W _w -Y _y - represents a linker component (LU), where in the specified linker component:

-A- is an extension component;

equal to 0 or 1,

each of -W- independently represents an amino acid component,

w is an integer from 0 to 12,

-Y- means spacer component

y is 0, 1 or 2;

p ranges from 1 to about 20; and

—D is a “drug” component having the formulas D _E and D _F ;

where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , - (CH ₂ ) _n —SO ₃ H, or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH;

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

where n is an integer from 0 to 6.

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

or their pharmaceutically acceptable salts or solvates,

where: R ² selected from hydrogen and C ₁ -C ₈ alkyl;

R ^{3 is} selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, -C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and -C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from hydrogen, -C ₁ -C ₈ alkyl, -C ₃ -C ₈ carbocycle, aryl, -C ₁ -C ₈ alkylaryl, -C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle), where R ^{5 is} selected from H and methyl, or R ⁴ and R ⁵ taken together with the carbon atom to which they are attached form a ring of the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from —H, —C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R ^{6 is} selected from H and —C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, -C ₁ -C ₈ alkyl, -C ₃ -C ₈ carbocycle, aryl, -C ₁ -C ₈ alkylaryl, -C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), A C ₃ -C ₈ heterocycle and a C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, -C ₁ -C ₈ alkyl, -C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and —C ₁ -C ₈ alkyl;

R ¹⁰ selected from an aryl group or a C ₃ -C ₈ heterocycle;

Z is —O—, —S—, —NH— or —NR ¹² , where R ¹² is —C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, -C ₁ -C ₂₀ alkyl, aryl, -C ₃ -C ₈ heterocycle, - (R ¹³ O) _m -R ¹⁴ or - (R ¹³ O) _m -CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents —C ₂ -C ₈ alkyl;

R ¹⁴ represents H or —C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, —COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , - (CH ₂ ) _n —SO ₃ H, or - (CH ₂ ) _n —SO ₃ —C ₁ - C ₈ alkyl;

each of R ¹⁶ independently represents H, -C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH, and

n is an integer from 0 to 6.

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

Ab - (A _a --W _w --Y _y --D) _P (Ia ')

or their pharmaceutically acceptable salts or solvates,

where: Ab is an antibody;

A is an extension component;

equal to 0 or 1,

each -W- independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer component

y is 0, 1 or 2,

p ranges from 1 to about 20; and

D is a drug selected from compounds of formulas D _E and D _F :

where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH;

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

n is an integer from 0 to 6.

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

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

(2) E16 (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (six-domain transmembrane prostate epithelial antigen, Genbank reg. No. NM_012449);

(4) 0772P (CA125, MUC16, Genbank reg. No. AF361486);

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

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 of the family of 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, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TK), and short cytoplasmic domain (semaphorine) 5B, Genbank Reg. No. AB040878);

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

(9) ETBR (type B endothelin receptor, 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, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein of the prostate Genbank Reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of 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 / Epstein-Barr virus receptor), or Hs. 73792, Genbank reg. No. M26004);

(15) CD79b (IGb (beta protein associated with immunoglobulin), B29, Genbank reg. No. NM_000626);

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

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

(18) NCA (Genbank Reg. No. M18728);

(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, immunoglobulin-associated alpha protein, B-cell-specific protein that covalently interacts with Ig beta (CD79B) and complexes with IgM molecules on the cell surface, transmitting a signal involved in the differentiation of B cells, Genbank reg. No. NP_001774.1);

(29) CXCR5 (Burkitt’s lymphoma receptor 1, a G-protein coupled receptor that is activated by the chemokine CXCL13, provides lymphocyte migration and humoral defense, and also plays a role in HIV-2 infection and, probably, in the development of AIDS, lymphomas, myelomas and leukemia, Genbank reg. No. NP_001707.1);

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

(31) P2X5 (the ion channel opened by the P2X 5 purinergic receptor ligand; the ion channel opened by extracellular ATP can participate in synaptic transmission and neurogenesis, and its deficiency can play a role in the pathophysiology of idiopathic urinary bladder dysfunction, Genbank reg. No. NP_0052 .2);

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

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

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

(35) IRTA2 (an immunoglobulin 2 superfamily receptor associated with translocation, a putative immunoreceptor that may play a role in B cell development and lymphogenesis; in some malignant B cells, gene regulation is disturbed by translocation, Genbank reg. No. NP_112571.1) .

In one embodiment, -Ww- is Val-Cit.

In another embodiment of the invention, R ³ , R ⁴ and R ⁷ independently represent isopropyl or sec-butyl, and R ⁵ represents —H. In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, R ⁵ is —H, R ⁷ is sec-butyl. In yet another embodiment of the invention, each of R ² and R ⁶ is methyl, and R ⁹ is —H.

In another embodiment of the invention, each R ⁸ is —OCH ₃ .

In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, each of R ² and R ⁶ is methyl, R ⁵ is H, R ⁷ is sec-butyl, each of R ⁸ is —OCH ₃ , and R ⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment of the invention, R ¹⁰ is aryl.

In a representative embodiment of the invention, R ¹⁰ is phenyl.

In a representative embodiment of the invention, if Z is —O—, then R ¹¹ is H, methyl or tert-butyl.

In one embodiment of the invention, if Z is —NH, then R ¹¹ is CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —N (R ¹⁶ ) ₂ and R ¹⁶ is —C ₁ -C ₈ alkyl; or - (CH ₂ ) _n -COOH.

In another embodiment, if Z is NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —SO ₃ H.

In one aspect of the invention, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAc10, hBR96, hS2C6, h1F6 and h2F2.

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

or

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

The drug load is determined by the number p, that is, the average number of drug molecules per antibody in the molecule (for example, compounds of formula Ia, Ia 'and Ic). The drug load can be from 1 to 20 drug molecules (D) per ligand (e.g., Ab or mAb). Compositions of compounds of formulas Ia and Ia ′ include antibody kits conjugated to 1-20 drug molecules. The average number of drug molecules per antibody in a preparation obtained by conjugation can be determined by standard methods such as mass spectroscopy, ELISA and HPLC. The quantitative distribution of the ligand-drug conjugates can also be expressed by the symbol p . In some cases, the isolation, purification and identification of homogeneous ligand-drug conjugates, where p has a certain value, from homogeneous ligand-drug conjugates with a different drug load can be achieved by methods such as reverse phase HPLC or electrophoresis .

4.2.2. FORMULA COMPOUNDS OF FORMULA (Ib)

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

or their pharmaceutically acceptable salts or solvates,

where: R ² selected from hydrogen and-C ₁ -C ₈ alkyl;

R ^{3 is} selected from hydrogen, -C ₁ -C ₈ alkyl, -C ₃ -C ₈ carbocycle, aryl, -C ₁ -C ₈ alkylaryl, -C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), - A C ₃ -C ₈ heterocycle and a C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from hydrogen, -C ₁ -C ₈ alkyl, -C ₃ -C ₈ carbocycle, aryl, -C ₁ -C ₈ alkylaryl, -C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), - C ₃ -C ₈ heterocycle and-C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle), where R ^{5 is} selected from H and methyl, or R ⁴ and R ⁵ taken together with the carbon atom to which they are attached, form a ring of the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, -C ₁ -C ₈ alkyl and -C ₃ -C ₈ carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R ^{6 is} selected from —H and —C ₁ -C ₈ alkyl;

R ^{7 is} selected from —H, —C ₁ -C ₈ alkyl, —C ₃ -C ₈ carbocycle, aryl, —C ₁ -C ₈ alkylaryl, —C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle) , -C ₃ -C ₈ heterocycle and -C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from —H, —OH, —C ₁ -C ₈ alkyl, —C ₃ -C ₈ carbocycle, and —O— (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from an aryl group or a C ₃ -C ₈ heterocycle;

Z is —O—, —S—, —NH— or —NR ¹² , where R ¹² is —C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, —C ₁ -C ₂₀ alkyl, aryl, —C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or - (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents —C ₂ -C ₈ alkyl;

R ¹⁴ represents H or —C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, —COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , - (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ - C ₈ alkyl;

each of R ¹⁶ independently represents —H, —C ₁ -C ₈ alkyl or - (CH ₂ ) _n —COOH, and

n is an integer from 0 to 6.

In one embodiment of the invention, R ³ , R ⁴ and R ⁷ independently represent isopropyl or sec-butyl, and R ⁵ represents —H. In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, R ⁵ is —H, R ⁷ is sec-butyl.

In another embodiment of the invention, each of R ² and R ⁶ is methyl, and R ⁹ is —H.

In yet another embodiment of the invention, each R ⁸ is —OCH ₃ .

In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, each of R ² and R ⁶ is methyl, R ⁵ is —H, R ⁷ is sec-butyl, each R ⁸ is —OCH ₃ and R ⁹ represents —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment of the invention, R ¹⁰ is aryl.

In a representative embodiment of the invention, R ¹⁰ is phenyl.

In a representative embodiment of the invention, if Z is —O—, then R ¹¹ is H, methyl or tert-butyl.

In one embodiment of the invention, if Z is —NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —N (R ¹⁶ ) ₂ and R ¹⁶ is - C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH.

In another embodiment of the invention, if Z is NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —SO ₃ H.

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

and their pharmaceutically acceptable salts or solvates.

COMPOUNDS OF FORMULA (Ic)

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

Ab - (A _a --W _w --Y _y --D) _P (Ic)

containing an antibody covalently linked to one or more components (molecules) of the drug. Said antibody-drug conjugate compounds include pharmaceutically acceptable salts or solvates thereof.

In the compounds of formula Ic:

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

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

(2) E16 (LAT1, SLC7A5, Genbank reg. No. NM_003486);

(3) STEAP1 (six-domain transmembrane prostate epithelial antigen, Genbank reg. No. NM_012449);

(4) 0772P (CA125, MUC16, Genbank reg. No. AF361486);

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

(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 of the family of 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, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorine) 5B, Genbank Reg. No. AB040878);

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

(9) ETBR (type B endothelin receptor, 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, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein of the prostate Genbank Reg. No. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of 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 / Epstein-Barr virus receptor), or Hs. 73792, Genbank reg. No. M26004);

(15) CD79b (CD79B, CD79β, IGb (beta protein associated with immunoglobulin), B29, Genbank reg. No. NM_000626);

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

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

(18) NCA (Genbank Reg. No. M18728);

(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 (B-cell activation factor receptor, BLys 3 receptor, BR3, NP_443177.1);

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

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

(29) CXCR5 (Burkitt’s lymphoma receptor 1, a G-protein bound receptor activated by chemokine CXCL13, provides lymphocyte migration and humoral defense, and also plays a role in HIV-2 infection and, probably, in the development of AIDS, lymphomas, myelomas and leukemia, Genbank reg. No. NP_001707.1);

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

(31) P2X5 (the -5 ion channel opened by the P2X purinergic receptor ligand; the extracellular ATP opening channel can participate in synaptic transmission and neurogenesis, and its deficiency can play a role in the pathophysiology of idiopathic bladder dysfunction, Genbank reg. No. NP_002552.2);

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

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

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

(35) IRTA2 (an immunoglobulin 2 superfamily receptor associated with translocation, a putative immunoreceptor that possibly plays a role in the development of B cells and lymphogenesis; moreover, in some malignant B cells, gene regulation is disturbed by translocation, Genbank reg. No. NP_112571.1).

A is an extension component;

equal to 0 or 1,

each -W- independently represents an amino acid component,

w is an integer from 0 to 12,

Y means spacer component

y is 0, 1 or 2,

p ranges from 1 to about 8; and

D is a drug selected from compounds of formulas D _E and D _F :

where the wavy line in D _E and D _F means the site of covalent binding to A, W or Y and where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or - (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , - (CH ₂ ) _n —SO ₃ H or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH,

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

n is an integer from 0 to 6.

In one embodiment, -Ww- is -Val-Cit.

In another embodiment of the invention, R ³ , R ⁴ and R ⁷ independently represent isopropyl or sec-butyl, and R ⁵ represents —H. In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, R ⁵ is —H, R ⁷ is sec-butyl.

In yet another embodiment of the invention, each of R ² and R ⁶ is methyl, and R ⁹ is —H.

In another embodiment of the invention, each R ⁸ is —OCH ₃ .

In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, each of R ² and R ⁶ is methyl, R ⁵ is —H, R ⁷ is sec-butyl, each R ⁸ is —OCH ₃ and R ⁹ represents —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment of the invention, R ¹⁰ is aryl.

In a representative embodiment of the invention, R ¹⁰ is phenyl.

In a representative embodiment of the invention, if Z is —O—, then R ¹¹ is —H, methyl or tert-butyl.

In one embodiment of the invention, if Z is —NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —N (R ¹⁶ ) ₂ and R ¹⁶ is C ₁ -C ₈ alkyl; or - (CH ₂ ) _n -COOH.

In another embodiment, if Z is —NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —SO ₃ H.

Representative ADC variants of formula Ic have the following structures:

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

The drug load is determined by the number p, that is, the average number of drug molecules per antibody in a molecule of formula I. The drug load can be from 1 to 20 drug molecules (D) per antibody (Ab or mAb). Compositions of ADCs of Formula I include antibody kits conjugated to 1-20 drug molecules. The average number of drug molecules per antibody in ADC preparations obtained by conjugation can be determined by standard methods, such as UV / VIS spectroscopy, mass spectroscopy, ELISA and HPLC. The quantitative distribution of ADC can also be expressed by the symbol p . In some cases, isolation, purification and identification of homogeneous ADC conjugates, where p has a specific value, from ADC conjugates with a different drug load can be achieved by methods such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. So, for example, if the attachment site is a cysteine thiol, as defined above in representative embodiments of the invention, then the antibody may have only one or more cysteine thiol groups, or it may have only one or more sufficiently reactive thiol groups through which the linker can be attached .

Usually, in the course of the conjugation reaction, less than theoretically the maximum number of drug molecules can be conjugated to the antibody. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate or with the linker reagent. Only the most reactive lysine groups can interact with the linker reagent that reacts with the amine. Generally speaking, antibodies do not contain many, if any, free and reactive cysteine thiol groups that can be associated with the drug molecule. Most thiol groups of cysteine residues in the antibodies of the compounds of the invention are present as disulfide bridges and must be reduced by a reducing agent such as dithiothreitol (DTT). In addition, in order to detect reactive nucleophilic groups, such as lysine or cysteine groups, such an antibody must be placed under denaturing conditions. The load (drug / antibody ratio) of the ADC can be controlled in several different ways, including: (i) limiting the molar excess of the drug-linker intermediate or linker reagent to the antibody, (ii) limiting the time or temperature of the conjugation reaction, and ( iii) the creation of partial or limiting reducing conditions for the modification of the thiol group of cysteine.

It should be noted that in the event that more than one nucleophilic group reacts with the drug-linker intermediate or with the linker reagent behind which the drug molecule is located, the resulting product is a mixture of ADC compounds with an appropriate distribution of one or several drug molecules attached to an antibody. The average number of drug molecules per antibody can be calculated by ELISA analysis of the mixture using two antibodies, one of which (the “second” antibody) is specific for the antibody and the other is specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectroscopy and separated by HPLC, such as hydrophobic chromatography (“Effect of drug loading on the pharmacology, pharmacokinetics and toxicity of an anti-CD30 antibody-drug conjugate”, Hamblett KJ 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 SC 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, a homogeneous ADC with a single load can be isolated from a mixture of conjugates by electrophoresis or chromatography.

4.3. Linker component

A “linker component” (LU) is a bifunctional compound that can be used to bind a drug molecule and a ligand to form drug-linker-ligand conjugates, or which can be used to produce immunoconjugates directed against tumor-associated antigens. Such immunoconjugates can provide selective delivery of toxic drugs to tumor cells. In one embodiment of the invention, the linker component of the drug-linker compound and the drug-linker-ligand conjugate has the formula:

-A _a --W _w --Y _y -

where: -A- is an extension component;

equal to 0 or 1,

each -W- independently represents an amino acid component,

w is independently an integer from 0 to 12,

-Y- means spacer component

y is 0, 1 or 2.

In the drug-linker-ligand conjugate, the linker is able to bind the drug molecule and the ligand.

4.3.1. Extension component

An extension component (-A-), if present, is capable of binding the ligand component to 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 can be either natural or chemically synthesized include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of the carbohydrate, and carboxyl. In one aspect of the invention, the functional groups of the ligand are sulfhydryl and amino. Sulfhydryl groups can be formed by reducing the intramolecular disulfide bond of the ligand. Alternatively, sulfhydryl groups can be formed by reacting the amino group of the lysine ligand molecule in the presence of 2-iminothiolane (Trout reagent) or another sulfhydryl-forming reagent.

In one embodiment of the invention, said extension component forms a bond with the sulfur atom of the ligand component. This sulfur atom may be derived from the sulfhydryl group of the ligand. A representative extension component of this embodiment of the present invention is indicated by square brackets in formulas IIIa and IIIb, where L-, -W-, -Y-, -D, w and y are defined above and R ^{17 is} selected from -C ₁ -C ₁₀ alkylene, -C ₃ -C ₈ carbocyclo-, -O- (C ₁ -C ₈ alkyl) -, arylene, -C ₁ -C ₁₀ alkylene arylene, -arylene-C ₁ -C ₁₀ alkylene, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ carbocyclo) -, - (C ₃ -C ₈ carbocyclo) -C ₁ -C ₁₀ alkylene, -C ₃ -C ₈ heterocyclo, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ heterocyclo ) -, - (C ₃ -C ₈ heterocyclo) -C ₁ -C ₁₀ alkylene, - (CH ₂ CH ₂ O) _r and - (CH ₂ CH ₂ O) _r -CH ₂ -; and r is an integer from 1 to 10. It should be noted that all representative variants of the compounds of formula Ia, such as III-VI, which, even if not specifically specified, contain from 1 to 20 drug molecules, are associated with the ligand (p = 1-20).

A representative example of an extension component is a component of formula IIIa, where R ¹⁷ represents - (CH ₂ ) ₅ -:

Another representative example of an extension component is a component of formula IIIa, where R ¹⁷ represents - (CH ₂ CH ₂ O) _r —CH ₂ - and r is 2:

Another representative example of an extension component is a component of formula IIIb, where R ¹⁷ represents - (CH ₂ ) ₅ -:

In another embodiment of the invention, the extension component is bonded to the ligand component via a disulfide bond between the sulfur atom of the ligand component and the sulfur atom of the extension component. A representative extension component of this embodiment of the present invention is indicated by square brackets in formula IV, where R ¹⁷ , L-, -W-, -Y-, -D, w and y are defined above.

L - [- SR ¹⁷ -C (O) -] - W _w -Y _y -D (Iv)

In yet another embodiment of the invention, the reactive group of said extension component comprises a reactive site that can form a bond with the primary or secondary amino group of the ligand. Examples of such reactive sites include, but are not limited to, activated esters such as succinimido esters, 4-nitrophenyl ethers, pentafluorophenyl ethers, tetrafluorophenyl ethers, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. A representative extension component of this embodiment of the present invention is indicated by square brackets in formulas Va and Vb, where R ¹⁷ , L-, -W-, -Y-, -D, w and y are defined above;

In yet another embodiment of the invention, the reactive group of said extension component comprises a reactive site capable of reacting with a modified carbohydrate group (—CHO) that may be present on the ligand. For example, a carbohydrate may be weakly oxidized with a reagent such as sodium periodate, and the resulting (-CHO) group of oxidized carbohydrate may be condensed with an extension component containing a functional group, such as hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone hydrazine carboxylate and aryl hydrazide described by T. Kaneko et al. (1991) Bioconjugate Chem. 2: 133-41. A representative extension component of this embodiment of the present invention is indicated by square brackets in formulas VIa, VIb and VIc, where —R ¹⁷ , L—, —W—, —Y—, —D, w and y are defined above.

4.3.2. Amino Acid Component

The amino acid component (-W-), if present, binds the extension component to the spacer element, if such a spacer component is present, binds said extension component to the drug molecule, if the specified spacer component is absent, and binds the ligand component to the drug molecule if said extension component and spacer component are absent.

W _w - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide component. Each of the components -W- independently has the formula shown below in square brackets, and w means an integer from 0 to 12:

where R ¹⁹ represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH ₂ OH, —CH (OH) CH ₃ , —CH ₂ CH ₂ SCH ₃ , —CH ₂ CONH ₂ , -CH ₂ COOH, -CH ₂ CH ₂ CONH ₂ , -CH ₂ CH ₂ COOH, - (CH ₂ ) ₃ NHC (= NH) NH ₂ , - (CH ₂ ) ₃ NH ₂ , - (CH ₂ ) ₃ NHCOCH ₃ , - (CH ₂ ) ₃ NHCHO, - (CH ₂ ) ₄ NHC (= NH) NH ₂ , - (CH ₂ ) ₄ NH ₂ , - (CH ₂ ) ₄ NHCOCH ₃ , - (CH ₂ ) ₄ NHCHO, - (CH ₂ ) ₃ NHCONH ₂ , - (CH ₂ ) ₄ NHCONH ₂ , -CH ₂ CH ₂ CH (OH) CH ₂ NH ₂ , 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, phenyl, cyclohexyl.

The amino acid component can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to release a drug molecule (-D), which, in one embodiment, is released after protonation in vivo to form a drug (D). Representative components of W _{w are} represented by formulas (VII) - (IX):

where R ²⁰ and R ²¹ have the following meanings:

R ²⁰ R ²¹ benzyl (CH ₂ ) ₄ NH ₂ methyl (CH ₂ ) ₄ NH ₂ isopropyl (CH ₂ ) ₄ NH ₂ isopropyl (CH ₂ ) ₃ NHCONH ₂ benzyl (CH ₂ ) ₃ NHCONH ₂ Isobutyl (CH ₂ ) ₃ NHCONH ₂ sec-butyl (CH ₂ ) ₃ NHCONH ₂

(CH ₂ ) ₃ NHCONH ₂ benzyl methyl benzyl (CH ₂ ) ₃ NHC (= NH) NH ₂

where R ²⁰ , R ²¹ and R ²² have the following meanings:

R ²⁰ R ²¹ R ²² benzyl benzyl (CH ₂ ) ₄ NH ₂ isopropyl benzyl (CH ₂ ) ₄ NH ₂ N benzyl (CH ₂ ) ₄ NH ₂

where R ²⁰ , R ²¹ , R ²² and R ²³ have the following meanings:

R ²⁰ R ²¹ R ²² R ²³ N benzyl Isobutyl N methyl Isobutyl methyl Isobutyl

Representative amino acid components are, but are not limited to, those of formula (VII), wherein: R ²⁰ is benzyl and R ²¹ is - (CH ₂ ) ₄ NH ₂ ; R ²⁰ represents isopropyl, and R ²¹ represents - (CH ₂ ) ₄ NH ₂ ; R ²⁰ represents isopropyl, and R ²¹ represents - (CH ₂ ) ₃ NHCONH ₂ . Another representative amino acid component is a component of formula (VIII), where R ²⁰ represents benzyl, R ²¹ represents benzyl, and R ²² represents - (CH ₂ ) ₄ NH ₂ .

Suitable components -W _w - can be obtained and optimized for their selectivity for enzymatic digestion with specific enzymes, for example, tumor-associated protease. In one embodiment of the invention, the -W _w - component is a component whose cleavage is catalyzed by cathepsin B, C and D or a plasmin protease.

In one embodiment of the invention, the -W _w - component is a dipeptide, tripeptide, tetrapeptide or pentapeptide.

If R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ are not hydrogen, then the carbon atom through which R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ is bonded is chiral.

Each carbon atom through which R ¹⁹ , R ²⁰ , R ²¹ , R ^22, or R ²³ is bonded independently has a (S) or (R) configuration.

In one embodiment of the amino acid component, said amino acid component is valine-citrulline. In another embodiment, the amino acid component is phenylalanine-lysine (i.e., fk). In another embodiment of the amino acid component, said amino acid component is N-methylvalin-citrulline. In yet another aspect, the amino acid component is 5-aminovalerianic acid, homophenylalanine-lysine, tetraisoquinolinocarboxylate-lysine, cyclohexylalanine-lysine, isonipecotinic acid-lysine, beta-alanine-lysine, glycine-serine-valine-glutamine, and isonipecot.

In some embodiments of the invention, the amino acid component may contain naturally occurring amino acids. In other embodiments of the invention, the amino acid component may contain unnatural amino acids.

4.3.3. Spacer component

The spacer component (-Y-), if present, binds the amino acid component to the drug molecule, if such an amino acid component is present. Alternatively, said spacer component binds an extension component to a drug molecule if such an amino acid component is absent. Said spacer component also binds the drug molecule to the ligand component if both the amino acid component and the extension component are absent.

Spacer components are components of two general types: self-eliminating and non-self-eliminating. A non-self-eliminating spacer component is a component where all or part of the spacer component remains bound to the drug molecule after cleavage, and in particular enzymatic cleavage of the amino acid component from the drug-linker-ligand conjugate or from the drug-linker compound " Examples of a non-self-eliminating spacer component are, but are not limited to, a glycine-glycine spacer component and a glycine spacer component (both of which are shown in Scheme 1) (see below). If a representative compound containing a glycine-glycine spacer component or glycine spacer component is enzymatically digested by a tumor-associated protease associated with protease cancer cells or protease-associated lymphocytes, then the glycine-glycine-drug molecule or the glycine-drug molecule is cleaved from LA _a -W _w . In one embodiment of the invention, an independent hydrolysis reaction occurs in the target cells and leads to cleavage of the bond in the glycine-drug molecule with the release of the drug.

In another embodiment, —Y _y — is p-aminobenzyl alcohol (RAB) (see Schemes 2 and 3), the phenylene portion of which is substituted with Q _m , where Q is —C ₁ -C ₈ alkyl, —O— (C ₁ -C ₈ alkyl), halogen, nitro or cyano, and m is an integer from 0 from 4.

Scheme 1

In one embodiment of the invention, the non-self-eliminating spacer component (-Y-) is -Gly-Gly-. In another embodiment of the invention, the non-self-eliminating spacer component (-Y-) is -Gly-.

In one embodiment, the present invention relates to a drug-linker compound or a drug-linker-ligand conjugate where the spacer component is absent (y = 0), or a pharmaceutically acceptable salt or solvate thereof.

Alternatively, a representative compound containing a self-eliminating spacer component can release -D without the need for a separate hydrolysis step. In this embodiment, —Y— is a PAB group which is bonded to W _w through a nitrogen atom of the amino group of PAB and which is directly bonded to —D via a carbonate, carbamate or ether group. Not limited to any particular theory or mechanism, the authors present below Scheme 2, which illustrates a possible mechanism for cleavage of a drug from the RAB group, which is directly attached to -D via a carbamate or carbonate group, and this mechanism is described in Toki et al. , (2002) J. Org. Chem. 67: 1866-1872.

Scheme 2

where Q is C ₁ -C ₈ alkyl, -O (C ₁ -C ₈ alkyl), 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 below Scheme 3, which illustrates a possible mechanism for cleavage of a drug from a RAB group that is attached to -D via an ether or amine bond.

Scheme 3

where Q is —C ₁ -C ₈ alkyl, —O (C ₁ -C ₈ alkyl), halogen, nitro or cyano; m is an integer from 0 to 4, and p is from about 1 to 20.

Other examples of self-eliminating spacers include, but are not limited to, aromatic compounds which, by their electronic properties, are similar to the RAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett 9: 2237) and ortho- or para-aminobenzylacetal. Spacers that cyclize after hydrolysis of the amide bond can also be used, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., Chemistry Biology, 1995, 2, 223), suitably substituted with bicyclo [2.2.1] - and bicyclo [2.2.2] cyclic systems (Storm et al., J. Amer. Chem. Soc. 1972, 94, 5815) and amides of 2-aminophenylpropionic acid (Amsberry et al., J. Org. Chem., 1990 55, 5867). Eliminating amine-containing drugs that are substituted at the α-position of glycine (Kingsbury et al., J. Med. Chem. 1984, 27, 1447) are also examples of self-eliminating spacers used in representative compounds.

In one embodiment of the invention, the spacer component is branched bis (hydroxymethyl) styrene (BHMS), shown in Scheme 4, and can be used to incorporate and release a variety of drugs.

Scheme 4

where Q is —C ₁ -C ₈ alkyl, —O (C ₁ -C ₈ alkyl), halogen, nitro or cyano; m is an integer from 0 to 4, n is 0 or 1, and p is about 1 to 20.

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

In one aspect of the invention, the spacer components (-Y _y -) are represented by formulas (X) - (XII):

where Q is —C ₁ -C ₈ alkyl, —O (C ₁ -C ₈ alkyl), halogen, nitro or cyano; and m is an integer from 0 to 4;

and

Variants of the antibody-drug conjugate compounds of formulas 1a 'and Ic are:

where each of w and y is 0.

и

and

4.4. Component “drug” (molecule)

The drug molecule (D) of the antibody-drug conjugate (ADC) is a type of dolastatin / auristatin (US Pat. No. 5,635,483; 5,780,588), which has been shown to adversely affect the dynamics of microvasculature, GTP hydrolysis, and nuclear fission and cells (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and have anti-cancer (US Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents. Chemother 42: 2961-2965).

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

In one embodiment of the invention, —D is a compound of formulas D _E or D _F :

where in each position independently:

R ^{2 is} selected from H and C ₁ -C ₈ alkyl;

R ^{3 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{4 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ^{5 is} selected from H and methyl;

or R ⁴ and R ⁵ taken together form a carbocyclic ring and have the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle , and n is selected from 2, 3, 4, 5, and 6;

R ^{6 is} selected from H and C ₁ -C ₈ alkyl;

R ^{7 is} selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkylaryl, C ₁ -C ₈ alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

each of R ^{8 is} independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, and O- (C ₁ -C ₈ alkyl);

R ^{9 is} selected from H and C ₁ -C ₈ alkyl;

R ¹⁰ selected from aryl or C ₃ -C ₈ heterocycle;

Z represents O, S, NH or —NR ¹² , where R ¹² represents C ₁ -C ₈ alkyl;

R ^{11 is} selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, - (R ¹³ O) _m —R ¹⁴ or (R ¹³ O) _m —CH (R ¹⁵ ) ₂ ;

m is an integer from 1 to 1000;

R ¹³ represents C ₂ -C ₈ alkyl;

R ¹⁴ represents H or C ₁ -C ₈ alkyl;

each of R ¹⁵ independently represents H, COOH, - (CH ₂ ) _n —N (R ¹⁶ ) ₂ , - (CH ₂ ) _n —SO ₃ H, or - (CH ₂ ) _n —SO ₃ —C ₁ —C ₈ alkyl;

each of R ¹⁶ independently represents H, C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH,

R ^{18 is} selected from —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ -aryl, —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ - (C ₃ -C ₈ heterocycle), and —C (R ⁸ ) ₂ -C (R ⁸ ) ₂ - (C ₃ -C ₈ carbocycle); and

n is an integer from 0 to 6.

In one embodiment of the invention, R ³ , R ⁴ and R ⁷ independently represent isopropyl or sec-butyl, and R ⁵ represents —H. In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, R ⁵ is —H, and R ⁷ is sec-butyl.

In another embodiment of the invention, each of R ² and R ⁶ is methyl, and R ⁹ is —H.

In yet another embodiment of the invention, each of R ⁸ is —OCH ₃ .

In a representative embodiment of the invention, each of R ³ and R ⁴ is isopropyl, each of R ² and R ⁶ is methyl, R ⁵ is H, R ⁷ is sec-butyl, each of R ⁸ is —OCH ₃ , and R ⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment of the invention, R ¹⁰ is aryl.

In a representative embodiment of the invention, R ¹⁰ is phenyl.

In a representative embodiment of the invention, if Z is —O—, then R ¹¹ is H, methyl or tert-butyl.

In one embodiment of the invention, if Z is —NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —N (R ¹⁶ ) ₂ and R ¹⁶ is - C ₁ -C ₈ alkyl or - (CH ₂ ) _n -COOH.

In another embodiment, if Z is —NH, then R ¹¹ is —CH (R ¹⁵ ) ₂ , where R ¹⁵ is - (CH ₂ ) _n —SO ₃ H.

Representative “drug” components (-D) are drug molecules having the following structures:

and their pharmaceutically acceptable salts or solvates.

In one aspect of the invention, hydrophilic groups, such as, but not limited to, triethylene glycol esters (TEG), as shown above, may be attached to the “drug” component represented by R ¹¹ . Without claiming to be a specific theory, the authors believe that hydrophilic groups contribute to internalization and prevent the agglomeration of the “drug” component.

4.5. Ligand component

The ligand component (L-) includes any ligand (L) component that binds, either associates through a reaction, or complexes with a receptor, antigen, or other susceptible molecule associated with this population of target cells. A ligand is a molecule that binds, forms complexes, or reacts with a part of the cell population that is analyzed for a therapeutic or some other biological modification. In one aspect of the invention, the ligand component delivers the drug component to a particular population of target cells with which the ligand component reacts. Such ligands are, but are not limited to, high molecular weight proteins, such as, for example, full-sized antibodies, antibody fragments, proteins with lower molecular weight, polypeptides or peptides, lectins, glycoproteins, non-peptides, vitamins, trace elements (such as, but not limited to, transferrin), or any other molecule or any other substance that binds to a cell.

The ligand component may form a bond with the extension component, the amino acid component, the spacer component, or the “drug” component. The ligand component can form a bond with the linker component through the heteroatom of the ligand. The heteroatoms that 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 the carbonyl, carboxyl or hydroxyl group of the ligand) and nitrogen (In one embodiment of the invention, from the primary or secondary amino group of the ligand). These heteroatoms can be present on the ligand in its natural state, for example, in a 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 this ligand is attached to the linker via a sulfur atom of the sulfhydryl group.

In yet another aspect of the invention, the ligand has one or more lysine residues that can be chemically modified to include one or more sulfhydryl groups. The specified ligand component is linked to the linker component via a sulfur atom of a sulfhydryl group. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl-S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Trout reagent).

In another embodiment of the invention, the ligand may have one or more carbohydrate groups, which can be chemically modified with the introduction of one or more sulfhydryl groups. Said ligand component is bonded to a linker component, such as an extension component, through a sulfur atom of a sulfhydryl group.

In yet another embodiment of the invention, the ligand may have one or more carbohydrate groups that can be oxidized to form an aldehyde group (—CHO) (see, for example, Laguzza et al., J. Med. Chem. 1989, 32 (3), 548-55). The corresponding aldehyde may form a bond with the reactive site on the extension component. Reactive sites on 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 modifying proteins for attachment to or binding to drug components are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), which is incorporated herein by reference.

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

Polyclonal antibodies suitable for use are heterogeneous populations of antibody molecules isolated from the serum of immunized animals. Various procedures well known to those skilled in the art can be performed to produce polyclonal antibodies against the antigen of interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection of the antigen of interest or its derivative, and such animals include, but are not limited to, rabbits, mice, rats and guinea pigs. Various adjuvants may be used to enhance the immune response, depending on the type of host, and such adjuvants include, but are not limited to, Freund's adjuvant (complete or incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin , pluronic polyols, polyanions, peptides, oil emulsions, cochlear lymphocytes hemocyanins, dinitrophenol and potentially human adjuvants such as BCG (Calmette-Guerin bacillus) and Corynebacterium parvum . Such adjuvants are also well known in the art.

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

Suitable monoclonal antibodies for use include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or human-mouse chimeric monoclonal antibodies (or other species). Human monoclonal antibodies can be obtained by any various methods known to those skilled 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 may also be bespecifically antibody. Methods for producing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305: 537-539). Due to the presence of a randomized set of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one molecule has the “correct” bispecific structure. Similar procedures are described in International Application Publication No. WO 93/08829 and in Traunecker et al. EMBO J. 10: 3655-3659 (1991).

According to another approach, the variable domains of an antibody with the desired binding specificity (antibody-antigen binding sites) are attached to immunoglobulin constant domain sequences. A preferred hybrid is an immunoglobulin heavy chain constant domain hybrid comprising at least a portion of a hinge region, a C _H 2 and C _H 3 region. Moreover, it is preferable that this hydride has a first constant region of the heavy chain (C _H 1) containing the site necessary for the binding of the light chain and present in at least one of the hybrids. Nucleic acids with sequences encoding the immunoglobulin heavy chain hybrids, and if necessary, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. This allows for high flexibility in correcting the ratios of three polypeptide fragments in those embodiments of the invention in which there are unequal ratios of the three polypeptide chains used in this design, and to obtain optimal yields. However, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector if the expression of at least two polypeptide chains in equal proportions gives high yields or if these relations are not significant.

In one embodiment of this approach, the bispecific antibodies have a hybrid immunoglobulin heavy chain with a first binding specificity on one branch and an immunoglobulin heavy chain-light chain hybrid pair (providing a second binding specificity) on another branch. Such an asymmetric structure facilitates the separation of the desired bispecific compound from undesired combinations of immunoglobulin chains, since such separation facilitation is due to the presence of the immunoglobulin light chain in only one half of the bispecific molecule (see International Application Publication No. WO94 / 04690, which is incorporated herein in its entirety by links).

A more detailed description of the preparation of bispecific antibodies can be found, for example, in 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. Using such methods, bispecific antibodies can be prepared for the treatment or prophylaxis of the diseases indicated herein.

Bifunctional antibodies are also described in European Patent Application Publication No. EPA 0105360. As described in this publication, hybrid or bifunctional antibodies can be obtained by biological methods, i.e. cell fusion methods, or chemical methods, in particular using cross-linking agents or reagents forming disulfide bridges, and these antibodies can be whole antibodies or fragments thereof. Methods for producing such hybrid antibodies are described, for example, in International Publication No. WO 83/03679 and in European Patent Application Publication No. EPA 0217577, each of which is incorporated herein by reference.

An antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, microbial antigens, or other antibodies associated with tumor cells or with a matrix. Accordingly, the term “functionally active” means that a given fragment, derivative or analogue has the ability to produce anti-anti-idiotypic antibodies that recognize the same antigen as the antibody from which the fragment, derivative or analogue is derived. In particular, in a representative embodiment of the invention, the antigenicity of the idiotype of an immunoglobulin molecule can be increased by deletion of the framework sequences and CDR sequences that are located on the C-terminus of the CDR sequence that specifically recognizes this antigen. In order to determine which CDR sequences bind to an antigen, antigen binding assays can be performed using synthetic peptides containing CDR sequences by any known method commonly used by those skilled in the art for binding analysis (e.g., analysis using the BIA CORE program) (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 are antibody fragments, such as, but not limited to, F (ab ') ₂ fragments that contain a variable region, a constant region of the light chain and the CH1 domain of the heavy chain and which can be produced by hydrolysis of the antibody molecule with pepsin, and Fab fragments that can be produced by reducing the disulfide bridges of the F (ab ') ₂ fragments. Other suitable antibodies are antibody heavy and light chain dimers, or any minimal fragment thereof, such as Fv, or single chain antibodies (scAb) (for example, as described in US Pat. No. 4,946,778; 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 same specificity as this antibody.

In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing human and non-human parts, which can be obtained using standard recombinant DNA methods, are suitable antibodies. A chimeric antibody is a molecule in which different parts are derived from animals of various species, namely, parts containing a variable region derived from constant regions of a murine monoclonal antibody and human immunoglobulin (see, for example, Cabilly et al., US Patent No. 4816567 and Boss et al., US patent No. 4816397, which in their entirety are introduced into the present description by reference). Humanized antibodies are antibody molecules derived from non-human animals and having one or more hypervariable regions (complementarity-determining regions, CDRs) derived from non-human animals and a framework region derived from a human immunoglobulin molecule (see ., for example, Queen, US patent No. 5585089, which in its entirety is introduced into the present description by reference). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA methods known to those skilled in the art, for example by the methods described in International Application Publication No. WO 87/02671; European Patent Application Publication No. 184187; European Patent Application Publication No. 171496; in European Patent Application Publication No. 173494; in the publication of International application No. WO 86/01533; US Pat. No. 4,816,567; European Patent Application Publication 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; in US 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 incorporated into this description by reference.

Full-sized human antibodies are particularly preferred and can be produced in a transgenic mouse that is unable to express endogenous immunoglobulin heavy and light chain genes, but is capable of expressing human immunoglobulin heavy and light chain genes. Transgenic mice are immunized with the selected antigen, for example, the whole polypeptide according to the invention or part thereof, in the usual way. Monoclonal antibodies directed against this antigen can be obtained using standard hybridoma technology. Human immunoglobulin transgenes present in transgenic mice rearrange in the process of B-cell differentiation, and then the class of immunoglobulins and somatic mutation are switched. Thus, the use of such a technique allows the production of therapeutically effective antibodies of IgG, IgA, IgM and IgE. A general overview of this technology for producing human antibodies can be found in Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93).

A detailed discussion of such technology for the production of human antibodies and human monoclonal antibodies, as well as protocols for the production of such antibodies can be found, for example, in US patent No. 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, by Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

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

In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, in which, for example, the antibody is linked via a covalent bond (e.g., peptide bond) at either the N-terminus or C-terminus to the amino acid sequence of another protein (or part thereof, preferably having at least 10, 20 or 50 amino acids), which is not an antibody. Moreover, the antibody or its fragment is preferably covalently linked to another protein at the N-terminus of the constant domain.

The term “antibodies” includes analogs and derivatives that are modified, that is, by covalent binding of any type of molecule, provided that such covalent binding allows the antibody to retain its immunospecificity of binding to the antigen. For example, derivatives and analogs of antibodies include, but are not limited to, derivatives and analogs that have been further modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protective / blocking groups, proteolytic cleavage, binding to cellular antibody or other protein, etc. Any of a variety of chemical modifications can be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, such an analogue or such derivative may contain one or more non-natural amino acids.

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

In a specific embodiment of the invention, known antibodies can be used to treat or prevent cancer. Antibodies immunospecific to a cancer cell antigen can be obtained from commercial sources, or they can be produced by any methods known to those skilled in the art, such as, for example, recombinant DNA expression methods. The nucleotide sequence encoding antibodies that are immunospecific for a cancer cell antigen can be obtained, for example, from the Genbank database or a similar database by methods described in the literature, or by routine cloning and sequencing. Examples of antibodies that can be used to treat cancer include, but are not limited to, a humanized monoclonal antibody against HER2, Herceptin® (trastuzumab; Genentech) for the treatment of patients with breast cancer giving metastases; Rituxan® antibody (rituximab; Genentech), which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex antibody (AltaRex Corporation, MA), which is a murine antibody for treating ovarian cancer; Panorex antibody (Glaxo Wellcome, NC), which is a mouse IgG _2a antibody for treating colon cancer; Cetuximab Erbitux antibody (Imclone Systems Inc., NY), which is an anti-EGFR chimeric IgG antibody for treating a cancer expressing positive for epidermal growth factor such as head and neck cancer; Vitaxin antibody (MedImmune, Inc., MD), which is a humanized antibody for treating sarcoma; Campath I / H antibody (Leukosite, MA), which is a humanized IgG1 antibody for treating chronic lymphocytic leukemia (CLL); Smart MI95 antibody (Protein Design Labs, Inc., CA), which is an anti-CD33 IgG antibody for treating acute myeloid leukemia (AML); LymphoCide antibody (Immunomedics, Inc., NJ), which is a humanized anti-CD22 IgG antibody for treating non-Hodgkin lymphoma; Smart ID10 antibody (Protein Design Labs, Inc., CA), which is an anti-HLA-DR antibody for treating non-Hodgkin lymphoma; an Oncolym antibody (Techniclone, Inc., CA), which is a radioactively labeled anti-HLA-Dr10 antibody for treating non-Hodgkin lymphoma; Allomune antibody (BioTransplant, CA), which is a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin antibody (Genentech, Inc., CA), which is a humanized anti-VEGF antibody for treating lung cancer and colon cancer; an Epratuzumab antibody (Immunomedics, Inc., NJ and Amgen, CA), which is an anti-CD22 antibody for treating non-Hodgkin lymphoma; and a CEAcide antibody (Immunomedics, NJ), which is an anti-CEA antibody for treating colon cancer.

Other antibodies that can be used to treat cancer include, but are not limited to, antibodies against the following antigens, such as CA125 (ovarian cancer), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma) , Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colon cancer), placental alkaline phosphatase (carcinoma), prostate specific antigen (prostate cancer), acid prostate phosphatase (prostate cancer), epidermal growth factor (carcinoma), MAGE-1 (carcinoma ohm), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), antitransferrin receptor (carcinoma), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colon cancer) , gp100 (melanoma), MART1 (melanoma), PSA (prostate cancer), IL-2 receptor (T-cell leukemia and T-cell lymphoma), CD20 (non-Hodgkin lymphoma), CD52 (leukemia), CD33 (leukemia ), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma) and the oncogenic product Neu (carcinoma). Some specific and effective antibodies are, but are not limited to, mAb BR96 (Trail PA, Willner D. Lasch SJ Henderson AJ Hofstead, SJ Casazza AM Firestone, RA Hellstrom, I., Hellstrom, KE, “Cure of Xenografted Human Carcinomas by BR96 -Doxorubicin Immunoconjugates ”Science 1993, 261, 212-215), BR64 (Trail PA, Willner D. Knipe J. Henderson AJ Lasch, SJ Zoeckler, ME, Trailsmith MD Doyle, TW King, HD, Casazza, AM Braslawsky, GR Brown JP Hofstead, SJ (Greenfield RS Firestone, RA Mosure K. Kadow, DF, Yang, MB Hellstrom, KE & Hellstrom, I. “Effect of Linker Variation on the Stability, Potency and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105), anti-CD40 antigen mAbs, such as S2C6 mAb (Francisco, JA, Donaldson KL Chace D. Siegall, CB & Wahl AF “Agonistic pr operties and in vivo antitumor activity of the anti-CD40 antibody, SGN-14 ”Cancer Res. 2000, 60, 3225-3231), anti-CD70 antigens mAbs, such as mAb 1F6 and mAb 2F2, and anti-CD30 mAbs, such as AC10 (Bowen MA, Olsen KJ 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). Many other internalizing antibodies that bind to tumor-associated antigens can also be used, and these antibodies are described in the literature (Franke AE, Sievers EL & Scheinberg DA, “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review“ Cancer Biother Radiopharm. 2000, 15, 459-76; Murray JL “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling F. & Dubel S. Recombinant Antibodies, John Wiley & Sons, New York, 1998).

In some embodiments of the invention, said antibody is not trastuzumab (a full-sized humanized anti-HER2 antibody (MW 145167)), herceptin-F (ab ') ₂ (derived from an enzymatically cleaved anti-HER2 antibody, MW 100000), 4D5 (full-sized mouse anti-HER2 hybridoma-produced antibody), rhu4D5 (temporarily expressed full-sized humanized antibody), rhuFab4D5 (recombinant humanized Fab, MW 47738), 4D5Fc8 (full-sized mouse anti-HER2 antibody with mutated Hr sizeless mismatch) or g ovannoe antibody 4D5, wherein the heavy chain hinge region cysteines were mutated to serines and which was expressed in E.coli (and therefore it is non-glycosylated)).

In another specific embodiment of the invention, known antibodies intended for the treatment or prophylaxis of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies that are immunospecific for the antigen of the cell responsible for the production of autoimmune antibodies can be obtained from any organization (for example, a Research Institute or company), or they can be produced by any known method, such as, for example, chemical synthesis or expression of recombinant DNA In another embodiment of the invention, suitable antibodies are immunospecific antibodies intended for the treatment of autoimmune diseases, and such antibodies include, but are not limited to, an antinuclear antibody, an anti-dsDNA antibody, an anti-ssDNA antibody, an anti-cardiolipin IgM and IgG antibody, an IgM antibody and Anti-phospholipid IgG, anti-SM antibody, anti-mitochondrial antibody, thyroid-specific antibody, microsomal antibody, anti-thyroglobulin antibody, anti-SCL-70 antibody, anti-Jo antibody; anti-U ₁ RNP antibody; anti-La / SSB antibody; anti-SSA antibody; anti-SSB antibody; antibody against peritoneal cells; anti-histone antibody; anti-RNP antibody; anti-C-ANCA antibody; anti-P-ANCA antibody; anti-centromere antibody; anti-fibrillarin antibody; and anti-GBM antibody.

In some embodiments of the invention, antibodies suitable for use may bind to both the receptor and the receptor complex expressed on the activated lymphocyte. The receptor or receptor complex may comprise a member of the immunoglobulin gene superfamily, a TNF receptor superfamily member, integrin, a cytokine receptor, a chemokine receptor, a histocompatibility complex protein, 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 TNF receptor superfamily are CD27, CD40, CD95 / Fas, CD134 / ОX40, CD137 / 4-IBB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / 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, CD103 and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectin.

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

In another specific embodiment of the invention, suitable ligands immunospecific for a viral or microbial antigen are monoclonal antibodies. Such antibodies may be chimeric, humanized or human monoclonal antibodies. The term “viral antigen” as used herein includes, but is not limited to, any viral peptide, polypeptide, protein (eg, HIV gp120, HIV nef , RSV glycoprotein F, influenza virus neuraminidase, influenza virus hemaglutinin, tax HTLV, herpes simplex virus glycoprotein ( for example, gB, gC, gD and gE) and the surface antigen of hepatitis B virus), which are capable of producing an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any molecule of a microbial peptide, polypeptide, protein, saccharide, polysaccharide or lipid (eg, a bacterial, fungal, pathogenic protozoa or yeast polypeptide, including, for example, LPS and capsular polysaccharide 5 / 8), which is capable of producing an immune response.

Antibodies that are immunospecific for a viral or microbial antigen can be supplied from commercial companies, 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 chemical synthesis method or expression by recombinant DNA method. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, for example, from the Genbank database or a similar database and the method described in the literature, or it can be obtained by routine cloning and sequencing.

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

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, Clostridium botulinum, Clostridium perfringens, 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, Treponem a carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagie, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasmaiatsi tsippsi tsippsi tsippsippsi tsippsi tsippsippsi tsippsi tsippsippsi tsippsi tsippsippsjtmsippsippsi tsipptsippsjtmsippsippsjtstsippsjtsi tsippsjtstjspptsipptsippsjtstjspptsippsippsipptsippsjtstjsppt pathogenic fungi ( Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa ( Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Pliymomium pliymomias plia mium pomiasia, Brachis pompius or helminths ( Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworms).

Other antibodies that can be used in the present invention for the treatment of viral disease are, 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), measles virus, respiratory syncytial virus, rubella virus, arboviruses, rabd virus, arenaviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A / non-B hepatitis, rhinoviruses, coronaviruses, rotaviruses and human immunodeficiency virus.

To identify effective cell targets for the diagnosis and treatment of cancer, researchers have attempted to identify transmembrane or any other tumor-associated polypeptides that are specifically expressed on the surface of one or more cancer cells of specific type (s) compared to one or more normal non-cancerous cells. In most cases, such tumor-associated polypeptides are expressed in excess on the surface of cancer cells, and not on the surface of non-cancer cells. Identification of such tumor-associated polypeptides of cell surface antigens will allow for specific destruction of target cancer cells by antibody therapy.

Antibodies that are Ab in the antibody-drug conjugates of formula Ic (ADC) and that can be used to treat cancer are, but are not limited to, antibodies against tumor associated antigens (TAA). Such tumor-associated antigens are known to those skilled in the art and can be prepared to produce antibodies using methods and data well known to those skilled in the art. Examples of TAA are (1) - (35), but are not limited to, TAA (1) - (35), listed below. For convenience, data relating to all of these known antigens are presented below and include names, alternative names, Genbank registration numbers, and primary source (s). Tumor-associated antigens to which antibodies are directed include all variants of amino acid sequences and isoforms having sequences that are at least about 70%, 80%, 85%, 90% or 95% identical to the sequences identified in the corresponding sequence list ( SEQ ID NO: 1-35), or sequences identified in the cited works. In some embodiments of the invention, TAAs having amino acid sequence variants have substantially the same biological properties or characteristics as TAAs having the sequence shown in the corresponding sequence listing (SEQ ID NOS: 1-35). So, for example, a TAA having a modified sequence is essentially capable of specifically binding to an antibody that specifically binds to a TAA having the corresponding sequence indicated in the sequence list. These sequences and the descriptions specifically cited herein are hereby incorporated by reference in their entirety.

Tumor-associated antigens (1) - (35)

(1) BMPR1B (type IB bone morphogenesis receptor, 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 (claim 12); US2003134790-AI (pages 38-39); WO2002102235 (Claim 13; Page 296); WO2003055443 (Pages 91-92); WO200299122 (Example 2; Pages 528-530); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; page 183); WO200254940 (Page 100-101); WO200259377 (Page 349-350); WO200230268 (Claim 27; page 376); WO200148204 (example; FIG. 4); NP_001194 receptor for bone morphogenesis protein type IB / pid = NP_001194.1. Cross reference: MIM: 603248; NP_001194.1; NM_001203_1; 502 amino acids:

(2) E16 (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 (claim 12); WO2003016475 (claim 1); WO200278524 (Example 2); WO200299074 (claim 19; pages 127-129); WO200286443 (Claim 27, pages 222, 393); WO2003003906 (claim 10; page 293); WO200264798 (Claim 33; pages 93-95); WO200014228 (claim 5; pages 133-136); US2003224454 (Fig. 3); WO2003025138 (claim 12; page 150); NP_003477 member 5 of the family of soluble carriers 7 (carrier 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 (six-domain transmembrane prostate epithelial antigen, 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 (claim 6); WO2004027049 (Fig. 1L); EP1394274 (Example 11); WO2004016225 (claim 2); WO20030442661 (claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Fig. 2); WO200289747 (Example 5; Pages 618-619); WO2003022995 (Example 9; FIG. 13A, Example 53, Page 173, Example 2; FIG. 2A); NP_036581, six domain prostate epithelial antigen. Cross Reference: MIM: 604415; NP_036581.1; NM_012449_1, 339 amino acids:

(4) 0772P (CA125, MUC16, Genbank reg. No. AF361486 J. Biol. Chem. 276 (29): 27371-27375 (2001)); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; claims 116-121); US2003124140 (Example 16); US2003091580 (claim 6); 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, Yamaguchi N. 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 (claim 14); WO2002102235 (Claim 13; pages 287-288); WO2002101075 (claim 4; pages 308-309); WO200271928 (pages 320-321); WO9410312 (Pages 52-57). Cross reference: MIM: 601051; NP_005814.2; NM_005823_1; 622 amino acids:

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, member 2 of the family of soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transporting protein 3b type II, Genbank reg. No. NM_006424; J. Biol. Chem. 277 (22 ): 19665-19672 (2002), Genomics 62 (2): 281-284 (1999), Feild JA et al. (1999) Biochem. Biophys. Res. Commun. 258 (3): 578-582); WO2004022778 (claim 2); EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569 (claim 1; pages 17-19); WO200157188 (claim 20; page 329); WO2004032842 (Example IV); WO200175177 (claim 24; claims 139-140). Cross Reference: MIM: 604217; NP_006415.1; NM_006424_1, 690 amino acids:

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

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, cDNA RIKEN 2700050C12, cDNA of the RIKEN gene 2700050C12, Genbank reg. No. AY358628); US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (Example 5); WO2003105758 (claim 12); US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148 (claim 20). Cross References: GI: 37182378; AAQ88991.1; AY358628_1; 141 amino acids:

(9) ETBR (type B endothelin receptor, 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 NA, et al. J. Biol. Chem. 268, 3873-3879, 1993 ; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg RL, 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; Hofstra RMW, 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, 2407-24 09, 1995; Auricchio A., et al. Hum. Mol. Genet. 5: 351-354, 1996; Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., 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); WO2004048938 (Example 2); WO2004040000 (Claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (FIG. 1); WO2003016494 (Fig. 6); WO2003025138 (Claim 12; Page 144); WO200198351 (claim 1; pages 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; pages 297-299); US2003109676; US6518404 (FIG. 3); US5773223 (claim 1A; column 31-34); WO2004001004, 442 amino acids:

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank reg. No. NM_017763); WO2003104275 (claim 1); WO2004046342 (Example 2); WO20030442661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6). Cross Reference: LocusID: 54894; NP_060233.2; NM_017763_1, 783 amino acids:

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein of the prostate Genbank Reg. No. AF455138; Lab. Invest. 82 (11): 1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; FIG. 1); WO200272596 (Claim 13; pages 54-55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (Claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10). Cross references: GI: 22655488, AAN04080.1; AF455138_1, 490 amino acids:

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cationic channel of the transient receptor, member of subfamily M, Genbank reg. No. NM_017636; Xu XZ et al., Proc. Natl. Acad. Sci., USA, 98, USA (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J. Biol. Chem. 278 (33): 30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; claims 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIGS. 1A-D). Cross reference: 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); WO2003083041 (Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; pages 52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; pages 94-95, 105); WO200222808 (claim 2; FIG. 1); US5854399 (Example 2; Columns 17-18); US5792616 (FIG. 2). Cross References: MIM: 187395; NP_003203.1; NM_003212_1, 188 amino acids:

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs. 73792, Genbank reg. No. M26004; Fujisaku et al. (1989) J. Biol. Chem. 264 (4) : 2118-2125); Weis J.J. et al., J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al., Proc. Natl. Acad. Sci., USA, 84, 9194-9198, 1987; Barel M., et al., Mol. Immunol. 35, 1025-1031, 1998; Weis J.J. 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 (claim 9); WO2004045520 (Example 4); WO9102536 (Figs. 9.1-9.9); WO2004020595 (claim 1); registration numbers: P20023; Q13866; Q14212, EMBL; M26004; AAA35786.1, 1033 amino acids:

(15) CD79b (CD79B, CD79β, IGb (beta protein associated with immunoglobulin), B29, 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. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 1, page 15); US5644033, WO2003048202 (claim 1, pages 306 and 309); WO99 / 558658, US6534482 (claim 13; FIG. 17A / B); WO200055351 (claim 11, pages 1145-1146). Cross reference: MIM: 147245; NP_000617.1; NM_000626_1; 229 amino acids:

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a containing the 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 MJ et al. (2001) Biochem. Biophys. Res. Commun. 280 (3): 768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; Fig. 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25) Cross-references: 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); Yamamoto, T. et al., Nature 319, 230-234, 1986; Semba K. 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; Ehsani A. et al., (1993) Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIGS. 1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Pages 95-107); WO200212341 (claim 68; FIG. 7); WO200213847 (Pages 71-74); WO200214503 (pages 114-117); WO200153463 (claim 2; pages 41-46); WO200141787 (page 15); WO200044899 (Claim 52; FIG. 7); WO200020579 (Claim 3; FIG. 2); US5869445 (claim 3 of the claims; columns 31-38); WO9630514 (claim 2; pages 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); reg. No. PO4626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1. 1255 amino acids:

(18) NCA (CEACAM6, Genbank Reg. No. M18728); 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 (claim 7); WO2004044178 (Example 4); WO2004031238; WO20030442661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; claims 427); WO200260317 (claim 2); reg. No. P40199; Q14920; EMBL; M29541; AAA59915.1, EMBL; M18728; 344 amino acids:

(19) MDP (DPEP1, Genbank Reg. No. BC017023, Proc. Natl. Acad. Sci., USA, 99 (26): 16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (Claim 33; pages 85-87); JPO5003790 (Figs. 6-8); WO9946284 (Fig. 9). Cross reference: MIM: 179780; AAN17023.1; BC017023_1, 411 amino acids:

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank Reg. No. AF184971); Clark H.F. et al., Genome Res. 13, 2265-2270, 2203; Mungall A.J. 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; EP139274 (Example 11); US2004005320 (Example 5); WO2003029262 (pages 74-75); WO2003002717 (claim 2; page 63); WO200222153 (pages 45-47); WO2003002717 (claim 2), US2002042366 (pages 20-21); WO200146261 (Pages 57-59); WO200146232 (Pages 63-65); WO9837193 (claim 1; 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 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1); 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 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (Pages 128-132); WO200053216 (claim 1; page 42). Cross reference: MIM: 600997; NP_004433.2; NM_004442_1, 987 amino acids:

(23) ASLG659 (B7h, Genbank reg. No. AX092328), US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3); US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (Fig. 60); WO2002102235 (Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (claim 6; FIG. 10); WO2001194641 (claim 12; FIG. 7b); WO200202624 (claim 13, FIGS. 1A-1B); US2002034749 (claim 54 of the claims; pages 45-46); WO200206317 (Example 2; Pages 320-321; Claim 34; Claims 321-322); WO200271928 (pages 468-469); WO200202587 (Example 1, FIG. 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Pages 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-234, 452-453); WO0116318; 282 amino acids:

(24) PSCA (Prostate Stem Cell Antigen Precursor, 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; EP1394274 (Example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO2003003906 (claim 10; page 288); WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; Fig. 1b); WO20032752 (Claim 18; FIG. 1); WO9851805 (claim 17; claims 97); WO9851824 (claim 10; page 94); WO98440403 (claim 2; FIG. 1B); reg. No. O43653; EMBL; AF043498; AAC39607.1, 123 amino acids

(25) GEDA (Genbank Reg. No. AY260763); protein similar to the partner “hybrid HMGIC lipomas AAP14954-Homo sapiens: View: Homo sapiens (human) WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (Claim 45). Cross reference: GI: 30102449; AAP14954.1; AY260763_1, 236 amino acids

(26) BAFF-R (B-cell activation factor receptor, BLyS 3 receptor, BR3, Genbank reg. No. NP_443177.1); NP_443177 BAFF receptor / pid = NP_443177.1-Homo sapiens Thompson J.S. et al. Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (example; pages 32-33); WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; pages 615-616); WO200294852 (columns 136-137); WO200238766 (claim 3 of the claims; page 133); WO200224909 (Example 3; FIG. 3). Cross reference: 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 (claim 9); WO2003072036 (claim 1; FIG. 1); WO200278524 (Example 2). Cross references: MIM: 107266; NP_001762.1; NM_001771_1, 847 amino acids:

(28) CD79a (CD79A, CD79α, immunoglobulin-bound alpha protein, B-cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with IgM molecules on the cell surface, transmitting a signal involved in the differentiation of B- cell) 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 (claim 9); US2002150573 (claim 4, pages 13-14); WO99558658 (claim 13, FIG. 16); WO9207574 (FIG. 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 (Burkitt’s lymphoma receptor 1, a G-protein bound receptor activated by chemokine CXCL13, provides lymphocyte migration and humoral defense, and also plays a role in HIV-2 infection and, probably, in the development of AIDS, lymphomas, myelomas 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 (FIG. 1); WO200157188 (claim 20 of the claims; page 269); WO200172830 (Pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38); US5440021 (Example 2, columns 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7; FIG. 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 the MHC class II molecule (antigen Ia), which binds to 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: 6p21.3, 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 (claim 13; FIG. 15); US6153408 (columns 35-38); US5976551 (columns 168-170); US6011146 (columns 145-146); Kashara et al., (1989) Immunogenetics 30 (1): 66-68; Larhammar et a l. (1985) J. Biol. Chem. 260 (26): 14111-14119; 273 amino acids:

(31) P2X5 (the -5 ion channel opened by the P2X purinergic receptor ligand; the extracellular ATP opening channel can participate in synaptic transmission and neurogenesis, and its deficiency can play a role in the pathophysiology of idiopathic bladder dysfunction) PROTEIN SEckENCE Fullq mg ... lephrst (1..422; 422 amino acids), pI: 7.63; MW 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Reg. No. NP_002552.2; Le et al. (1997) FEBS Lett. 418 (1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10: 165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); W02003029277 (Page 82); 422 amino acids:

(32) CD72 (CD72 lineage antigen, Lyb-2) PROTEIN SEQUENCE Full maeaity ... tafrfpd (1..359; 359 amino acids), pI: 8.66, MW: 40255 TM: 1 [P] Gene Chromosome : 9p13.3, reg. No. NP_001773.1; WO2004042346 (Claim 65); 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 (lymphocytic antigen 64 (RP105), a membrane protein belonging to the leucine-rich repeat (LRR) family of proteins, type I, which regulates B cell activation and apoptosis, and the loss of function of this protein is associated with systemic progression in patients lupus erythematosus) PROTEIN SEQUENCES Full mafdvsc ... rwkyqhi (1..661; 661 amino acids), pI: 6.20, MW: 74147 TM 1 [P] Gene Chromosome 5q12, Genbank reg. No. NP_005573.1; US2002193567; WO9707198 (claim 11; pages 39-42); Miura et al. (1996) Genomic 38 (3): 299-304; Miura et al., (1998) Blood 92: 2815-2822; WO2003083047; WO9744452 (claim 8; pages 57-61); WO200012130 (Pages 24-26); 661 amino acids:

(34) FCRH1 (Fc receptor-like protein 1, putative receptor for the immunoglobulin Fc domain, which contains Ig type C2 domains 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 (claim 6, FIG. 18E-1-18-E2); Davis et al. (2001) Proc. Natl. Acad. Sci., USA, 98 (17): 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7); 429 amino acids:

(35) IRTA2 (a translocation-associated immunoglobulin 2 superfamily receptor, a putative immunoreceptor that may play a role in B cell development and lymphogenesis; moreover, in some malignant B cells, gene regulation is disturbed 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. 97); Nakayama et al., (2000) Biochem. Biophys. Res. Commun. 277 (1): 124-127; WO2003077836; WO200138490 (claim 3 of the claims; FIG. 18B-1-18B-2); 977 amino acids:

See also: WO04 / 045516 (June 3, 2004); WO03 / 000113 (January 3, 2003); WO02 / 016429 (February 28, 2002); WO02 / 16581 (February 28, 2002); WO03 / 024392 (March 27, 2003); WO04 / 016225 (February 26, 2004); WO01 / 40309 (June 7, 2001) and US Provisional Application Reg. No. 60/520842 “COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN”, filed November 17, 2003, each of which is hereby incorporated by reference in its entirety.

In one embodiment of the invention, the ligand-linker-drug conjugate has the formula IIIa, wherein the ligand is an Ab antibody, including an antibody that binds to at least one of the antigens CD30, CD40, CD70, Lewis Y, w = 0, y = 0, and D has the formula Ib. Representative conjugates of formula IIIa are conjugates in which R ¹⁷ is - (CH ₂ ) ₅ -. The present invention also includes those 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, that is, conjugates of formula Ia, where p has a value ranging from about 3 to 8 for example about 3-5. The scope of the compounds of the invention also includes conjugates having combinations of the structural features indicated in this section.

In another embodiment of the invention, the ligand-linker-drug conjugate has the formula IIIa, wherein the ligand is an Ab antibody, including an antibody that binds to one of the antigens CD30, CD40, CD70, Lewis Y, w = 1, y = 0, and D has the formula Ib. The present invention also includes those conjugates of formula IIIa in which R ¹⁷ is - (CH ₂ ) ₅ -. The present invention also includes those conjugates of formula IIIa in which W is 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, that is, conjugates of formula Ia, where p has a value ranging from about 3 to 8, for example about 3 -5. Representative conjugates are also conjugates having combinations of the structural features indicated in this section.

In another embodiment of the invention, the ligand-linker-drug conjugate has the formula IIIa, wherein the ligand is an Ab antibody, including an antibody that binds to one of the antigens CD30, CD40, CD70, Lewis Y, w = 1, y = 1, and D has the formula Ib. The present invention also includes those conjugates of formula IIIa in which R ¹⁷ is - (CH ₂ ) ₅ -. The present invention also includes those conjugates of formula IIIa in which W is 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. Conjugates having combinations of the structural features mentioned in this section are also included in the scope of the compounds of the invention.

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

In another embodiment, the present invention relates to a TAA compound or a pharmaceutically acceptable salt or solvate thereof, which are present in isolated and purified form.

In another embodiment, the present invention relates to a method for preventing or inhibiting the growth of tumor or cancer cells, comprising administering to a patient, for example, a person suffering from hyperproliferative disorder, a TAA compound or a pharmaceutically acceptable salt or solvate thereof in an amount effective to prevent or inhibit the growth of tumor or cancer cells.

In another embodiment, the present invention relates to a method for treating cancer, comprising administering to a patient, for example, a person suffering from hyperproliferative disorder, a TAA compound or a pharmaceutically acceptable salt or solvate thereof in an amount effective for treating cancer, alone or in combination with an effective amount of another anti-cancer agent .

In another embodiment, the present invention relates to a method for treating an autoimmune disease, comprising administering to a patient, for example, a person suffering from hyperproliferative disorder, a TAA compound or a pharmaceutically acceptable salt or solvate thereof in an amount effective to treat an autoimmune disease.

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

4.5.1. Recombinant Antibody Production

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

For recombinant expression of antibodies or fragments thereof, derivatives or analogs, it is necessary to construct a nucleic acid encoding such an antibody. If the nucleotide sequence for a given antibody is known, then a nucleic acid assembly encoding such an antibody can be assembled from chemically synthesized oligonucleotides (for example, as described by Kutmeier et al., 1994, BioTechniques 17: 242), and such assembly involves the synthesis of overlapping oligonucleotides containing parts of an antibody coding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides, for example, by PCR.

Alternatively, the nucleic acid molecule encoding the antibody can be obtained from a suitable source. If the clone containing the nucleic acid encoding the specific antibody is not commercially available, but the sequence of the antibody is known, then the nucleic acid encoding the antibody can be obtained from a suitable source (for example, from an antibody cDNA library or from a cDNA library produced from any tissue or any cells expressing immunoglobulin), for example, by PCR amplification using synthetic primers hybridizing at the 3'- and 5'-ends of the sequence, or by clo nucleation using an oligonucleotide probe specific for a particular gene sequence.

If an antibody that specifically recognizes a particular antigen (or source of a cDNA library for cloning a nucleic acid encoding such an immunoglobulin) is not commercially available, then antibodies specific for that antibody can be obtained by any known method, for example, by immunizing a patient or a suitable animal models, 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 (19 75, Nature 256: 495-497) or as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p. 77-96). Alternatively, a clone encoding at least a Fab fragment of an antibody can be obtained by screening Fab-expressing libraries (for example, as described by Huse et al., 1989, Science 246: 1275-1281) for clones of Fab fragments that bind with a specific antigen, or by screening antibody libraries (see, for example, Clackson et al., 1991, Nature 352: 624; Hane et al. 1997, Proc. Natl. Acad. Sci., USA, 94: 4937).

After obtaining a nucleic acid sequence encoding at least the variable domain of an antibody, it can be introduced into a vector containing a nucleotide sequence encoding the constant regions of an antibody (see, for example, International Application Publication No. WO86 / 05807; WO89 / 01036 and Patent US No. 5122464). Vectors containing a full-sized light or heavy chain and allowing expression of a full-sized antibody molecule are commercially available. Then, the nucleic acid encoding the antibody can be used to introduce nucleotide substitutions or deletions necessary to replace (or deletion) one or more cysteine residues of the variable region involved in the formation of an intrachain disulfide bond with an 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 into the nucleotide sequence, and such methods are, but are not limited to, for example, the method of chemical mutagenesis and site-directed mutagenesis in vitro (Hutchinson et al. 1978, J. Biol. Chem. 253: 6551).

In addition, methods 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 having corresponding antigen specificity, together with the genes of a human antibody molecule having corresponding biological activity. A chimeric antibody is a molecule in which different parts are derived from animals of various species, such as parts having a variable region derived from a murine monoclonal antibody and a constant region of a human immunoglobulin, for example a humanized antibody.

Alternatively, known methods used to produce such antibodies can be adapted to produce single chain antibodies (US Pat. No. 4,694,778; 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 are produced by binding fragments of the Fv region of the heavy and light chains via an amino acid bridge, resulting in the formation of a single chain polypeptide. Can also be applied methods of assembly of functional Fv fragments in E. coli (Skerra et al., 1988, Science 242: 1038-1041).

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

After obtaining the nucleic acid sequence encoding the antibody, a vector can be obtained for producing the antibody by well-known recombinant DNA methods. Methods well known in the art can be used to construct expression vectors containing antibody coding sequences and corresponding transcriptional and translational regulation signals. Such methods are, for example, in vitro recombinant DNA methods, synthesis methods, and in vivo genetic recombination methods. See, for example, the 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 itself, can be transferred to the host cell by standard methods (for example, by electroporation, liposomal transfection and precipitation with calcium phosphate), after which the transfected cells can be cultured using standard methods for producing antibodies. In specific embodiments, the expression of an antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

The host cells used to express the recombinant antibody can be either bacterial cells, such as Escherichia coli , or preferably eukaryotic cells, in particular cells suitable for expression of a full-length recombinant immunoglobulin molecule. In particular, mammalian cells, such as Chinese hamster ovary (CHO) cells in combination with a vector, such as a promoter element of the main early gene derived from human cytomegalovirus (Foecking et al., 198, Gene 45: 101; Cockett et al., 1990, BioTechnology 8: 2).

For the expression of immunoglobulins (antibodies), various host-expression vector systems can be used. Such host-expression vector systems are carriers through which sequences encoding antibodies can be produced and then purified, however, such systems can also be cells which, when transformed or transfected with appropriate nucleotide coding sequences, express an immunoglobulin molecule (antibodies) in situ . Such cells include, but are not limited to, microorganisms, such as bacteria (eg, E. coli and B. subtilis ), transformed with vectors expressing recombinant bacteriophage DNA, plasmid DNA or cosmid DNA and containing sequences encoding immunoglobulin; yeast (eg, Saccharomyces Pichia ) transformed with recombinant vectors for expression in yeast containing sequences encoding immunoglobulin; insect cell systems infected with recombinant viral expression vectors (eg, baculoviruses) containing immunoglobulin coding sequences; insect cell systems infected with recombinant viral 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 immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing recombinant expression constructs including promoters derived from the mammalian cell genome (e.g., metallothionein promoter) or from mammalian viruses (e.g., late adenovirus promoter ; 7.5K promoter of vaccinia virus).

In bacterial systems, a number of expression vectors can be selected mainly depending on the purpose of the expressed antibody. So, for example, if large quantities of such a protein are produced, it is desirable that vectors that aim to express high levels of fusion protein products can be easily purified. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody coding sequence can be separately ligated into a vector, in the same frame reading, with the coding region of lac Z, so that as a result of this produced a hybrid protein; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster 1989, J. Biol. Chem. 24: 5503-5509) and the like. PGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix spheres coated with glutathione agarose, followed by elution in the presence of free glutathione. PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites, whereby the desired cloned gene product can be released from the GST molecule.

In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or a similar Drosophila Melanogaster virus is used as a vector for the expression of foreign genes . This virus is produced in Spodoptera frugiperda cells. The antibody coding sequence can be cloned separately into minor regions (e.g., the polyhedrin gene region) of the virus and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as the expression vector, the antibody-coding sequence of interest can be ligated with a complex that regulates the transcription / translation of adenovirus, for example, with a late promoter and a three-component leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Integration into a minor region of the viral genome (e.g., E1 or E3 region) results in the formation of a recombinant virus that is viable and can express an immunoglobulin molecule in infected hosts (see, for example, Logan & Shenk, 1984, Proc. Natl. Acad. Sci . , USA, 81: 355-359). Efficient translation of the inserted antibody coding sequences may also require specific initiation signals. Such signals are the initiation ATG codon and adjacent sequences. In addition, to ensure translation of the entire insert, the initiating codon must be in the same reading frame as the desired coding sequence. These exogenous translation control signals and initiating codons can come from various sources, that is, they can be natural and synthetic. Expression efficiency can be increased by incorporating appropriate enhancer elements of transcription, transcription terminators, and the like. (see Bittner et al., 1987, Methods in Enzymol. 153: 51-544).

In addition, to modulate the expression of the inserted sequences or to modify and specifically process the desired gene product, an appropriate strain of the host cell can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can play an important role in the functioning of the protein. Various host cells have characteristic and specific mechanisms of post-translational processing and modification of proteins and gene products. To ensure the necessary modification and processing of the expressed foreign protein, appropriate cell lines or host systems can be selected. To achieve this, eukaryotic host cells can be used that have a cellular mechanism for the corresponding processing of the primary transcript, for glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For continuous, highly efficient production of recombinant proteins, stable expression is preferred. Thus, for example, cell lines stably expressing an antibody can be constructed. For this purpose, instead of expression vectors containing viral origin of replication, host cells transformed with DNA under the control of the corresponding expression regulation elements (for example, promoter sequences, enhancer, transcription terminators, polyadenylation sites, etc.) and a selective marker can be used . After the introduction of foreign DNA, the constructed cells can be placed for 1-2 days in an enriched growth medium, and then this medium can be replaced by a selective medium. A selectable marker in a recombinant plasmid reports resistance to the agent selected for selection and allows cells to stably integrate this plasmid into their chromosomes and grow with the formation of loci, which, in turn, can be cloned and amplified into cell lines. This method can advantageously be used to construct cell lines expressing the antibody. Such engineered cell lines can be particularly effective for screening and evaluating tumor antigens that directly or indirectly interact with an antibody.

Various selection systems, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Szybalska, 192, Proc. Natl. Acad. Sci. USA, 48: 202) and adenine phosphoribosyl transferase (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 a basis for screening for the following genes: DHFR, which reports 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 reports resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci., USA, 78: 2072); neo, which reports resistance to 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 reports hygromycin resistance (Santerre et al., 1984, Gene 30: 147). Well-known methods used in recombinant DNA technology are described by 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 chapters 12 and 13 of Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

Antibody expression levels can be increased by vector amplification (for a review see, e.g., 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 the antibody is amplifiable, then as the level of inhibitor present in the culture of the host cell increases, the number of copies of the marker gene will increase. Since the amplified region is associated with the nucleotide sequence of an antibody, the level of production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3: 257).

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

After recombinant expression of an antibody, it can be purified by any known method used to purify antibodies, for example by chromatography (ion exchange, affinity, and in particular chromatography by affinity for a specific antigen on a protein A column, and size exclusion chromatography), centrifugation, separation based on different solubility or any other standard methods of protein purification.

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

In any case, hybrid antibodies may have double specificity, preferably due to the presence of one or more binding sites specific for the desired hapten, or one or more binding sites specific for the target antigen, for example, an antigen associated with a tumor, an autoimmune disease, and an infectious a microorganism or with another pathological condition.

4.5.2. Antibody production

Antibody production will be illustrated below with anti-CD30 antibodies, however, it will be apparent to those skilled in the art that antibodies against other members of the TNF receptor family can be produced and modified in a similar manner. The use of CD30 for antibody production is for illustrative purposes only and is not specifically limited.

The CD30 antigen used to produce antibodies may, for example, have a soluble form of the extracellular domain of CD30 or part thereof containing the desired epitope. Alternatively, cells expressing CD30 on their surface (e.g., L540 (cell line derived from Hodgkin lymphoma with a T-cell phenotype) and L428 (cell line derived from Hodgkin lymphoma with B-cell can be used to generate antibodies) phenotype)). Other forms of CD30 that can be used to produce antibodies are known to those skilled in the art.

In another representative embodiment of the invention, the ErbB2 antigen used to produce antibodies may, for example, have a soluble form of the extracellular domain of ErbB2 or part thereof containing the desired epitope. Alternatively, cells expressing ErbB2 on their surface (e.g., transformed NIH-3T3 cells expressing excessive levels of ErbB2; or a carcinoma cell line, such as SK-BR-3 cells, see Stancovski et al., Can be used to generate antibodies). Proc. Natl. Acad. Sci. USA, 88: 8691-8695 (1991)). Other forms of ErbB2 that can be used to produce antibodies are known to those skilled in the art.

(i) Polyclonal antibodies

Polyclonal antibodies are preferably produced in animals after multiple subcutaneous (sc) or intraperitoneal (ip) injections of the corresponding antigen and adjuvant. They can be used to conjugate the corresponding antigen with a protein that is immunogenic for the species being immunized, for example with cochlear lymphocyte hemocyanin, serum albumin, bovine thyroglobulin or a soybean trypsin inhibitor using a bifunctional or derivatizing agent (maleimidobenzoyne aminosulfide) N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic acid anhydride, SOC l ₂ or R ¹ N = C = NR, where R and R ¹ are different alkyl groups.

Animals are immunized with antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and by subcutaneous injection of the solution into multiple sites. After a month, the animals are re-immunized at multiple sites by subcutaneous injection of 1 / 5-1 / 10 of the initial amount of the peptide or conjugate in Freund's complete adjuvant. After 7-14 days, the animals were bled and the serum was analyzed for antibody titer. The animals are then re-immunized until a titer plateau is reached. Re-immunization of animals is preferably carried out by injection of a conjugate of the same antigen, but conjugated to another protein and / or through another cross-linking reagent. Conjugates can also be obtained in recombinant cell culture as fusion proteins. In addition, aggregating agents such as alum can also be used to enhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, individual antibodies that make up a given population that are identical, with the exception of antibodies having naturally occurring mutations that may be present in small amounts. Thus, the adjective “monoclonal” indicates a type of antibody that is not a mixture of discrete antibodies.

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

To produce hybridomas, a mouse or other suitable host animal, such as a hamster, is immunized as described above to produce lymphocytes in them that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro . The lymphocytes are then fused to myeloma cells using a suitable fusion agent such as polyethylene glycol, resulting in hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, p. 59-103 (Academic Press, 1986)).

The hybridoma cells thus obtained are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused parental myeloma cells. For example, if the parent myeloma cells do not contain the hypoxanthine guanine phosphoribosyltransferase enzyme (HGPRT or HPRT), then the hybridoma culture medium usually includes hypoxanthine, aminopterin and thymidine (NAT medium), that is, substances that prevent the growth of HGPRT-deficient cells .

Preferred myeloma cells are cells that are able to undergo efficient fusion, maintain stable production of high levels of antibodies by selected antibody-producing cells, and are sensitive to a medium, such as NAT medium. Of these cells, the preferred myeloma cell lines are murine myeloma lines, such as cell lines derived from murine tumor cells MORS-21 and MPC-11 available at the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells or X63-Ag8-653, available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines are also used to produce human monoclonal antibodies (Kozbor J. Immunol., 133: 3001 (1984) & Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p.51- 63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium for the growth of hybridoma cells is analyzed for the production of monoclonal antibodies against antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is preferably determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107: 220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, clones can be subcloned by limiting dilution procedures and cultured using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, p. 59-103 (Academic Press, 1986)). Suitable culture media designed to achieve this goal are, for example, D-MEM or RPMI-1640. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

Monoclonal antibodies secreted by subclones are appropriately isolated from culture medium, ascites fluid or serum by standard antibody purification procedures, such as, for example, protein chromatography on A-Sepharose, chromatography on hydroxyapatites, gel electrophoresis, dialysis or affinity chromatography.

DNA encoding monoclonal antibodies can be easily isolated and sequenced in accordance with standard procedures (for example, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The preferred source of such DNA are hybridoma cells. Once isolated, this DNA can be inserted into expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which otherwise do not produce an antibody protein, as a result, monoclonal antibodies are synthesized in these recombinant host cells. A discussion of the recombinant expression of antibody-coding DNA in bacteria can be found in 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 a phage antibody library created 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) describe the isolation of murine and human antibodies, respectively, using phage libraries. More recent publications describe the production of high-affinity (about nM) human antibodies through gene permutation of antibody chains (Marks et al. Bio / Technology, 10: 779-783 (1992)), as well as by combined infection and in vivo recombination, used as strategies for constructing very large phage libraries (Waterhouse et al. Nuc. Acids. Res. 21: 2265-2266 (1993)). Thus, these methods are an acceptable alternative to traditional hybridoma technology used to isolate 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 murine antibodies (US patent No. 4816567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 : 6851) or by covalently attaching to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides are used to replace constant domains of an antibody or are used to replace the variable domains of one antigen binding site, to produce a chimeric bivalent antibody having specificity for one antigen, or another antigen binding site having specificity for another antigen.

(iii) Humanized antibodies

A humanized antibody may have one or more amino acid residues introduced therein, originating from a non-human source. These non-human amino acid residues are often referred to as “import” residues, which are usually taken from the “import” variable domain. Humanization can be carried out mainly by the method of Winter (Winter) and collaborators (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 the sequences of the hypervariable region with the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), in which a substantially smaller portion than the variable domain of an intact human antibody is replaced by the corresponding sequence from a non-human antibody. In fact, humanized antibodies are typically human antibodies in which some residues of the hypervariable region and, possibly, some residues of the framework region (FR) are replaced by residues derived from similar regions of rodent antibodies.

To reduce antigenicity when creating humanized antibodies, it is very important to choose the variable domains of both the light and heavy chains of a human antibody. According to the so-called “fitting” method, the sequence of the variable domain of a rodent antibody is screened throughout the library of known variable domain sequences of a human antibody. The human sequence, which is most similar to the rodent sequence, is then taken as the human framework region (FR) to create a humanized antibody (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol Biol. 196: 901 (1987)). Another method uses a specific framework region, derived from the consensus sequence of all human antibodies of a particular subgroup of light and heavy chains. The same framework region can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci., USA, 89: 4285 (1992); Presta et al., J. Immunol, 151: 2623 ( 1993)).

In another embodiment of the invention, the antibodies can be humanized while maintaining high affinity for this antigen and other favorable biological properties. Humanized antibodies can be obtained by analysis of the source sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are well known in the art. Computer programs exist that illustrate and represent probable three-dimensional conformational structures of selected immunoglobulin candidate sequences. The study of these ideas allows us to analyze the likely role of these residues in the functioning of the candidate immunoglobulin sequences, that is, residues that affect the ability of the candidate immunoglobulin to bind to its antigen. Thus, FR residues can be selected from recipient and “import” sequences and combined, resulting in the desired antibody with the desired properties, such as increased affinity for the antigen target (s). In general terms, residues of the hypervariable region directly affect binding to the antigen, and are mainly involved in such binding.

Various forms of humanized antibodies have been examined. So, for example, a humanized antibody may be an antibody fragment, such as Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

The examples describe the production of a representative, humanized anti-ErbB2 antibody. Such a humanized antibody may, for example, contain residues of the hypervariable region of a non-human antibody driven into the variable domain of the heavy chain of a human antibody, and may further comprise a substitution in the frame region (FR) at a position selected from the group consisting of 69H, 71H and 73H according to the variable domain numbering system described by Kabet (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, the humanized antibody includes FR substitutions at two or all of 69H, 71H and 73H positions. Another example describes the preparation of purified trastuzumab antibody from HERCEPTIN®.

(iv) Human antibodies

As an alternative to humanization, human antibodies can be produced. Thus, for example, transgenic animals (eg, mice) can be obtained which, after immunization, will be able to produce a complete repertoire of human antibodies without producing endogenous immunoglobulin. For example, it was reported that a homozygous deletion of the gene of the junction region in the heavy chain (J _H ) of an antibody in chimeric and mutant germ line mice leads to complete inhibition of the production of endogenous antibodies. Transferring a set of human germline immunoglobulin genes to such mutant germline mice can lead to the production of human antibodies after antigen stimulation. 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 Immuno, 7:33 (1993) and US Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.

Alternatively, phage presentation technology (McCafferty et al. Nature 348: 552-553 (1990)) can be used to produce human antibodies and in vitro antibody fragments from a set of immunoglobulin variable domain (V) gene genes derived from non-immunized donors. In accordance with this technology, the V domain genes of an antibody are cloned with a reading frame into the main or minor protein of the coat of a filamentous bacteriophage, such as M13 or fd, and present as functional fragments of the antibody on the surface of the phage particle. Since the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also allows selection of a gene encoding an antibody possessing these properties. Thus, the phage mimics some of the properties of b-cells. Phage presentation can be carried out in various formats; see, for example, a review by Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). For the implementation of phage representation can be used several sources of V-gene segments. Clackson et al., Nature, 352: 624-628 (1991) isolated a different set of diverse antibodies against oxazolone from a small randomized combinatorial library of V genes originating from the spleen of immunized mice. In this case, a set of V genes from non-immunized human donors can be constructed, and then antibodies can be isolated against a set of different 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). See also US Patent Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies can also be produced in vitro by activated B cells (see US Patent Nos. 5,567,610 and 5,229,275). Human anti-CD30 antibodies are described in US Patent Application Reg. No. 10/338366.

(v) Antibody Fragments

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments are formed by proteolytic cleavage of intact antibodies (see, for example, 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 host cells. Thus, for example, antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can be directly isolated from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ′) ₂ fragments can be isolated directly from a culture of a recombinant host cell. Other methods for producing antibody fragments are known to those skilled in the art. In other embodiments, the selected antibody is a single chain Fv fragment (scFv). See WO 93/16185, US patent No. 5571894 and US patent No. 5587458. An antibody fragment can also be a “single chain antibody”, for example, the antibody described in US patent No. 5641870. Such single chain antibody fragments can be monospecific or bespecifically.

(vi) Bispecific antibodies

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

The traditional production of full-length bispecific antibodies is based on coexpression of two pairs of the immunoglobulin heavy chain-light chain, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). These hybridomas (quadromas), due to the randomized set of immunoglobulin heavy and light chains, produce a potential mixture of 10 different antibody molecules, of which only one molecule has the “correct” bispecific structure. The purification of such a “correct” molecule, which is usually carried out by stepwise affinity chromatography, is rather difficult and gives a low yield of the product. Similar procedures are described in WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). According to another approach, the variable domains of an antibody with the desired binding specificities (combined antibody-antigen sites) are attached to immunoglobulin constant domain sequences. Such a fusion is preferably carried out with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge region, CH2 and CH3. Moreover, it is preferable that this hybrid has a first constant region of the heavy chain (CH1) containing the site necessary for binding to the light chain present in at least one of the hybrids. DNA encoding immunoglobulin heavy chain hybrids and, if necessary, immunoglobulin light chain hybrids are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides a high degree of flexibility in correcting the ratios of three polypeptide fragments in those embodiments of the invention in which unequal contents of the three polypeptide chains used in this design give optimal yields. However, coding sequences for two or all three polypeptide chains can be inserted into one expression vector if expression of at least two polypeptide chains in equal proportions gives high yields or if such ratios are not critical.

In one embodiment of this approach, bispecific antibodies consist of a hybrid immunoglobulin heavy chain having a first binding specificity in one branch, and an immunoglobulin hybrid heavy chain-light chain pair (providing a second binding specificity) in another branch. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from undesirable combinations of immunoglobulin chains, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides an easy way to isolate it. This method is described in WO 94/04690. For a more detailed description of the preparation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

In accordance with the second approach described in US patent No. 5731168, the boundary between a pair of antibody molecules can be constructed in order to maximize the percentage of heterodimers isolated from recombinant cell culture. Such a boundary preferably contains at least a portion of the CH3 domain of the constant domain of an antibody. In this method, the small side chains of one or more amino acids in the boundary region of the first antibody molecule are replaced by larger side chains (e.g. tyrosine or tryptophan). In the boundary region of the second molecule of the antibody, compensating “cavities” are created having a size identical or similar to the size of the larger side chain (s) by replacing the large side chains of amino acids with smaller side chains (for example, alanine or threonine) . This allows you to increase the yield of heterodimers in relation to other undesirable end products, such as homodimers.

Methods for producing bispecific antibodies from antibody fragments have also been described in the literature. So, for example, bespecifically antibodies can be obtained by chemical binding. Brennan et al., Science, 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to form F (ab ') ₂ fragments. These fragments are reduced in the presence of a dithiol complex forming agent, such as sodium arsenite, to stabilize adjacent dithiols and prevent the formation of an intermolecular disulfide bond. Then the obtained Fab'-fragments are converted into derivatives of thionitrobenzoate (TNB). Thereafter, one of the Fab'-TNB derivatives is again converted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative, whereby a bispecific antibody is obtained. Such bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent advances in this area allow direct isolation of Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of a fully humanized F (ab ') ₂ molecule of a bispecific antibody. Each Fab'fragment was separately secreted from E. coli and subjected to direct chemical binding in vitro with the formation of bespecifically antibodies.

Other methods for producing and isolating fragments of a bispecific antibody directly from a recombinant cell culture have also been described. For example, bispecific antibodies were produced using “leucine zippers”. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were coupled to the Fab ′ portions of two other antibodies by ligation of genes. Antibody homodimers were reduced in the hinge region to form monomers, and then oxidized again to form antibody heterodimers. This method can also be used to produce antibody homodimers. Dyatel technology described by Hollinger et al., Proc. Natl. Acad. Sci., USA, 90: 6444-6448 (1993), provides an alternative mechanism for producing bispecific antibody fragments. These fragments contain the heavy chain variable domain (V _H ) coupled to the light chain variable domain (V _L ) via a linker that is too short to pair between two domains of the same chain. Accordingly, V _H and V _L domains of one fragment are forced to pair with complementary V _L and V _H domains of another fragment, thereby forming two antigen-binding sites. Another strategy has also been described for producing bispecific antibody fragments using single chain Fv (sFv) dimers. See Gruber et al., J. Immunol., 152: 5368 (1994).

Antibodies with more than two valencies are also contemplated. So, for example, trispecific antibodies can also be obtained. Tutt et al., J. Immunol. 147: 60 (1991).

(vii) Other modifications of amino acid sequences

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

A method that is used to identify certain residues or regions of an antibody and which allows the localization of mutagenesis to be determined is called “alanine scanning mutagenesis” and is described by Cunningham & Wells, Science 244: 1081-1085 (1989). In this method, a residue or group of desired residues is identified (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) to influence the interaction of these amino acids with the antigen. Then, the localization of amino acids exhibiting functional sensitivity to such substitutions is specified by introducing additional or other options at a given substitution position or instead of this substitution. So, for example, the site of introduction of the amino acid substitution can be determined in advance, while the nature of the mutation per se need not be determined in advance. For example, to analyze the effectiveness of a mutation at a given site, Ala-scanning or nonspecific mutagenesis is performed in the desired codon or in the desired region and variants of the expressed antibody are screened for the desired activity.

Insertions in amino acid sequences are insertions at the amino and / or carboxy terminus, forming hybrids having a length from one residue to polypeptides containing one hundred or more residues, as well as inserts within a sequence consisting of one or multiple amino acid residues. Examples of terminal insertions are an antibody with an N-terminal methionyl residue or an antibody attached to a cytotoxic polypeptide. Other insertion variants of the antibody molecule are hybrids of the N- or C-terminus of the antibody with an enzyme (for example, for ADEPT) or with a polypeptide that helps increase the half-life of the antibody in serum.

Another type of variant is the amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by another residue. Hypervariable regions are sites of greatest interest for mutagenesis by substitution, but substitutions in FR can also be considered.

Significant changes in the biological properties of antibodies can be achieved by selecting substitutions that have a significant effect on (a) the structure of the polypeptide backbone in the region of substitution, for example, folding and helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (c) the volume of the side chain . Natural residues are divided into the following groups according to the general 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 affect chain orientation: Gly, Pro and

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

Non-conservative substitutions result in replacements of a member of one of these classes with a member of another class.

A particularly preferred type of replacement option is the replacement of one or more residues of the hypervariable region of the parent antibody (e.g., a humanized or human antibody). In general terms, the options obtained, selected for further development, will contribute to the improvement of biological properties compared with the parent antibody from which they originate. The standard way to generate such variants with substitutions involves the maturation of affinity using the phage representation method. For this, several positions of the hypervariable region (for example, 6-7 positions) are mutated to generate amino acid substitutions at each position. Antibody variants generated in this way will be presented as monovalent molecules on filamentous phage particles in the form of hybrids with gene III product packaged in each phage M13 particle. Then, the variants presented on the phage are screened for their biological activity (for example, binding affinity), as described in this application. To identify sites of the hypervariable region that are candidates for modification, alanine scanning mutagenesis can be carried out, which allows the identification of residues of the hypervariable region that play a significant role in binding to the antigen. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and antigen. Such contact residues and neighboring residues are candidates for replacement carried out in accordance with the method developed here. After generating such variants, a panel of these variants is subjected to the screening described in this application, and antibodies showing excellent properties in one or more relevant assays can be selected for further study.

It may be desirable to modify the antibody of the present invention in terms of their effector function, 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 into the Fc region of the antibody. Alternatively or additionally, cysteine (s) residue (s) may be introduced into this Fc region (s), which will contribute to the formation of interchain disulfide bonds in this region. The homodimeric antibody thus generated may have increased internalization ability and / or increased complement dependent cytotoxicity (ADCC). See Caron et al. J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B.J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using the heterobifunctional cross-linking agents described by Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has two Fc regions, and therefore may have enhanced complement dependent lysis and ADCC. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

To increase the half-life of an antibody in serum, a rescue receptor binding epitope can be introduced into a given antibody (and in particular an antibody fragment), as described, for example, in US Pat. No. 5,739,277. As used herein, the term “rescue receptor binding epitope” means an Fc epitope. -regions of an IgG molecule (e.g., IgG1, IgG2, IgG3 or IgG4), which is responsible for increasing the half-life of an IgG molecule in serum in vivo .

(viii) Glycosylation Options

Antibodies in the ADCs of the invention can be glycosylated at conserved positions in their constant regions (Jefferis & Lund (1997) Chem. Immunol. 65: 111-128; Wright & Morrison (1997) TibTECH 15: 26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al. (1996) Mol. Immunol. 32: 1311-1318; Wittwe & Howard (1990) Biochem. 29: 4175-4180) and the intramolecular interaction between parts of the glycoprotein, which can affect the conformation and the presented three-dimensional surface of a given glycoprotein (Hefferis & Lund, see above; Wyss & Wagner (1996) Current Opin. Biotech. 7: 409-416). Oligosaccharides can also serve to direct a given glycoprotein to certain molecules that have specific recognition structures. For example, it was reported that in a non-galactosylated IgG, the oligosaccharide moiety “falls out” of the space between CH2 and the terminal N-acetylglucosamine residues become available for binding to the mannose binding protein (Malhorta et al. (1995) Nature Med. 1: 237 -243). Removal, under the action of glycopeptidase, of oligosaccharides from CAMPATH-1H (recombinant humanized mouse monoclonal antibody IgG1 that recognizes the CDw52 antigen of human lymphocytes) produced in Chinese hamster ovary cells (CHO) led to a complete cessation of complement (CM) dependent al. (1996) Mol. Immunol. 32: 1311-1318), while the selective removal of sialic acid residues by neuraminidase did not result in loss of DMCL. It has also been reported that glycosylation of antibodies affects antibody-dependent cell cytotoxicity (ADCC). In particular, it was reported that CHO cells with tetracycline-regulated expression of β (1,4) -N-acetylglucosaminyl transferase III (GnTIII), i.e. glycosyltransferase, which catalyzes the formation of GlcNAc, which separates the sequence in half, has an increased ADCC activity (Umana et al. (1999) Mature Biotech. 17: 176-180).

Glycosylation of antibodies is usually either N-linked or O-linked. N-linked glycosylation means the attachment of a carbohydrate group to the side chain of an asparagine residue. The tripeptide sequences “asparagine-X-serine” and “asparagine-X-threonine”, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide contributes to the creation of a potential glycosylation site. O-linked glycosylation means the addition of one of the sugars, such as N-acetylgalactosamine, galactose or xylose, to a hydroxy amino acid, mainly to serine or threonine, although they may also be 5-hydroxyproline or 5-hydroxylysine.

Variants of glycosylation of antibodies are those in which the type of glycosylation of the antibody is changed. By such a “change” is meant a deletion of one or more carbohydrate groups present in an antibody, addition of one or more carbohydrate groups to an antibody, a change in glycosylation composition (such as glycosylation), degree of glycosylation, and the like.

Attachment of glycosylation sites to an antibody is usually accomplished by modifying the amino acid sequence such that the amino acid sequence will contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such a modification can also be carried out by adding one or more serine or threonine residues to the sequence or replacing them in the sequence of the original antibody (for O-linked glycosylation sites). Similarly, the removal of glycosylation sites can be accomplished by modifying the amino acids in the natural glycosylation sites of the antibody.

Amino acid sequence is usually modified by changing the nucleic acid sequence encoding it. Such methods include, but are not limited to, isolation from a natural source (in the case of natural variants of the amino acid sequence) or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis of a previously obtained variant or unmodified antibody variant.

Glycosylation (including the type of glycosylation) of antibodies can also be changed without modifying the amino acid sequence or its nucleotide sequence. Glycosylation mainly depends on the host cell used to express the antibody. Since the cells used to express recombinant glycoproteins, such as antibodies that are potential therapeutic agents, are rarely natural cells, a significant change in the type of glycosylation of antibodies can be expected. See, for example, Hse et al., (1997) J. Biol. Chem. 272: 9062-9070. In addition to the choice of host cells, factors influencing glycosylation in the process of recombinant production of antibodies include the growth pattern, medium composition, culture density, oxygenation, pH, purification schemes, etc. Various methods have been proposed to change the type of glycosylation achieved in a particular host organism, including the administration or overexpression of certain enzymes involved in the production of oligosaccharides (US Pat. Nos. 5,047,335; 5,510,261; 5,278,299). Glycosylation (or a specific type of glycosylation) of a glycoprotein can be prevented, for example, using endoglycosidase H (endo-H). In addition, the recombinant host cell can be genetically engineered, for example, it can be made defective in the processing of certain types of polysaccharides. These and similar methods are well known in the art.

The glycosylation structure of antibodies can be easily analyzed by standard carbohydrate analysis methods, including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, stepwise enzymatic hydrolysis, and HPAEC-PAD, which use high pH anion exchange chromatography to separate oligosaccharides based on their charge. Methods for the release of oligosaccharides used for analytical purposes are also known, and such methods include, but are not limited to, enzymatic treatment (usually carried out using peptide-N-glycosidase F / endo-β-galactosidase), elimination, mainly, O- bound structures under severe alkaline conditions; and chemical methods for the release of N- and O-linked oligosaccharides using anhydrous hydrazine.

4.5.2a. Antibody Drug Conjugate Screening (ADC)

Transgenic animals and cell lines are particularly suitable for screening for antibody-drug conjugate (ADC) conjugates that can be used for the prophylactic or therapeutic treatment of diseases or disorders associated with overexpression of proteins, including Lewis Y, CD30, CD40 and CD70 antigens . Transgenic animals and cell lines are particularly suitable for screening for antibody-drug conjugate (ADC) conjugates that can be used for the prophylactic or therapeutic treatment of diseases or disorders associated with overexpression of HER2 (US6632979). Screening for effective ADC may include administering the ADC candidate at different dose ranges to transgenic animals and analyzing the effectiveness (s) of ADC in treating the disease and disorder in question at different time intervals. Alternatively or additionally, the drug may be administered prior to treatment or at the same time as treatment with a disease-inducing factor, if used. An ADC candidate can be screened stepwise and separately or simultaneously in an environment or in a high-performance screening format. The speed with which ADC can be screened for its effective use in the prophylactic or therapeutic treatment of diseases or disorders is limited only by the speed of synthesis or by a screening method, including detection / measurement / analysis of data.

In one embodiment of the invention, a screening method comprises (a) transplanting cells from a stable kidney cancer cell line to a non-human animal, (b) administering an ADC candidate to a non-human animal, and (c) determining the ability of said candidate to inhibit tumor formation from transplanted cell line.

Another embodiment of the invention is a screening method comprising (a) contacting cells derived from a stable Hodgkin disease cell line with an ADC candidate and (b) evaluating the ability of such an ADC candidate to block CD40 ligand activation.

Another embodiment of the invention is a screening method comprising (a) contacting cells derived from a stable Hodgkin disease cell line with an ADC candidate and (b) evaluating the ability of such an ADC candidate to induce cell death. In one embodiment of the invention, the ability of an ADC candidate to induce apoptosis is assessed.

One embodiment of the invention is a screening method comprising (a) transplanting cells from a stable cancer cell line to a non-human animal, (b) administering an ADC candidate to a non-human animal, and (c) determining the ability of said candidate to inhibit tumor formation in transplanted cell line. The present invention also relates to a method for screening ADC candidates for use in treating a disease or disorder characterized by overexpression of HER2, wherein said method comprises (a) contacting the cells derived from a stable breast cancer cell line with a drug candidate and ( b) an assessment of the ability of such an ADC candidate to inhibit the growth of a stable cell line.

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

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

Another embodiment of the invention is a screening method comprising (a) administering an ADC candidate to a transgenic non-human mammal in which overexpression of the native human HER2 protein or fragment thereof is observed in breast cells, wherein said transgenic animal exhibits stable integration of the nucleic acid sequence into the genome an acid encoding native human HER2 protein or a fragment thereof having biological activity of native human HER2 and functionally linked oh to the transcriptional regulation sequences directing its expression into the mammary gland, a breast tumor develops that is not susceptible or poorly susceptible to treatment with anti-HER2 antibody; or administering said candidate to a non-human mammal that has a tumor developed from transplanted cells taken from said non-human transgenic mammal; and (b) assessing the effect of said ADC candidate on the disease or disorder in question. These diseases or disorders may include, but are not limited to, cancer associated with overexpression of HER2, such as cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, and bladder. Said cancer is preferably breast cancer, in which HER2 is expressed in an amount of at least about 500,000 copies per cell, and more preferably at least about 2,000,000 copies per cell. ADC candidates can, for example, be evaluated for their ability to induce cell death and / or apoptosis by analytical methods well known in the art and described below.

In one embodiment of the invention, the ADC candidate is screened by administering the transgenic animal at various doses and evaluating the physiological response of the animal to these compounds versus time. Administration can be done orally or by appropriate injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be desirable to administer the compound in combination with cofactors, which should increase the effectiveness of the compound. If cell lines derived from the considered transgenic animals are used to screen compounds that can be used to treat various disorders, the test compounds are added to the cell culture medium at an appropriate time interval and the cellular response to this compound is evaluated over a certain time interval using appropriate biochemical and / or histological analyzes. In some cases, it may be desirable to add a compound of interest to the culture medium in combination with cofactors, which should increase the effectiveness of the compound.

Thus, the present invention considers assays conducted to identify ADC that specifically targets and binds to a target protein, the presence of this protein correlating with abnormal cell function, and in the pathogenesis of cell proliferation and / or differentiation, it is causally associated with tumor development.

To identify an ADC that blocks activation of an ErbB receptor ligand (e.g., ErbB2), the ability of said compound to block the binding of ErbB ligand to cells expressing an ErbB receptor (ErbB2) can be evaluated (e.g., by conjugation to another ErbB receptor of interest ErbB receptor forms an ErbB hetero-oligomer). For example, cells isolated from a transgenic animal in which HER2 is overexpressed and transfected to express another ErbB receptor (with which HER2 forms a hetero-oligomer) can be incubated, that is, cultured with ADC, and then treated with the labeled ErbB ligand. After that, the ability of the indicated compound to block the binding of the ligand to the ErbB receptor in the ErbB hetero-oligomer can be evaluated.

For example, inhibition of binding of heregulin (HRG) to mammary tumor cell lines overexpressing HER2 and developed in non-human transgenic mammals (e.g. mice) by the action of the ADC candidate according to the invention can be carried out using monolayer ice cultures 24-well plate format. Monoclonal antibodies against ErbB2 can be added to each well and incubated for 30 minutes. Then ¹²⁵ I-labeled rHRGβ1 _177-224 (25,000 cpm) can be added, and incubation can be continued for 4-16 hours. After this, dose-response curves can be plotted and the IC ₅₀ value (cytotoxic activity) can be calculated for the compound of interest.

Alternatively or additionally, the ability of ADC to block ErbB ligand-stimulated tyrosine phosphorylation of the ErbB receptor present in the ErbB hetero-oligomer can be evaluated. For example, cell lines developed in transgenic animals of the invention can be incubated with the tested ADC, and then they can be analyzed for ErbB ligand-dependent tyrosine phosphorylation activity using a monoclonal anti-phosphotyrosine antibody (which is, but is not necessarily conjugated to detectable label). The kinase receptor activation assay described in US Pat. No. 5,766,863 can also be used to determine ErbB receptor activation and block this activation with this compound.

In one embodiment of the invention, an ADC screening can be performed that inhibits HRG stimulation of tyrosine p180 phosphorylation in MCF7 cells, mainly as described below. For example, a cell line produced in an HER2 transgenic animal can be cultured in 24-well plates, and a compound can be added to each well, followed by incubation for 30 minutes at room temperature, after which each well can be added rHRGβ _1177-224 to a final concentration of 0.2 nM, and incubation can be continued for about 8 minutes. Then the medium is aspirated from each well, after which the reaction can be stopped by adding 100 μl of SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl) can be electrophoresed in a 4-12% gradient gel (Novex), and then it can be electrophoretically transferred to a polyvinylidene difluoride membrane. After that, immunoblots of antibodies against phosphotyrosine (at 1 μg / ml) can be manifested and the intensity of the predominant reactive band at M _r ~ 180,000 can be quantified using reflective densitometry. An alternative method for assessing receptor phosphorylation inhibition is the KIRA assay (for kinase receptor activation) described by Sadick et al. (1998) Jour. of Pharm. and Biomed. Anal. Some of the well-identified anti-HER2 monoclonal antibodies that are known to inhibit HRG stimulation of tyrosine p180 phosphorylation can be used as a positive control in this assay. A dose-response curve can then be plotted to inhibit HRG stimulation of tyrosine p180 phosphorylation, which can be evaluated by reflection densitometry, after which an IC ₅₀ for the compound of interest can be calculated.

Can also be evaluated for the tumor-inhibitory effect of the test ADC on cell lines derived from a HER2 transgenic animal, for example, mainly as described by Schaefer et al. (1997) Oncogene 15: 1385-1394. In accordance with this analysis, cells can be treated with the test compound at various concentrations for 4 days and stained with crystalline violet or dye Alamar blue, which has a redox effect. By incubating with this compound, it is possible to demonstrate a tumor-inhibitory effect on a given cell line similar to that of 2C4 monoclonal antibody on MDA-MB-175 cells (Schaefer et al., Supra). In another embodiment of the invention, exogenous HRG does not have a significant inverse effect on such inhibition.

To identify tumor-inhibiting compounds that specifically target an antigen of interest, a screening can be made for compounds that inhibit the growth of cancer cells overexpressing an antigen of interest and originating from transgenic animals, and such screening analysis is described in US Pat. No. 5,677,171. According to this analysis, cancer cells overexpressing the antigen of interest are cultured in a 1: 1 mixture of F12 medium and DMEM, to which 10% fetal bovine serum was added, with glutamate and penicillin-streptomycin. Cells are then plated at a density of 20,000 cells in a 35 mm cell culture dish (2 ml / 35 mm plate) and test compound is added at various concentrations. After six days, the number of cells was counted compared to untreated cells using a COULTER ^™ electronic cell counter. Compounds that inhibit cell growth by about 20-100% or about 50-100% can be selected as compounds that inhibit cell growth.

To select compounds that induce cell death, an assessment can be made of the loss of membrane integrity, as indicated, for example, by the absorption of PI, trypan blue or 7AAD compared to the control. The PI uptake assay utilizes cells isolated from the tumor tissue of interest in a transgenic animal. In accordance with this analysis, the cells were cultured in a mixture of Dulbecco's modified Eagle medium (D-MEM): Hams F-12 medium (50:50), to which 10% thermally inactivated FBS (Hyclone) and 2 mM L-glutamine were added. Thus, this analysis is carried out in the absence of complement and immune effector cells. The cells are then plated in 100 × 20 mm plates at a density of 3 × 10 ⁶ per plate and allowed to fuse overnight. After this, the medium is removed and replaced with only one fresh medium or one containing various concentrations of the compound. Cells are incubated for 3 days. After each treatment, the monolayers are washed with PBS and separated by trypsinization. After that, the cells are centrifuged at 1200 rpm for 5 minutes at 4 ° C and the pellet is resuspended in 3 ml of cold Ca ²⁺ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ) and divided into 35 mm aliquots, which are distributed into 12 × 75 ml tubes with a strainer (1 ml per tube, 3 tubes per treatment group) to remove accumulations of cells. Then PI (10 μg / ml) was added to the tubes. Samples can be analyzed on a FACSCAN ^™ flow cytometer and using the FACSCONVERT ^™ CellQuest computer program (Becton Dickinson). Compounds that induce statistically significant levels of cell death, as determined by the uptake of PI, can be selected as compounds that induce cell death.

To select compounds that induce apoptosis, annexin binding assays are performed using cells obtained from the tumor tissue of interest in transgenic animals. Cells are then cultured and plated in plates as described in the previous paragraph. Then the medium is removed and replaced with only one fresh medium or medium containing 10 μg / ml antibody-drug conjugate (ADC). After incubation for 3 days, the monolayers are washed with PBS and separated by trypsinization. The cells are then centrifuged, resuspended in a Ca ²⁺ binding buffer and aliquoted, as discussed above in the description of the cell death assay. After this, labeled annexin (e.g., annexin V-FITC) (1 μg / ml) is added to the tubes. Samples can be analyzed on a FACSCAN ^™ flow cytometer and using the FACSCONVERT ^™ CellQuest computer program (Becton Dickinson). Compounds that induce statistically significant levels of annexin binding, compared to the control, are selected as apoptosis inducing compounds.

4.5.3. In Vitro Cell Proliferation Assays

In general terms, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is measured by treating mammalian cells having receptor proteins with an ADC conjugate antibody in a cell culture medium; culturing cells for a period of time from about 6 hours to 5 days; and determining cell viability. Such in vitro cell assays are used to assess cell viability (proliferation), cytotoxicity and induce apoptosis (caspase activation) of cells by the ADC of the invention.

The in vitro efficacy of the antibody-drug conjugates is measured by analysis of cell proliferation (Example 18, FIGS. 7-10). Luminescence-based cell viability analysis (CellTiter-Glo®) is commercially available (Promega Corp., Madison, WI) and is a uniform assay performed by recombinant expression of Coleoptera luciferase (US Pat. Nos. 5,583,024; 5,674,713 and 5,700,670). This analysis of cell proliferation allows you to determine the number of viable cells in culture based on the quantification of the presence of ATP, which is an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160: 81-88, US patent No. 6602677) . CellTiter-Glo® analysis was performed in a 96-well plate format, which allows automatic high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6: 398-404). A homogeneous analytical procedure involves the addition of one reagent (CellTiter-Glo® reagent) directly to cells cultured in serum supplemented medium. It does not require the stages of washing the cells, removing the medium and many stages of pipetting. This system allows the detection of at least 15 cells / well in a 384-well plate 10 minutes after reagent addition and mixing. These cells can be continuously treated with ADC, or they can be processed and separated from ADC. In general, treating cells for a short period of time, that is, for 3 hours, gives the same effect as continuous treatment of cells.

Homogeneous analysis carried out in the “add-mix-measure” format leads to cell lysis and to the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. In the CellTiter-Glo® assay, a “luminescence” type luminescent signal is generated by the luciferase reaction, which has a half-life of more than five hours, depending on the type of cells and the medium used. Viable cells are determined in relative luminescence units (RLU). The substrate, beetle luciferin, undergoes oxidative decarboxylation under the influence of recombinant firefly luciferase with the simultaneous conversion of ATP to AMP and the generation of photons. The long half-life of luciferase eliminates the need for injectors to introduce reagents and provides the possibility of continuous or batch processing of multiple tablets. Such cell proliferation assays can be performed in various multi-well plates, for example, in a 96-well or 384-well plate. Data can be recorded using a luminometer or a CCD-based imaging camera. The luminescence yield is presented in relative light units (RLU), measured with time.

The antiproliferative effects of antibody-drug conjugates are measured by cell proliferation in the above-described in vitro assay for cell cytolysis of four different mammary tumor cell lines (FIGS. 7-10). For SK-BR-3 and BT-474 cells, in which, as you know, overexpression of the HER2 receptor protein is observed, IC ₅₀ values were determined. Table 2a shows the efficacy measurements (IC ₅₀ ) of representative antibody-drug conjugates in the SK-BR-3 cell proliferation assay. Table 2b presents the efficacy measurements (IC ₅₀ ) of representative antibody-drug conjugates in the BT-474 cell proliferation assay.

Antibody drug conjugates: trastuzumab-MS-vc-RAV-MMAF, 3.8 MMAF / Ab, trastuzumab-MS- (N-Me) vc-RAB-MMAF, 3.9 MMAF / Ab, trastuzumab-MS-MMAF, 4.1 MMAF / Ab; trastuzumab-MS-vc-RAB-MMAE, 4.1 MMAE / Ab; trastuzumab-MS-vc-RAB-MMAE, 3.3 MMAE / Ab; and trastuzumab-MS-vc-RAB-MMAF, 3.7 MMAF / Ab did not inhibit the proliferation of MCF-7 cells (Fig. 9).

Antibody drug conjugates: trastuzumab-MS-vc-RAB-MMAE, 4.1 MMAE / Ab, trastuzumab-MS-vc-RAB-MMAE, 3.3 MMAE / Ab; trastuzumab-MS-vc-RAV-MMAF, 3.7 MMAF / Ab; trastuzumab-MS-vc-RAB-MMAF, 3.8 MMAF / Ab; trastuzumab-MS- (N-Me) vc-RAB-MMAF, 3.9 MMAF / Ab; and trastuzumab-MS-MMAF, 4.1 MMAF / Ab did not inhibit cell proliferation of MDA-MB-468 (figure 10).

MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor protein. It follows that the anti-HER2 antibody-drug conjugates of the invention have selectivity for inhibiting cells that express HER2.

Table 2a

Cells SK-BR-3

Table 2b

BT474 cells

H = trastuzumab.

7C2 = a murine anti-HER2 antibody that binds to an epitope different from the epitope to which trastuzumab binds.

Fc8 = mutant that does not bind to FcRn.

Hg = “Hinge-free” is a full-sized humanized 4D5 antibody in which the cysteines of the hinge region of the heavy chain are replaced by serines. This antibody was expressed in E. coli (and therefore it is non-glycosylated).

Anti-TF Fab = anti-tissue factor antibody fragment.

* activity against MDA-MB-468 cells.

Surprising and unexpected were the results obtained for ADC activity directed against cell proliferation in vitro , and are presented in tables 2a and 2b, which in general terms showed that ADC with a low average content of drug molecules in relation to the antibody had significant efficiency, for example IC ₅₀ <0.1 μg ADC / ml. These results suggest that, at least for ADC with trastuzumab, the optimal ratio of drug molecules to antibody molecules can be less than 8 or even about 2-5.

4.5.4. In vivo excretion of plasma ADC and its stability

The pharmacokinetics of the elimination of ADC from plasma and its stability were studied in rats and dog-like monkeys. Plasma concentration was determined over a period of time. Table 2c presents the pharmacokinetic data for the antibody-drug conjugates and other doses of samples in rats. Rats are nonspecific models for the production of antibodies against the ErbB receptor, since it is not yet known whether their HER2 receptor proteins are expressed.

Table 2c

Pharmacokinetics in rats

H = trastuzumab bound by cysteine [Cys], unless otherwise indicated; dose of 2 mg / kg, unless otherwise specified

AUCinf is the area under the curve of plasma concentration versus time of dose administration to infinity and is a measure of the total exposure of the measured molecule (drug, ADC). CL is defined as the volume of plasma purified from the measured molecule per unit time, and expressed as a value normalized by body weight. T ^1/2 period means the half-life of the drug in the body, measured in the phase of its elimination. % conjugate means the relative amount of ADC compared to the total amount of antibody detected in individual immunoaffinity tests using ELISA (“Analytical Methods for Biotechnology Products”, Ferraiolo et al, p.85-98 in Pharmacokinetics of Drugs (1994) PG Welling & LP Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110, Springer-Verlag). % conjugate is calculated by the formula: AUCinf ADC: AUCinf total Ab, and this% is a general indicator of linker stability, although, in fact, other factors and mechanisms may be such indicators.

Figure 11 presents a graph of the excretion of the test concentration from plasma after administration to rats of Sprague-Dawley conjugates of the antibody-drug: H-MS-vc-RAB-MMAF-TEG and H-MS-vc-RAV-MMAF. Concentrations of total antibodies and ADC were measured versus time.

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

In vivo efficacy

In vivo, the efficacy of the ADCs of the invention was measured in a mouse model with a transgenic explant expressing high levels of HER2. An allograft was cultured in transgenic, MMTV-infected Fo5 mice that are unresponsive or poorly susceptible to Herceptin® therapy. The mice were treated once with ADC and monitored for 3-6 weeks to determine the period of time that elapses until the tumor volume doubles, log the number of killed cells; and the degree of decrease in tumor volume. Then, a dose-response graph was constructed and experiments were performed using multiple doses.

Tumors easily developed in transgenic mice that expressed the mutated form of neu , the rat homologue of HER2, but HER2, which was overexpressed in a breast cancer tumor, was not mutated, and therefore tumor formation was much less intense in transgenic mice in which overexpression of unmuted HER2 (Webster et al., (1994) Semin. Cancer Biol. 5: 69-76).

To accelerate the formation of a tumor with unmuted HER2, transgenic mice were obtained using a plasmid cDNA encoding HER2 in which the upstream ATG was deleted to prevent translation initiation in such upstream ATG codons, which should somehow reduce the translation initiation frequency from the downstream authentic initiating HER2 codon ( see, for example, Child et al. (1999) J. Biol. Chem. 274: 24335-24341). In addition, a chimeric intron was added to the 5'-end, which should also increase expression, as previously reported (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 is derived from the Promega vector, the pCI neo vector used for expression in mammals (890-1022 bp). The 3'-end of the cDNA is flanked by exons 4 and 5 of human growth hormone and polyadenylation sequences. In addition, FVB mice were used because this strain is more susceptible to tumor development. To ensure tissue-specific expression of HER2 in the mammary gland, a promoter derived from MMTV-LTR was used. To increase the susceptibility to tumor formation, animals were fed AIN 76A (Rao et al. (1997) Breast Cancer Res. And Treatment 45: 149-158).

Table 2d

Tumor measurement in nude mice with MMTV-HER2-Fo5 breast tumor allograft model;

one dose per day 1 (T = 0), unless otherwise specified;

H = trastuzumab bound by cysteine [Cys] unless otherwise noted.

7C2 = a murine anti-HER2 antibody that binds to an epitope different from the epitope to which trastuzumab binds.

Fc8 = mutant that does not bind to FcRn.

Hg = “Hinge-free” is a full-sized humanized 4D5 antibody in which the cysteines of the hinge region of the heavy chain are replaced by serines. This antibody was expressed in E. coli (and therefore it is non-glycosylated).

2H9 = anti-EphB2R antibody

11D10 = anti-0772P antibody.

The term “Ti” means the number of animals with a tumor in the study group at T = 0: the total number of animals in the group. The term HR means the number of animals in which partial tumor remission was achieved: the number of animals with a tumor in the group at T = 0. The term CR means the number of animals in which complete tumor remission was achieved: the number of animals with a tumor in the group at T = 0. The term log of the number of killed cells means the time in days during which the tumor volume doubles - the time in days for which the tumor volume doubles in the control group divided by 3.32 × the time during which the tumor volume doubles times in control animals (to which the carrier dose was administered). The log value of the number of killed cells is calculated taking into account the time of tumor growth retardation as a result of treatment and the time of doubling the tumor volume in the control group. The antitumor activity of ADC was classified by log values of the number of destroyed cells, namely:

++++ ≥3.4 (high activity) +++ = 2.5-3.4 ++ = 1.7-2.4 + = 1.0-1.6

Inactivity = 0

On Fig illustrated the change in the average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER1 Fo5, which on day 0 was administered: carrier, trastuzumab-MS-vc-RAV-MAAE (1250 μg / m ² ) and trastuzumab-MS-vc-RAB-MAAF (555 μg / m ² ) (H = trastuzumab). Tumor growth slowed down after ADC treatment compared with the growth level in the control group (vehicle). On Fig illustrates the change in the average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which were administered on day 0: 10 mg / kg (660 μg / m ² ) conjugate trastuzumab-MS -MMAE and 1250 μg / m ² conjugate of trastuzumab-MS-vc-RAV-MMAE. On Fig illustrated the change in average tumor volume depending on time in nude mice with allografts of breast tumors MMTV-HER2 Fo5, which were injected with 650 μg / m ² trastuzumab-MS-MMAF. Table 2d and FIGS. 13-15 show that ADC has strong antitumor activity in the allograft of the HER2-positive tumor (Fo5), which originally developed in MMTV-HER2-transgenic mice. In this model, a single antibody (e.g., trastuzumab) did not have significant antitumor activity (Erickson et al., US Patent No. 6,632,979). As illustrated in FIGS. 13-15, after ADC treatment, tumor growth was slowed down compared to the growth level of the control group (vehicle).

Surprising and unexpected were the results obtained for the in vivo antitumor activity of ADC, illustrated in Table 2d, which generally showed that ADC with a low average drug molecule content with respect to the antibody had noticeable efficacy, for example, after ADC treatment, time doubling the tumor volume was> 15 days, and the average log value of the number of destroyed cells was> 1.0. 16, it is illustrated that for the antibody-drug conjugate, that is, trastuzumab-MS-vc-RAB-MMAF, the average tumor volume decreased and did not increase when the ratio of MMAF: trastuzumab was 2 and 4, whereas when the ratio 5.9 and 6, the tumor progressed, but its progression rate was lower than that of the group to which the carrier was introduced (buffer). The tumor progression rate in these mice with a xenograft model in the vehicle-administered group and the trastuzumab-administered group was approximately the same, i.e., 3 days. These results suggest that, at least for ADC with trastuzumab, the optimal ratio of drug molecules to antibody should be less than 8 and may be from about 2 to 4.

4.5.5. Toxicity in rodents

Antibody drug conjugates and ADC negative control (“vehicle”) were evaluated for acute toxicity for the rat model. The toxicity of ADC was evaluated by treating male and female Sprague-Dawley rats administered with ADC and then examining and analyzing its effect on various organs. Empirical observations included an assessment of changes in body weight and signs of damage and bleeding. Evaluation of the clinical parameters of the pathology (chemical and hematological analysis of serum), histopathological analysis and autopsy were performed in animals after a dose.

It is believed that weight loss or weight change in animals that were administered ADC, compared with animals that were administered only the carrier, is an approximate and general indicator of systemic or local toxicity. 17-19 illustrate the effect of various ADCs and controls (vehicle) on rat body weights.

Hepatotoxicity was measured by an increase in liver enzymes, an increase in the number of mitotic and apoptotic signs, and the degree of hepatocyte necrosis. Hematolymphoid toxicity was observed in the depletion of leukocytes, primary granulocytes (neutrophils) and / or platelets, and in lesions of the lymphoid organs, i.e., atrophy or apoptotic activity. Gastrointestinal lesions, such as an increase in the number of mitotic and apoptotic symptoms and degenerative enterocolitis, may also indicate toxicity.

Enzymes-indicators of the studied lesions of the liver are:

AST (aspartate aminotransferase):

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

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

- T _1/2 : 17 hours;

ALT (alanine aminotransferase):

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

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

- T _1/2 : 42 hours; daily change;

GGT (γ-glutamyl transferase):

- localization: the plasma membrane of cells with high secretory or absorption ability; liver, kidneys, small intestine;

- poor prognosis of liver damage; the content of this enzyme is usually increased in diseases of the bile duct.

The toxicity profiles of the conjugates trastuzumab-MS-val-cit-MMAF, trastuzumab-MS (Me) -val-cit-RAB-MMAF, trastuzumab-MS-MMAF and trastuzumab-MS-val-cit-RAV-MMAF were studied in female Sprague rats -Dawley (example 19). The humanized antibody trastuzumab did not show any noticeable binding to rat tissue, and any toxicity should be considered non-specific. Variants of MMAF at doses of 840 and 2105 μg / m ² were comparable with the conjugate trastuzumab-MS-val-cit-RAB-MMAF at a dose of 2105 μg / m ² .

In animals in groups 1, 2, 3, 4, 6, and 7 (vehicle, 9.94 and 24.90 mg / kg trastuzumab-MS-val-cit-MMAF, 10.69 mg / kg trastuzumab-MS (Me) -val-cit-RAV-MMAF and 10.17 and 25.50 mg / kg trastuzumab-MS-MMAF, respectively) an increase in body weight was observed during the study. Animals in groups 5 and 8 (26.78 mg / kg trastuzumab-MS (Me) -val-cit-PAB-MMAF and 21.15 mg / kg trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight study time. On day 5 of the study, the change in body weight of animals in groups 2, 6 and 7 did not differ significantly from the change in body weight of animals of group 1. The change in body weight of animals in groups 3, 4, 5, and 8 was statistically different from the change in body weight in animals in group 1 (example 19).

Rats treated with the trastuzumab-MS-MMAF conjugate (groups 6 and 7) at both doses did not differ from control animals treated with the vehicle, that is, in this model this conjugate showed the highest safety profile. Rats treated with the trastuzumab-MS-val-cit-MMAF conjugate (without the self-eliminating RAB group; groups 2 and 3) showed a dose-dependent change typical of MMAF conjugates; the degree of change was less in comparison with the action of the full-size conjugate “MS-val-cit-RAB-MMAF” (group 8). The platelet count on day 5 was approximately 30% of the baseline for group 3 animals (high dose “trastuzumab-MS-val-cit-MMAF”), in contrast to group 8 animals, which had a platelet count of 15% (high dose “Trastuzumab-MS-val-cit-RAB-MMAF”). The dose-dependent increase in the level of AST and ALT or bilirubin enzymes in the liver and the degree of thrombocytopenia were most noticeable in animals treated with the conjugate “trastuzumab-MS (Me) -val-cit-RAB-MMAF” (groups 4 and 5), and in animals of group 5 (the high-dose group), on day 5, ALT levels were approximately 10 times higher than baseline, and platelet counts were reduced by about 90% at the time of autopsy.

Sprague-Dawley female rats were also given high doses (Example 19, high-dose experiment: Groups 2, 3, 4) of the trastuzumab-MS-MMAF conjugate and vehicle (control group 1). In this case, weak toxicity signals were observed, including a dose-dependent increase in liver levels of ALT, AST, and GGT enzymes. On day 5, animals in the highest dose group showed a 2-fold increase in ALT and a 5-fold increase in AST; at the same time, the level of GGT also increased (6 units / l). On day 12, enzyme levels tended to normalize. On the 5th day, the groups showed weak granulocytosis for all three doses, but the platelet count was almost unchanged in all animals. Morphological changes were weak, that is, histological analysis of the liver, spleen, thymus, small intestine and bone marrow of animals treated with a dose of 4210 μg / m ² (group 2) did not reveal significant changes. Slightly increased apoptotic and mitotic activity was observed in the thymus and in the liver, respectively, in animals treated with a dose of 5500 μg / m ² (group 3). The bone marrow had normal cells, but showed granulocytic hyperplasia, which corresponded to the absolute granulocytosis observed when counting the formulaic blood elements in these animals. In animals of group 4, which were administered the highest dose, the same qualitative characteristics were observed, however, in comparison with animals of group 3, mitotic activity in the liver was somewhat increased. In addition, extramedullary hematopoiesis was observed in the spleen and liver of these animals.

EphB2R is a type 1 tyrosine kinase receptor, the mouse and human EphB2R receptors having a high degree of homology, and this receptor is overexpressed in colon cancer cells. 2H9 is an anti-EphB2R antibody. Taken separately, the antibody does not have any effect on tumor growth, while the 2H9-val-cit-MMAE conjugate destroys EphB2R-expressing cells and is effective in mice with the CXF1103 human colon tumor xenograft model (Mao et al. (2004 ) Cancer Res. 64: 781-788). 2H9 and 7C2 are mouse anti-HER2 IgG1 antibodies. Conjugates 2H9-MS-val-cit-PAB-MMAF (3.7 MMAF / Ab), 7C2-MS-val-cit-RAB-MMAF (4 MMAF / Ab) and trastuzumab-MS-val-cit-PAB-MMAF (5.9 MMAF / Ab) have comparable toxicity profiles. Differences in the structures of each immunoconjugate or drug component of such an immunoconjugate can affect the pharmacokinetics and ultimately the safety profile. The humanized antibody trastuzumab does not show any noticeable binding to rat tissue, and therefore any toxicity should be considered non-specific.

Toxicity / safety tested in dog-like monkeys

Similarly to the study of the toxicity level / degree of safety in rats, studies were conducted on dog-like monkeys, which included processing animals with ADC followed by measuring the level of enzymes in the liver, as well as evaluating and analyzing their effect on various organs. General observations included an assessment of changes in body weight and signs of damage and bleeding. Assessment of the clinical parameters of the pathology (chemical and hematological analysis of serum), histopathological analysis and autopsy were performed in animals after administration of the dose (example 19).

The antibody-drug conjugate, H-MS-vc-RAB-MMAE (H = trastuzumab, coupled via cysteine) did not show signs of any toxicity in the liver at any of the tested doses. After a single dose of 1100 mg / m ² was administered, peripheral blood granulocyte depletion was observed, and 14 days after this dose was administered, their complete recovery was observed. The antibody-drug conjugate, N-MS-vc-RAB-MMAE, showed an increase in liver enzymes at doses of 550 (short-term) and 880 mg / m ² , with no signs of granulocytopenia and dose-dependent short-term ( groups 2 and 3) decrease in platelet count.

4.6. The synthesis of compounds according to the invention

Representative compounds and conjugates can be obtained by carrying out the synthesis procedures described below in schemes 5-16. As will be described in more detail below, representative compounds or conjugates can be easily prepared using a linker having a reactive drug and ligand binding site. In one aspect of the invention, the linker has a reactive site that contains an electrophilic group that interacts with a nucleophilic group present on a ligand, such as, but not limited to, an antibody. Suitable nucleophilic groups on an antibody include, but are not limited to, sulfhydryl, hydroxyl, and amino groups. The heteroatom of the nucleophilic group of the antibody interacts with the 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 haloacetamide groups. Said electrophilic group provides a suitable site for binding to the antibody.

In another embodiment of the invention, the linker has a reactive site that contains a nucleophilic group that interacts with an electrophilic group present on the antibody. Suitable electrophilic groups on an antibody include, but are not limited to, the carbonyl groups of the aldehyde and ketone. The heteroatom of the nucleophilic group of the linker can interact with the 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 aryl hydrazide. Specified electrophilic group on the antibody provides a suitable site for binding to the linker.

The functional groups of the carboxylic acid and the functional groups of chloroformate are also suitable reactive sites for linking to the linker, since they can interact with the secondary amino groups of the drug and form an amide bond. A carbonate functional group on a linker, such as, but not limited to, p-nitrophenyl carbonate, which can interact with an amino group of a drug, such as, but not limited to, N-methylvaline, and form a carbamate bond, can also be used as a reactive site. . Typically, peptide drugs can be prepared by forming 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), which is a well-known method, used in the field of peptide synthesis.

The synthesis of a representative extension component having an electrophilic maleimide group is illustrated below in Schemes 8-9. General synthesis methods used for linker synthesis are described in Scheme 10. Scheme 11 shows the construction of the linker component having a Val-Cit group, an electrophilic maleimide group, and a self-eliminating spacer group RAB. Scheme 12 illustrates the synthesis of a linker having a Phe-Lys group, an electrophilic maleimide group with and without a self-eliminating spacer group PAB. Scheme 13 illustrates a general method for synthesizing a drug-linker compound, and Scheme 14 presents an alternative method for preparing a drug-linker compound. Scheme 15 shows the synthesis of a branched linker containing a BHMS group. Scheme 16 illustrates a method for attaching an antibody to a drug-linker compound to form a drug-linker-antibody conjugate, and scheme 14 illustrates the synthesis of drug-linker-antibody conjugates having, for example, but not limited to them, 2 or 4 drug molecules per antibody.

As will be described in more detail below, representative conjugates are usually prepared using a linker having two or more reactive sites for binding to a drug and a ligand. In one aspect of the invention, the linker has a reactive site that contains an electrophilic group that interacts with a nucleophilic group present on a ligand, such as an antibody. Suitable nucleophilic groups on an antibody include, but are not limited to, sulfhydryl, hydroxyl, and amino groups. The heteroatom of the nucleophilic group of the antibody interacts with the 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 haloacetamide groups. Said electrophilic group provides a suitable site for binding to the antibody.

In another embodiment of the invention, the linker has a reactive site that contains a nucleophilic group that interacts with an electrophilic group present on a ligand, such as an antibody. Suitable electrophilic groups on an antibody include, but are not limited to, the carbonyl groups of the aldehyde and ketone. The heteroatom of the nucleophilic group of the linker can interact with the 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 aridhydrazide. Specified electrophilic group on the antibody provides a suitable site for binding to the linker.

4.6.1 Drug Synthesis

Typically, peptide drugs can be prepared by forming 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), which is a well-known method, used in the field of peptide synthesis.

Medicines auristatin / dolastatin can be obtained by the General methods described in US patent No. 5635483, in US patent No. 5780588; Pettit et al., (1989) J. Am. Chem. Soc. 111: 5463-5465; from 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 embodiment, the drug is prepared by combining approximately the stoichiometric equivalent of a dipeptide and a tripeptide, and preferably by conducting a reaction in one vessel under suitable condensation conditions. This method is illustrated below in schemes 5-7.

Scheme 5 illustrates the synthesis of the N-terminal tripeptide molecule F, which is a suitable intermediate for the synthesis of drug compounds of formula 1b.

Scheme 5

As illustrated in Scheme 5, the protected amino acid A (where PG is an amine protecting group, R ^{4 is} selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, -O- (C ₁ -C ₈ alkyl), aryl, alkylaryl, alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle, alkyl (C ₃ -C ₈ heterocycle), where R ^{5 is} selected from H and methyl; or R ⁴ and R ⁵ taken together to the carbon atom to which they are bonded form a ring having the formula - (CR ^a R ^b ) _n -, where R ^a and R ^{b are} independently selected from hydrogen, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle, and n is selected from 2, 3, 4, 5, and 6) are coupled with tert-buty In ovym ether (wherein R ⁶ is selected from -H and -C ₁ -C ₈ alkyl, and R ⁷ is selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, -O- (C ₁ -C ₈ alkyl), aryl, alkylaryl, alkyl- (C ₃ -C ₈ heterocycle), C ₃ -C ₈ heterocycle and alkyl- (C ₃ -C ₈ heterocycle)) under suitable conditions of the coupling reaction, for example, in the presence of PyBrop and diisopropylethylamine or using DCC (see, for example, Miyazaki K. et al., Chem. Pharm. Bull. 1995, 43 (10), 1706-1718).

Suitable PG protecting groups and suitable synthesis methods for protecting an amino group with a protecting group are well known in the art. See, for example, Greene T.W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Representative protected amino acids A are PG-Ile, and in particular PG-Val, and other suitable protected amino acids are, but are not limited to, PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine and PG-tert-butyl glycine. Z represents a representative protecting group. Fmoc is another representative protecting group. A representative tert-butyl ether B is dolaisoleucine tert-butyl ether.

Dipeptide C can be purified, for example, by chromatography, and then it can be subjected to a deprotection reaction, for example, using H ₂ and 10% Pd-C in ethanol, if PG is benzyloxycarbonyl, or using diethylamine to remove the protective Fmoc groups. The resulting amine D readily forms a peptide bond with amino acid BB (where R ^{1 is} selected from —H, —C ₁ -C ₈ alkyl and —C ₃ -C ₈ carbocycle, R ^{2 is} selected from —H and —C ₁ -C ₈ alkyl; or R ¹ and R ² taken together with the nitrogen atom to which they are bonded form a ring of the formula - (CR ^a R ^b ) _n , where R ^a and R ^{b are} independently selected from —H, —C ₁ -C ₈ alkyl, and -C ₃ -C ₈ carbocycle, and n is selected from 2, 3, 4, 5 and 6, and R ^{3 is} selected from hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, -O- (C ₁ - C ₈ alkyl), aryl, alkylaryl, alkyl- (C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and alkyl- (C ₃ -C ₈ heterocycle)). Representative N, N-dialkyl amino acids are BB amino acids, such as commercially available N, N-dimethylvaline. Other N, N-dialkyl amino acids can be obtained by reductive bis-alkylation in accordance with standard procedures (see, for example, Bowman RE Stroud HHJ Chem. Soc. 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two representative BB amino acids that can be used to synthesize N-monoalkyl derivatives. Amine D and amino acid BB are reacted in the presence of a combination reagent DEPC and triethylamine as a base to give tripeptide E. Then the C-terminal protecting group E is removed using HCl to give a tripeptide compound of formula F.

An exemplary method for conducting a coupling reaction using DEPC and a coupling reaction using PyBrop shown in Scheme 5 is described below in General Procedure A and General Procedure B, respectively. An exemplary method for deprotecting a Z-protected amine by catalytic hydrogenation is described below in General Procedure C.

General Procedure A: Peptide Synthesis Using DEPC . The N-protected or N, N-disubstituted amino acid or peptide D (1.0 eq.) And amine BB (1.1 eq.) Are diluted with an aprotic organic solvent such as dichloromethane (0.1-0.5 M). An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) Is then added, followed by DEPC (1.1 eq.). The resulting solution was preferably stirred under argon for up to 12 hours and monitored by HPLC or TLC. The solvent was removed in vacuo at room temperature and the crude product was purified by, for example, HPLC or flash column chromatography (on a silica gel column). The appropriate fractions were combined and concentrated in vacuo to give tripeptide E, which was dried in vacuo overnight.

General Procedure B: Peptide Synthesis Using PyBrop . Amino acid B (1.0 equiv.), Optionally having a carboxy protecting group, is diluted with an aprotic organic solvent, such as dichloromethane or DME, to give a solution with a concentration of 0.5 to 1.0 mM, after which diisopropylethylamine (1.5 eq.) Then, Fmoc or Z-protected amino acid A (1.1 eq.) as a solid is then added in one portion, after which PyBrop (1.2 eq.) is added to the resulting mixture. The reaction is monitored by TLC or HPLC followed by treatment similar to that described in General Procedure A.

General Procedure C: Deprotection of the Z-Group by Catalytic Hydrogenation The Z-protected amino acid or Z-protected peptide C is diluted with ethanol to give a solution with a concentration of from 0.5 to 1.0 mM in a suitable vessel, such as a round bottom flask with thick walls. Then add 10% palladium on carbon (5-10% wt./wt.) And the reaction mixture is placed in an atmosphere of hydrogen. The reaction is monitored by HPLC, and usually the reaction is completed in 1-2 hours. The reaction mixture is filtered through a pre-washed pad of celite, and then, after filtration, the celite is washed again with a polar organic solvent such as methanol. The eluent solution was concentrated in vacuo to give a residue, which was diluted with an organic solvent, and preferably toluene. The organic solvent is then removed in vacuo to give amine C without a protecting group.

Scheme 6 illustrates the method used to produce the C-terminal dipeptide of formula K and the method used in the reaction of combining a dipeptide of formula K with a tripeptide of formula F to produce drug compounds of formula 1b.

Scheme 6

The dipeptide K can be easily obtained by condensation of the modified amino acid Boc-dolaproin G (see, for example, Pettit, GR, et al., Synthesis, 1996, 719-725) with an amine of formula H using condensing agents well known to those skilled in the art peptide chemistry, such as, for example, DEPC , in the presence of triethylamine, as shown in scheme 5.

The dipeptide of formula K can then undergo a coupling reaction with the tripeptide of formula F in accordance with general procedure D to obtain Fmoc-protected compounds of the drug of formula L, in which, according to general procedure E, the protective group can then be removed, whereby compounds can be obtained a drug of formula (Ib).

General Procedure D: Drug Synthesis . A mixture of dipeptide K (1.0 equiv.) And tripeptide F (1 equiv.) Is diluted with an aprotic organic solvent, such as dichloromethane, to obtain a 0.1 M solution, and then a strong acid, such as trifluoroacetic acid (1/2, vol. ./vol.), and the resulting mixture was stirred under nitrogen for two hours at 0 ° C. Monitoring the reaction can be carried out using TLC or preferably HPLC. The solvent was removed in vacuo and the resulting residue was dried by double azeotropic distillation, preferably using toluene. The resulting residue was dried under high vacuum for 12 hours and then diluted with an aprotic organic solvent, such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) Is added, and then, depending on the chemical functional groups of the residue, either PyBrop (1.2 eq.) Or DEPC (1.2 eq.) Are added. . The reaction is monitored by TLC or HPLC and, after completion of the reaction, the reaction mixture is subjected to a treatment similar or identical to that described in General Procedure A.

General Procedure E: Removal of Fmoc Using Diethylamine . The Fmoc-protected drug L is diluted with an aprotic organic solvent, such as dichloromethane, and diethylamine (1/2, v / v) is added to the resulting solution. Monitoring the progress of the reaction is carried out using TLC or HPLC, and usually the reaction is completed after 2 hours. The reaction mixture was concentrated in vacuo and the resulting residue was dried by azeotropic distillation, preferably using toluene, and then dried under high vacuum to give drug 1b having an unprotected amino group.

Scheme 7 illustrates the method used to obtain the MMAF derivatives of formula (Ib).

Scheme 7

The dipeptide O can be easily obtained by condensation of the modified amino acid Boc-dolaproin G (see, for example, Pettit, GR, et al., Synthesis, 1996, 719-725) with a protected amino acid of formula M using condensing agents well known to those skilled in the art areas of peptide chemistry, such as, for example, DEPC , in the presence of triethylamine, as shown in schemes 5 and 6.

A dipeptide of formula O can then undergo a coupling reaction with a tripeptide of formula F in accordance with general procedure D to produce Fmoc-protected MMAF compounds of formula P, in which, according to general procedure E, the protecting group can then be removed, whereby MMAF can be obtained - compounds of the drug of formula (Ib).

So, for example, the above methods are suitable for the preparation of drugs that can be used in the present invention.

4.6.2. Synthesis of drug-linker conjugate

To obtain the drug-linker compound of the invention, the drug is reacted with a reactive site on the linker. In general terms, the linker may have the structure:

Jet Site 2 - A _a -W _w -Y _y - Jet Site 1

where a spacer component (-Y-) and an extension component (-A-) are present. Alternatively, the linker may have the structure:

Jet Site 2 - A _a -W _w - Jet Site 1

where there is no spacer component (-Y-).

 The linker may also have the structure:

Jet Site 2 -W _w - Jet Site 1

where there is no extension component (-A-) and spacer component (-Y-).

The linker may also have the structure:

Jet Site 2 - A _a - Jet Site 1

where there is no amino acid component (W) and spacer component (Y).

In general terms, a suitable linker has an amino acid component attached to an optional extension component and an optional spacer component. Reactive site 1 is present at the end of the spacer, and reactive site 2 is present at the end of the extension component. If there is no spacer component, then reactive site 1 is present at the C-terminus of the amino acid component.

In a representative embodiment of the present invention, the reactive site No. 1 undergoes a reaction with the nitrogen atom of the drug, and the reactive site No. 2 undergoes a 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

.

In another aspect of the invention, reactive site No. 1 is:

In yet another aspect of the invention, reactive site No. 1 is p-nitrophenyl carbonate having the formula:

In one aspect of the invention, reactive site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups are haloacetamide groups having the formula:

where X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl, —Br or —I; or a maleimide group having the formula:

Used linkers can be obtained from commercially available sources, such as Molecular Biosciences Inc. (Boulder, CO), or they can be obtained as described below in schemes 8-10.

Scheme 8

where X is —CH ₂ - or —CH ₂ OCH ₂ -; and n is an integer from 0 to 10 if X is —CH ₂ -; or from 1 to 10 if X is —CH ₂ OCH ₂ -.

In the method illustrated in Scheme 9, maleimide is reacted with glycol under the Mitsunobu reaction conditions, resulting in an extension component "polyethylene glycol-maleimide" (see, for example, Walker MAJ Org. Chem. 1995, 60, 5352-5), and then a p-nitrophenyl carbonate group is introduced into the reactive site.

Scheme 9

where E represents —CH ₂ - or —CH ₂ OCH ₂ -; and e is an integer from 0 to 8.

Alternatively, PEG maleimide and PEG haloacetamide extension components can be prepared as described by Frisch et al. Bioconjugate Chem. 1996, 7, 180-186. Scheme 10 illustrates the general synthesis of a representative linker component containing a maleimide extension group and, optionally, a self-eliminating spacer, which is a p-aminobenzyl ether.

Pattern 10

where Q is —C ₁ -C ₈ alkyl, —O— (C ₁ -C ₈ alkyl), halo, nitro or cyano; 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 by Molecular Biosciences (Boulder, CO), as described below in accordance with known methods of organic synthesis. The extension components of formula (IIIa) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid component, as shown in Schemes 11 and 12.

where n is an integer from 1 to 10, and T represents —H or —SO ₃ Na;

where n is an integer from 0 to 3;

и

and

.

.

Extension components of formula (IIIb) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid component:

where X represents Br or I; and

The extension components of formula (IV) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid component:

Extension components of formula (Va) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid component:

Other extension components used may be synthesized by known methods. The extending aminooxy components of the following formula can be prepared by treating the alkyl halides with N-Boc-hydroxylamine in accordance with the procedures described by Jones D.S. et al. Tetrahedron Letters, 2000, 41 (10), 1531-1533 and Gilon C. et al., Tetrahedron, 1967, 23 (11), 4441-4447.

where R ^{17 is} selected from —C ₁ -C ₁₀ alkylene, —C ₃ -C ₈ carbocyclo, —O— (C ₁ -C ₈ alkyl), arylene, —C ₁ -C ₁₀ alkylene arylene, arylene-C ₁ -C ₁₀ alkylene, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ carbocyclo) -, - (C ₃ -C ₈ carbocyclo) -C ₁ -C ₁₀ alkylene, -C ₃ -C ₈ heterocyclo, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ heterocyclo) -, - (C ₃ -C ₈ heterocyclo) -C ₁ -C ₁₀ alkylene, - (CH ₂ CH ₂ O) _r -, - (CH ₂ CH ₂ O) _r -CH ₂ -; and r is an integer from 1 to 10.

The isothiocyanate extension components of the following formula can be prepared from isothiocyanocarboxylic acid chlorides, as described by Angew. Chem. 1975, 87 (14): 517.

where R ¹⁷ is as defined in the description of the invention.

Scheme 11 illustrates a method for preparing a val-cit dipeptide linker having a maleimide extension component and, optionally, a self-eliminating spacer, which is p-aminobenzyl.

Scheme 11

where Q is —C ₁ -C ₈ alkyl, —O— (C ₁ -C ₈ alkyl), halo, nitro or cyano; and m is an integer from 0 to 4.

Scheme 12 illustrates the synthesis of a Phe-Lys (Mtr) dipeptide linker component having a maleimide extension component and a p-aminobenzyl self-eliminating spacer component. Source material AD (Lys (Mtr)) is commercially available (Bachem, Torrance, CA), or it can be obtained as described by Dubowchik et al. Tetrahedron Letters, (1997) 38: 5257-60.

Pattern 12

where Q is —C ₁ -C ₈ alkyl, —O— (C ₁ -C ₈ alkyl), halo, nitro or cyano; and m is an integer from 0 to 4.

As shown in Scheme 13, the linker can be reacted with the amino group of the drug compound of formula (Ib) to form a drug-linker compound that contains an amide or carbamate group linking the drug component to the linker component. If the reactive site No. 1 is a carboxylic acid group, as in the AJ linker, the coupling reaction can be carried out using HATU or PyBrop and the corresponding amine base to obtain the drug-linker AK compound containing the amide bond between the drug component and linker component. If reactive site No. 1 is carbonate, as in the AL linker, then the linker can be attached to the drug using HOBt in a DMF / pyridine mixture to obtain the drug-linker AM compound containing a carbamate linkage between the drug component and linker component.

Alternatively, if reactive site No. 1 is a good leaving group, such as in the AN linker, then the linker can be attached to the amino group of the drug by a nucleophilic substitution reaction, whereby a drug-linker compound having an amine bond can be obtained (AO) between the drug component and the linker component.

Representative methods used to attach the drug to the ligand to obtain the drug-linker compound are described in Scheme 13 and illustrated in the general G-H procedures.

Pattern 13

General Procedure G: Amide Formation Using HATU . The drug (Ib) (1.0 equiv.) And the N-protected linker containing the carboxylic acid reactive site (1.0 equiv.) Are diluted with a suitable organic solvent such as dichloromethane, and the resulting solution is treated with HATU (1.5 equiv. ) and an organic base, preferably pyridine (1.5 equiv). The reaction mixture is stirred in an inert atmosphere, preferably in an argon atmosphere, for 6 hours, during which the reaction mixture is monitored by HPLC. The reaction mixture was concentrated and the resulting residue was purified by HPLC to give an amide of the formula AK.

Procedure H: Carbamate Formation Using HOBt . A mixture of an AL linker having a p-nitrophenyl carbonate reactive site (1.1 eq.) And a drug (1b) (1.0 eq.) Is diluted with an aprotic organic solvent, such as DMF, to give 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 argon atmosphere. The reaction mixture was stirred for 15 minutes, and then an organic base such as pyridine (1/4, v / v) was added and the reaction was monitored by HPLC. Linker absorption usually occurs in 16 hours. Then the reaction mixture was concentrated in vacuo and the resulting residue was purified, for example, by HPLC to give carbamate AM.

An alternative method for preparing the drug-linker compounds is illustrated in Scheme 14. According to the method illustrated in Scheme 14, the drug is attached to a portion of the linker component (eg, ZA) to which the extension component is not attached. As a result, an intermediate AP can be obtained that has an amino acid component containing an Fmoc-protected N-terminal group. The Fmoc group is then removed, after which the resulting amine intermediate AQ is coupled to the extension component via a coupling reaction catalyzed using PyBrop or DEPC. The construction of drug-linker compounds containing either a bromoacetamide extension component AR or a PEG-maleimide extension component AS is illustrated in Scheme 14.

Scheme 14

М!

M!

where Q is —C ₁ -C ₈ alkyl, —O— (C ₁ -C ₈ alkyl), halo, nitro or cyano; and m is an integer from 0 to 4.

The methodology used to obtain the linker component containing a branched chain spacer is illustrated in scheme 15.

Scheme 15

Scheme 15 illustrates the synthesis of a val-cit dipeptide linker having a maleimide extension component and a bis (4-hydroxymethyl) styrene (BHMS) component. The synthesis of the BHMS intermediate (AW) has been improved over the synthesis previously described in the literature (see International Application Publication No. WO 9813059, Firestone et al. & Crozet MP; Archaimbault G. .; Vanelle P .; Nouguier R. Tetrahedron Lett. (1985) 26: 5133-5134), and commercially available diethyl (4-nitrobenzyl) phosphonate (AT) and commercially available 2,2-dimethyl-1,3-dioxan-5-one are used as starting materials in this synthesis. (AU). Linkers AY and BA can be obtained from the intermediate connection AW in accordance with the methodology illustrated in scheme 9.

4.6.3. Dendritic linkers

The linker may be a dendritic type linker used to covalently link more than one drug molecule to a ligand, such as, but not limited to, an antibody via a branching multifunctional linker molecule (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, that is, a load that corresponds to the effectiveness of the drug-linker-ligand conjugate. Thus, if a cysteine-based antibody contains only one reactive cysteine thiol group, then a large number of drug molecules can be attached via a dendritic linker.

In the following representative embodiments, dendritic linker reagents allow the formation of conjugates with nine nucleophilic reagents of a drug molecule through interaction with the chloroethyl 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 obtain the drug-linker-ligand conjugates having from about 2 to 4 drug molecules per antibody. The antibody is treated with a reducing agent such as dithiothreitol (DTT) to reduce some or all of the cysteine disulfide residues to form highly nucleophilic thiol groups of cysteine (-CH ₂ SH). Such a partially reduced antibody is reacted with drug-linker compounds or with linker reagents, that is, with their electrophilic functional groups, such as maleimide or α-halogenocarbonyl, according to the conjugation method described on page 766 of Klussman et al. (2004) Bioconjugate Chemistry 15 (4): 765-773.

Pattern 16

For example, an antibody, such as AC10, 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 37 ° C for 30 minutes, the buffer is replaced by elution with Sephadex G25 resin and PBS with 1 mM DTPA is eluted. The thiol / Ab ratio is calculated by determining the concentration of the reduced antibody based on the optical density at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the optical density at 412 nM. The reconstituted antibody is dissolved in PBS and cooled on ice. A drug-linker conjugate, for example MS-val-cit-RAB-MMAE in DMSO, dissolved in acetonitrile and water at known concentrations, is added to the cooled reconstituted antibody in PBS. After about one hour, an excess of maleimide is added to quench the reaction and “cap” the unreacted thiol groups of the antibody. The reaction mixture was concentrated by ultrafiltration in a centrifuge and ADC, for example AC10-MS-vc-RAB-MMAE, was purified, desalted by elution through G25 resin in PBS, filtered through 0.2 μm filters under sterile conditions and frozen for storage.

Various antibody-drug conjugates (ADCs) were prepared with various linkers and drug molecules, MMAE and MMAF. The following table shows representative ADC groups that were obtained according to the protocol described in Example 27 and were characterized by HPLC and drug loading analysis.

4.7. Compositions and methods of administration

In another embodiment, the present invention relates to a composition comprising an effective amount of a representative compound and / or a representative conjugate and a pharmaceutically acceptable carrier or excipient. For convenience, the “drug” components and the “drug-linker” compounds will be referred to as representative compounds, and the “drug-ligand” and “drug-linker-ligand” conjugates will be referred to as representative conjugates. These compositions can be used for administration to animals and humans.

Compositions according to the invention can be obtained in any form that allows these compositions to be administered to a patient. So, for example, the composition may have a solid, liquid or gaseous (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, intraocular, intratumoral, and intranasal administration. Parenteral administration includes subcutaneous, intravenous, intramuscular and intrasternal injections or infusions. In one aspect of the invention, the compositions are administered parenterally. In another aspect of the invention, representative compounds and / or representative conjugates or compositions are administered intravenously.

Pharmaceutical compositions may be formulated so that, after administration to a patient, representative compounds and / or representative conjugates become bioavailable. Compositions can be prepared in the form of one or more unit dosage forms, for example, in the form of a tablet, which may be a single unit dosage form, and a container containing a representative compound and / or a representative conjugate in the form of an aerosol, which may contain many unit dosage forms.

The materials used to prepare the pharmaceutical compositions may be non-toxic in the amounts used. For each specialist, it is obvious that the optimal dose of the active ingredient (s) in the pharmaceutical composition depends on a number of factors. Such factors include, but are not limited to, the type of animal (eg, human), the particular form of the representative compound or conjugate, route of administration, and composition used.

The pharmaceutically acceptable carrier or excipient may be a solid, which makes it possible to obtain compositions, for example, in the form of a tablet or powder. The carrier may be liquid, and therefore, compositions containing it may be, for example, prepared in the form of a syrup for oral administration or an injection. In addition, the carrier can be gaseous or powdery and is used to prepare an aerosol composition used, for example, for administration by inhalation.

For oral administration, the composition is preferably prepared in solid or liquid form, which may be semi-solid, semi-liquid, suspension and gel forms, which are incorporated herein in forms considered solid or liquid.

A solid composition for oral administration can be prepared in the form of a powder, granules, compressed tablets, dragees, capsules, chewing gum, cachet, or the like. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following agents may be present in this composition: binding agents such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose or gelatin; fillers such as starch, lactose or dextrins; disintegrating agents such as alginic acid, sodium alginate, primogel, corn starch and the like; lubricants such as magnesium stearate or sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; a fragrance such as peppermint, methyl salicylate or an orange flavor; and dye.

If the composition is prepared in the form of a capsule, for example in the form of a gelatin capsule, then, in addition to the above materials, it may contain a liquid carrier such as polyethylene glycol, cyclodextrin or fatty oil.

The composition may be prepared in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. Such a liquid can be used for oral administration or for delivery by injection. A composition for oral administration may contain one or more agents such as a sweetener, preservatives, a colorant / coloring agent, and an intensifier of taste and aroma. One or more agents such as a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonicity agent may be included in a composition intended for administration by injection.

Liquid compositions, whether they are solutions, suspensions, or have a different form, may also include one or more of the following agents, namely sterile diluents, such as water for injection, saline, and preferably physiological saline, Ringer's solution, isotonic sodium chloride, fatty oils, such as synthetic mono- or diglycerides, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solutions spectators; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents, such as sodium chloride or dextrose. The composition for parenteral administration may be enclosed in an ampoule, in a disposable syringe or in a receptacle for repeated administration made of glass, plastic or other material. A representative adjuvant is saline. The composition for injection is preferably sterile.

The amount of a representative compound and / or a representative conjugate that is effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical methods. In addition, in vitro or in vivo assays may be performed, but not necessarily, to determine optimal dose levels. The exact dose used in these compositions also depends on the route of administration, the severity of the disease or disorder, and must be prescribed individually by the attending physician for each patient.

The compositions contain a certain amount of a representative compound and / or a representative conjugate, effective to obtain the appropriate dose. Typically, this amount is at least about 0.01% of a representative compound and / or a representative conjugate by weight of the composition. For oral administration, this amount can vary 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 a representative compound and / or a representative conjugate by weight of the composition. In yet another aspect, compositions of the invention are formulated so that a parenteral dosage form contains about 0.01 to 2% by weight of a representative compound and / or a representative conjugate.

The composition for intravenous administration may contain from about 0.01 to 100 mg of a representative compound and / or a representative conjugate per kg of animal body weight. In one aspect of the invention, such a composition may contain from about 1 to 100 mg of a representative compound and / or a representative conjugate per kg of animal body weight. In another aspect of the invention, the administered amount of a representative compound and / or a representative conjugate is in the range of about 0.1 to 25 mg / kg body weight of the animal.

Typically, a dose of a representative compound and / or a representative conjugate administered to a patient is from about 0.01 to 2000 mg / kg body weight of the animal. In one aspect of the invention, the dose administered to the patient is from about 0.01 to 10 mg / kg of animal body weight, in another aspect of the invention, the dose administered to the patient is from about 0.1 to 250 mg / kg of animal body weight, B in yet another aspect of the invention, the dose administered to the patient is from about 0.1 to 20 mg / kg body weight of the animal. In yet another aspect of the invention, the dose administered is from about 0.1 to 10 mg / kg of body weight of the animal, and in another aspect of the invention, the administered dose is from about 1 to 10 mg / kg of animal body weight.

Representative compounds and / or a representative conjugate or compositions can be administered by standard methods, for example, by infusion or injection of a loading dose, by absorption through the epithelium or lining of the mucous membrane (for example, through the mucous membrane of the oral cavity, rectum, small intestine, etc.). Such an introduction may be systemic or local. Various delivery systems are known, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, and the like, and these systems can be used to introduce a representative compound and / or a representative conjugate or composition. In some embodiments of the invention, more than one representative compound and / or a representative conjugate or composition may be administered to a patient.

In particular embodiments of the invention, it may be desirable to locally administer one or more representative compounds and / or representative conjugates or compositions to a site of an organism in need of treatment. This can be achieved by such methods, but not limited to, such as, for example, local infusion during a surgical operation; external application, for example, in the form of a dressing on a wound after surgery; injection; catheter insertion; administration by suppository or administration of an implant, wherein said implant consists of a porous, non-porous or gel-like material, including membranes such as sialastic membranes or fibers. In one embodiment of the invention, administration may be by direct injection into a given area (or primary area) of the localization of the cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment of the invention, administration may be by direct injection into a given area (or primary area) of a manifestation of symptoms of an autoimmune disease.

In some embodiments of the invention, it may be desirable to administer one or more representative compounds and / or representative conjugates or compositions to the central nervous system in any suitable manner, including intraventricular and intrathecal injection. An intraventricular injection can be more easily administered via an intraventricular catheter, for example, attached to an Ommaya reservoir.

Pulmonary administration may also be effected, for example, by means of an inhaler or nebulizer, either by preparation of a preparation using a nebulizing agent, or by perfusion in fluorocarbon or in a synthetic surfactant suitable for administration to the lungs.

In yet another embodiment of the invention, representative compounds and / or representative conjugates or compositions may be administered using a controlled delivery system, such as, but not limited to, a pump or various polymeric materials. In another embodiment of the invention, a system for controlled delivery can be placed in the immediate vicinity of the target to which representative compounds and / or representative conjugates or compositions are directed, for example to the brain, and only part of the systemic dose may be required (see, for example, Goodson , in Medical Applications of Controlled Release, see above, vol. 2, p. 115-138 (1984)). Other controlled delivery systems discussed by Langer may be used (Science 249: 1527-1533 (1990)).

The term “carrier” means a diluent, adjuvant or excipient in combination with which a representative compound and / or a representative conjugate are administered. Such pharmaceutical carriers may be liquids such as water and oil, including liquid paraffin, animal and vegetable oil, or synthetic oil such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers may be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, additives that stabilize agents, thickeners, lubricants and colorants can be used. In one embodiment of the invention, a representative compound and / or a representative conjugate or compositions and pharmaceutically acceptable carriers administered to a patient are sterile. For intravenous administration of representative compounds and / or representative conjugates, a suitable carrier is water. As liquid carriers, and in particular to obtain solutions for injection, physiological solutions and aqueous solutions of dextrose and glycerol can also be used. Also suitable pharmaceutical carriers are excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, separated milk powder, glycerin, propylene glycol, water, ethanol and the like The compositions of the invention, if necessary, may also contain small amounts of wetting or emulsifying agents or pH buffering agents.

Compositions according to the invention can be prepared in the form of solutions, suspensions, emulsions, tablets, dragees, granules, capsules, capsules containing liquids, powders, sustained release preparations, suppositories, emulsions, aerosols, sprays, suspensions or any other form suitable for such application. Other examples of suitable pharmaceutical carriers are described in E.W. Martin “Remington's Pharmaceutical Sciences”.

In one embodiment of the invention, representative compounds and / or representative conjugates are prepared in accordance with routine procedures in the form of a pharmaceutical composition adapted for intravenous administration to animals, and in particular to humans. Typically, carriers or excipients for intravenous administration are sterile isotonic aqueous buffers. If necessary, such compositions may also include a solubilizing agent. Compositions for intravenous administration may, but not necessarily, contain a local anesthetic, such as lignocaine, to relieve pain at the injection site. Typically, the ingredients are added either individually or in a mixture in the form of a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, which indicates the amount of active substance. If representative compounds and / or a representative conjugate are administered by infusion, they can be dispensed, for example, in vials containing sterile water or physiological saline of pharmaceutical grade. If representative compounds and / or a representative conjugate are administered by injection, an ampoule of sterile water for injection can be prepared, or physiological saline for injection can be prepared so that the ingredients can be mixed prior to administration.

Compositions for oral delivery can be obtained, for example, in the form of tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Orally administered compositions may contain one or more optional agents, for example sweeteners, such as fructose, aspartame or saccharin; fragrances such as peppermint, wintergreen oil, cherry oil; dyes; and preservatives, to give such pharmaceuticals acceptable palatability. In addition, if the compositions are in the form of tablets or dragees, then they can be coated to slow the disintegration and absorption in the gastrointestinal tract and, thus, to ensure their effect over a long period of time. Selectively permeable membranes surrounding osmotically active stimulant compounds are also suitable for orally administered compounds. In these membranes, the liquid from the environment surrounding the capsule is impregnated with a stimulating compound that swells and displaces the agent or composition through the membrane opening. These delivery membranes can provide mainly a zero-order delivery profile, in contrast to the pulse profile of immediate release formulations. To slow down the delivery time, a substance such as glycerol monostearate or glycerol stearate can be used.

The compositions may be intended for topical use, in which case a carrier in the form of a solution, emulsion, ointment or gel base may be used. The composition for transdermal administration can be obtained in the form of a transdermal patch or device for iontophoresis. Topical compositions may contain a concentration of a representative compound and / or a representative conjugate of about 0.05% to 50% w / v. (mass per unit volume of the composition), and in another aspect, from 0.1% to 10% wt./about.

The composition may be administered rectally, for example in the form of suppositories, which melt in the rectum and release a representative compound and / or a representative conjugate.

This composition may include various materials that modify the physical form of a solid or liquid standardized drug. So, for example, this 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, from sugar, shellac and other enteric coating agents. Alternatively, the active ingredients may be enclosed in a gelatin capsule.

The compositions may consist of gaseous unit dosage forms, for example, they may be an aerosol. The term “aerosol” is used to refer to various systems, from colloidal systems to systems consisting of sealed packages. Delivery may be via liquefied or compressed gas, or through a suitable pump system dispensing the release of active ingredients.

The compositions of the invention, whether present in solid, liquid, or gaseous form, may include a pharmacological agent used to treat cancer, an autoimmune disease, or an infectious disease.

4.8. Therapeutic use of representative conjugates

Representative compounds and / or representative conjugates can be used to treat cancer, an autoimmune disease, or an infectious disease in a patient.

4.8.1. Cancer treatment

Representative compounds and / or representative conjugates can be used to inhibit the growth of tumor or cancer cells, induce apoptosis in tumor or cancer cells, or to treat cancer in a patient. Representative compounds and / or representative conjugates can be used respectively in various methods of treating cancer in animals. The drug-linker-ligand conjugate can be used to deliver a drug or drug component to a tumor or cancer cell. Without claiming to be a specific theory, it can only be noted that In one embodiment of the invention, the ligand component of a representative conjugate binds to or associates with an antigen of cancer cells or tumor cells, and this representative conjugate can be absorbed by a tumor or cancer cell via receptor-mediated endocytosis. The antigen may be attached to a tumor or cancer cell, or it may be an extracellular matrix protein associated with a tumor cell or cancer cell. After introduction into the cell, one or more specific peptide sequences in the linker component is hydrolytically cleaved by one or more proteases of the tumor or cancer cell, which leads to the release of the drug or drug-linker compound. Then, the released drug or drug-linker compound migrates freely into the cell and induces cytotoxic or cytostatic activity. In an alternative embodiment, the drug or drug component is cleaved from the representative conjugate and enters the external environment of the tumor or cancer cell, and then the drug or drug-linker compound enters the cell.

In one embodiment of the invention, the ligand component binds to a tumor or cancer cell.

In another embodiment of the invention, the ligand component binds to a tumor or cancer cell antigen that is located on the surface of the tumor or cancer cell.

In another embodiment of the invention, the ligand component binds to a tumor or cancer cell antigen, which is an extracellular matrix protein associated with a tumor cell or cancer cell.

The specificity of the ligand component for specific tumor or cancer cells can play an important role in determining the type of tumor or cancer cells that are best suited for effective killing. Thus, for example, representative conjugates containing BR96 ligand can be used to treat antigen-positive carcinomas, including carcinomas of the lung, breast, ovary, and pancreas. Representative conjugates containing a ligand, such as antibodies against CD30 or against CD40, can be used to treat hematologic malignant diseases.

Other specific types of cancers that can be treated with representative conjugates include, but are not limited to, the cancers indicated in Table 3.

Table 3

Solid tumors, including, but not limited to:

fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosarcoma
rhabdomyosarcoma colon cancer colon cancer kidney cancer pancreas cancer bone cancer mammary cancer ovarian cancer prostate cancer esophageal carcinoma stomach cancer oral cancer nasal cancer throat cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonic carcinoma Wilms tumor cervical cancer uterine cancer testicular cancer small cell lung carcinoma bladder carcinoma lungs' cancer epithelial carcinoma glioma glioblastoma multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma auditory nerve neuroma oligodendroglioma meningioma skin cancer melanoma neuroblastoma retinoblastoma

Blood cancer, including but not limited to:

acute lymphoblastic leukemia, “ALL” acute lymphoblastic b-cell leukemia acute lymphoblastic T-cell leukemia acute myeloid leukemia, “AML” acute promyelocytic leukemia, “OPL” acute monoblastic leukemia acute red blood cell leukemia acute megakaryoblastic leukemia acute myelomonocytic leukemia acute non-lymphocytic leukemia acute undifferentiated leukemia chronic myelocytic leukemia, “CML” chronic lymphocytic leukemia, “CLL” leukemic reticulosis multiple myeloma Acute and chronic leukemia lymphoblastic myelogenous lymphocytic myelocytic Lymphomas Hodgkin's disease non-Hodgkin lymphoma multiple myeloma Waldenstrom macroglobulinemia heavy chain disease true polycythemia

Representative conjugates provide the delivery of compounds to tumor or cancer cells specific for conjugation, which leads to a decrease in the overall toxicity of these compounds. Linker components stabilize representative conjugates in the blood, which, however, are cleaved by tumor-specific proteases of the cell, releasing the drug.

4.8.2. Cancer treatment

Cancers, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth, can be treated or prevented by the administration of a representative conjugate and / or representative compound.

In other embodiments of the invention, methods of treating or preventing cancer are described, comprising administering to a patient in need thereof an effective amount of a representative conjugate and a chemotherapeutic agent. In one embodiment of the invention, such a chemotherapeutic agent is one that has not been found to be effective in treating cancer. In another embodiment of the invention, such a chemotherapeutic agent is one that has been found to be effective in treating cancer. Representative conjugates may be administered to a patient who has also undergone surgery to radically treat cancer.

In one embodiment of the invention, an additional method of treating cancer is radiation therapy.

In a specific embodiment of the invention, a representative conjugate is administered simultaneously with a chemotherapeutic agent or simultaneously with radiation therapy. In another specific embodiment of the invention, the administration of a chemotherapeutic agent or radiation therapy is carried out before or after the introduction of representative conjugates, for example, in one aspect, such administration is carried out at least / after one hour, five hours, 12 hours, one day, one week , one month before / after administration of a representative conjugate, and in other aspects, within / after several months (for example, three months) before / after administration of this conjugate.

A chemotherapeutic agent may be administered in several sessions. In this case, one of the chemotherapeutic agents listed in table 4, or a combination of them, can be introduced. 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, X-ray therapy, and in particular for the treatment of deep tumors, high-energy radiation of several megavolts (radiation with an energy of more than 1 MV) can be used, and electron radiation and X-ray radiation can be used to treat skin cancer in several ortovolts. Gamma radiation emitted by radioisotopes such as radioactive isotopes of radium, cobalt and other elements may also be used.

In addition, methods of treating cancer using a representative compound and / or a representative conjugate can serve as an alternative to chemotherapy or radiation therapy in cases where such chemotherapy or radiation therapy is or may be too toxic, for example, causing unacceptable or intolerable side effects in an individual being treated. An animal in need of treatment may, but is not necessarily, undergo another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on which of these therapies has been established by experts to be acceptable or tolerated.

Representative compounds and / or representative conjugates can also be used in in vivo or ex vivo methods of treatment used to treat certain types of cancer, including, but not limited to, leukemia and lymphoma, where such treatment includes the introduction of autologous stem cell transplants. Such treatment can be carried out by conducting multistage procedures in which the animal’s autologous hematopoietic stem cells are collected and cleaned of all cancer cells, and then the residual population of animal bone marrow cells is treated by administering a high dose of a representative compound and / or a representative conjugate with simultaneous or without radiotherapy with high doses and the stem cell transplant is again administered to the animal. After restoration of bone marrow function and recovery of the animal, maintenance therapy is performed.

4.8.3. Multiple Drug Cancer Therapy

Methods of treating cancer are discussed below, including administering to a patient in need thereof an effective amount of a representative conjugate and another therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, topocarbazine, gasotancarbin, , etoposide, 5-fluorouracil, BCNU, irinotecan, capmotecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclaxetel and In one aspect of the invention, anti-cancer agents are, but are not limited to, the drugs listed in table 4.

Table 4 Alkylating agents: Nitrogen analogues of mustard gas: cyclophosphamide,
ifosfamide
trophosphamide
chlorambucil
melphalan Nitrosourea: carmustine (BCNU)
lomustine (CCNU) Alkylsulfonates: busulfan
threosulfan Triazines: decarbazine Compounds containing platinum: cisplatin
carboplatin Plant alkaloids: Vinca alkaloids: vincristine
vinblastine
vindesine
vinorelbine Taxoids: paclitaxel
docetaxol DNA Topoisomerase Inhibitors Epipodophyllins: etoposide
teniposide
topotecan
9-aminocamptothecin
camptothecin
crisnatol Mitomycin: mitomycin C Antimetabolites: Antifolates: DHFR Inhibitors: methotrexate
trimethrexate IMP Dehydrogenase Inhibitors: mycophenolic acid
thiazofurin
ribavirin
Eicar Ribonucleotide reductase inhibitors: hydroxyurea
deferoxamine Pyrimidine analogues: Uracil analogues: 5-fluorouracil
phloxuridine
doxyfluridine
ratitrexed Cytosine analogues: cytarabine (ara-C)
cytosine arabinoside
fludarabine Analogues of purine: mercaptopurine
thioguanine Hormonal therapeutic agents: Receptor antagonists: Antiestrogens: tamoxifen
raloxifene
megestrol LHRH agonists: goskrilin
leuprolide acetate Antiandrogens: flutamide
bicalutamide Retinoids / Deltoids: Vitamin D3 analogs: EB 1089
CB 1093
KN 1060 Photodynamic therapeutic agents: vertoporfin (BPD-MA)
phthalocyanine
photosensitizer Pc4
demethoxyhypocrelin A (2BA-2-DMHA) Cytokines: interferon-α
interferon-γ
tumor necrosis factor Other means: gemcitabine
velkad
revamide
thalamide Isoprenylation Inhibitors: lovastatin Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell cycle inhibitors: staurosporin Actinomycin: actinomycin D
dactinomycin Bleomycin: bleomycin A2
bleomycin B2
peplomycin Anthracyclines: daunorubicin
doxorubicin (adriamycin)
idarubicin
epirubicin
pyrarubicin
zorubicin
mitoxantrone MDR Inhibitors: verapamil Inhibitors of Ca ²⁺ ATPase: thapsigargin

4.8.4. Autoimmune Disease Treatment

Representative conjugates can be used to prevent or inhibit the proliferation of cells that induce an autoimmune disease, or to treat an autoimmune disease. Representative conjugates can be used, respectively, in various methods for treating an autoimmune disease in a patient. The drug-linker-ligand conjugates can be used to deliver the drug to the target cell. Without claiming to be a specific theory, it can only be noted that, in one embodiment of the invention, the drug-linker-ligand conjugate binds to an antigen on the surface of the target cell, and such a representative conjugate can be absorbed by the target cell via receptor-mediated endocytosis . After introduction into the cell, one or more specific peptide sequences in the linker component are enzymatically or hydrolytically cleaved to release the drug. Then, the released drug migrates freely into the cytosol and induces cytotoxic or cytostatic activity. In an alternative embodiment of the invention, the drug is cleaved from the representative conjugate and enters the external environment of the target cell, after which it enters the cell.

In one embodiment of the invention, the ligand component binds to an autoimmune antigen. In one aspect of the invention, the antigen is present on the surface of a cell involved in the induction of an autoimmune condition.

In another embodiment of the invention, the ligand component binds to an autoimmune antigen present on the surface of the cell.

In one embodiment of the invention, the ligand binds to activated lymphocytes, which are associated with an autoimmune pathological condition.

In another embodiment of the invention, representative conjugates prevent or inhibit the proliferation of cells producing an autoimmune antibody associated with a particular autoimmune disease.

The specific types of autoimmune diseases that can be treated with representative conjugates include, but are not limited to, disorders associated with Th2 lymphocytes (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omen syndrome, systemic sclerosis and transplant versus transplant the owner ”); disorders associated with Th1 lymphocytes (for example, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves disease, primary biliary cirrhosis, Wegener's granulomatosis and tuberculosis); disorders associated with activated B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, and type I diabetes); and the disorders listed in Table 5.

Table 5 Active chronic hepatitis Addison's Disease Allergic Alveolitis Allergic reaction Allergic rhinitis Alport Syndrome Anaphylaxis Ankylosing Spondylitis Antiphospholipid Syndrome Arthritis Ascariasis Aspergillosis Atopic allergy Atopic dermatitis Atopic rhinitis Behcet's Disease Lung disease of bird lovers Bronchial asthma Kaplan Syndrome Cardiomyopathy Celiac disease Chagas disease Chronic glomerulonephritis Kogan Syndrome Cold Agglutinin Synthesis Disease Congenital Infectious Measles Rubella CREST Syndrome Crohn's disease Cryoglobulinemia Cushing's Syndrome Dermatomyositis Discoid lupus erythematosus Dressler's Syndrome Eaton-Lambert Syndrome Echovirus infection Encephalomyelitis Endocrine ophthalmopathy Epstein-Barr Virus Infection Fuse in horses Erythematosis Evans Syndrome Felty syndrome Fibromyalgia Cyclite Fuchs Atrophy of the stomach Food allergy Giant cell arteritis Glomerulonephritis Goodpasture Syndrome Transplant versus host reaction Graves disease Guillain-Barré disease Hashimoto's thyroiditis Hemolytic anemia Purple Genoa-Shenlein Idiopathic adrenal atrophy Idiopathic pulmonary fibritis IgA nephropathy Inflammatory bowel disease Insulin dependent diabetes mellitus Juvenile arthritis Juvenile diabetes mellitus (type I) Lambert-Eaton Syndrome Laminitis Lichen planus Lupoid hepatitis Lupus erythematosus Lymphopenia Meniere's disease Mixed connective tissue disease Multiple sclerosis Severe myasthenia gravis Pernicious anemia Polyglandular syndromes Presenile Dementia Primary Agammaglobulinemia Primary biliary cirrhosis Psoriasis Psoriasis arthritis Raynaud phenomenon Habitual miscarriage Reiter's Syndrome Rheumatic fever Rheumatoid arthritis Sampler Syndrome Schistosomiasis Schmidt syndrome Scleroderma Schulman Syndrome Sjögren's syndrome Syndrome of “stiff person” Sympathetic Ophthalmia Systemic lupus erythematosus Arteritis Takayasu Temporal arthritis Thyroiditis Thrombocytopenia Thyrotoxicosis Toxic epidermal necrolysis Type B insulin resistance Type I diabetes Ulcerative colitis Uveitis Vitiligo Waldenstrom macroglobulinemia Wegener Granulomatosis

4.8.5. Complex therapy of autoimmune diseases using a variety of drugs

Methods for treating an autoimmune disease are discussed below, including administering to a patient in need thereof an effective amount of a representative conjugate and another therapeutic agent that is a known agent for treating an autoimmune disease. In one embodiment of the invention, such agents against autoimmune diseases are, but are not limited to, the agents listed in table 6.

Table 6 Cyclosporin Cyclosporin A Mycophenylate Mofetil Sirolim Tacrolim Enanercept Prednisone Azathioprine Cyclophosphamide methotrexate Prednisone Aminocaproic acid Chloroquine Hydroxychloroquine Hydrocortisone Dexamethasone Chlorambucil DHEA Danazole Bromocriptine Meloxicam Infliximab

4.8.6. Infectious Disease Treatment

Representative conjugates can be used to prevent or inhibit the proliferation of cells that induce an infectious disease, or to treat an infectious disease. Representative conjugates can be used respectively in various methods of treating an infectious disease in a patient. The drug-linker-ligand conjugates can be used to deliver the drug to the target cell. In one embodiment of the invention, the ligand component binds to an infected cell.

In one embodiment of the invention, said conjugates prevent or inhibit the proliferation of cells inducing a particular infectious disease.

The specific types of infectious diseases that can be treated with 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 epiglottis tracheitis adenoid hypertrophy pharyngeal abscess impetigo ecthyma pneumonia endocarditis septic arthritis pneumococcal infection peritonitis bacteremia meningitis acute purulent meningitis urethritis cervicitis proctitis pharyngitis salpingitis epididymitis gonorrhea syphilis listeriosis anthrax nocardiosis salmonellosis typhoid fever dysentery conjunctivitis sinusitis brucellosis tularemia cholera Bubonic plague tetanus necrosing enteritis actinomycosis mixed anaerobic infections syphilis relapse fever leptospirosis Lyme disease rat bite fever tuberculosis lymphadenitis leprosy chlamydia chlamydia pneumonia trachoma conjunctivitis with inclusions (pool conjunctivitis)

Systemic fungal diseases:

histoplasmosis coccidiodomycosis blastomycosis sporotrichosis cryptococcycosis systemic candidiasis aspergillosis mucomycosis mycetoma chromomycosis

Rickettsial diseases:

typhoid Rocky Mountain Spotted Fever ehrlichiosis oriental tick bite rickettsiosis rickettsia pox Q fever bartonellosis

Diseases caused by parasites:

malaria babeschioz african sleeping sickness Chagas disease leishmaniasis doom-doom fever toxoplasmosis meningoencephalitis keratitis entamebiasis giardiosis cryptosporidiosis isosporiosis cyclosporiosis microsporidiosis ascariasis trichocephalosis hookworm nematodosis “wandering larva” eye syndrome trichinosis dracunculiasis lymphatic filariasis loiasis River blindness dog filariasis caused by nematodes of the Onchocercidae family schistosomiasis bather's itch pulmonary paragonimiasis paragonimiasis of the liver fascioliasis fasciolopsiasis opisthorchiasis teniidosis echinococcosis alveolar echinococcosis

Viral diseases:

measles subacute sclerosing panencephalitis runny nose piggy rubella roseola fifth disease chicken pox respiratory syncytial virus infection croup bronchiolitis Infectious mononucleosis polio herpetic pharyngitis foot and mouth disease Bornholm disease genital herpes genital warts aseptic meningitis myocarditis pericarditis gastroenteritis acquired immunodeficiency syndrome (AIDS) human immunodeficiency virus (HIV) infection Reye syndrome kawazaki syndrome flu bronchitis viral “outpatient” pneumonia acute infectious respiratory disease acute pharyngoconjunctival fever epidemic keratoconjunctivitis herpes simplex virus 1 infection (HSV-1) herpes simplex virus 2 infection (HSV-2) shingles cytomegaly, inclusion disease rabies progressive multiple leukoencephalopathy kuru malignant hereditary insomnia Creutzfeldt-Jakob disease Gestmann-Straussler-Scheinker disease tropical spastic paraparesis eastern equine encephalitis California encephalitis St. Louis encephalitis yellow fever dengue virus disease lymphocytic choriomeningitis Lassa fever hemorrhagic fever hantavirus pulmonary syndrome infections caused by marburg virus Ebola infections smallpox

4.8.8. Complex treatment of infectious diseases using a variety of drugs

The following are methods of treating an infectious disease, including administering to a patient in need thereof an effective amount of a representative conjugate and another therapeutic agent that is an anti-infectious agent. In one embodiment of the invention, such anti-infective agents are, but are not limited to, the agents listed in table 8.

Table 8 β-lactam antibiotics: penicillin G penicillin V cloxacillin dicloxacillin methicillin nafcillin oxacillin ampicillin amoxicillin bacampicillin azlocillin carbenicillin meslocillin piperacillin ticarcillin

Aminoglycosides:

amikacin gentamicin kanamycin neomycin netilmicin streptomycin tobramycin

Macrolides:

azithromycin clarithromycin erythromycin lincomycin clindamycin

Tetracyclines:

demeclocycline doxycycline minocycline oxytetracycline tetracycline

Quinolones:

cinoxacin nalidixic acid

Fluoroquinolones:

ciprofloxacin enoxacin grefafloxacin levofloxacin lomefloxacin norfloxacin ofloxacin sparfloxacin trovafloxacin

Polypeptides:

bacitracin colistin polymyxin B

Sulfonamides:

sulfisoxazole sulfamethoxazole sulfadiazine sulfamethizole sulfacetamide

Other antibacterial agents:

trimethoprim sulfamethazole chloramphenicol vancomycin metronidazole quinupristine dalfopristine rifampin spectinomycin nitrofurantoin

Antiviral agents:

Common antiviral agents:

idoxuradine vidarabine trifluridine acyclovir famcicyclovir pencicyclovir valaciclovir gancicyclovir foscarnet ribavirin amantadine rimantadine cidofovir antisense oligonucleotides immunoglobulins interferons

Medicines for the treatment of HIV infections:

tenofovir emtricitabine zidovudine didanosine zalcitabine stavudine lamivudine nevirapine delavirdine saquinavir ritonavir indinavir nelfinavir

5. Examples

Example 1 — Preparation of Compound AB

Fmoc-val-cit-RAB-OH (14.61 g, 24.3 mmol, 1.0 eq. US Pat. No. 6,214,345, Firestone et al.) Was diluted with DMF (120 ml, 0.2 M), and to this diethylamine (60 ml) was added to the solution. The reaction was monitored by HPLC, and it was found that the reaction was completed after 2 hours. The reaction mixture was concentrated and the resulting residue was precipitated with ethyl acetate (approx. 100 ml), sonicated for 10 minutes. After this, ether (200 ml) was added and the precipitate was again sonicated for 5 minutes. The solution was left without stirring for 30 minutes, and then filtered and dried under high vacuum to give Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). The conjugate val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 ml) and the resulting solution was treated with MS-OSu (Willner et al. (1993) Bioconjugate Chem. 4: 521; 6.5 g, 21 mmol, 1.0 equiv.). The reaction was completed after 2 hours, as indicated by HPLC. The reaction mixture was concentrated and the resulting oil was precipitated with ethyl acetate (50 ml). After sonication for 15 minutes, ether (400 ml) was added and the mixture was sonicated again until all large particles were completely destroyed. After that, the solution was filtered and the solid was dried to obtain an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m / z 757.9 [M-H].

Fmoc-val-cit-RAB-OH (14.61 g, 24.3 mmol, 1.0 eq. US Pat. No. 6,214,345, Firestone et al.) Was diluted with DMF (120 ml, 0.2 M), and to this diethylamine (60 ml) was added to the solution. The reaction was monitored by HPLC, and it was found that the reaction was completed after 2 hours. The reaction mixture was concentrated and the resulting residue was precipitated with ethyl acetate (approx. 100 ml), sonicated for 10 minutes. After this, ether (200 ml) was added and the precipitate was again sonicated for 5 minutes. The solution was left without stirring for 30 minutes, and then filtered and dried under high vacuum to give Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). The conjugate val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 ml) and the resulting solution was treated with MS-OSu (Willner et al. (1993) Bioconjugate Chem. 4: 521; 6.5 g, 21 mmol, 1.0 equiv.). The reaction was completed after 2 hours, as indicated by HPLC. The reaction mixture was concentrated and the resulting oil was precipitated with ethyl acetate (50 ml). After sonication for 15 minutes, ether (400 ml) was added and the mixture was sonicated again until all large particles were completely destroyed. After that, the solution was filtered and the solid was dried to obtain an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m / z 757.9 [M-H].

The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 ml, 0.12 M) and bis (4-nitrophenyl) carbonate (8.5 g, 28.0 mmol) was added to the resulting solution. , 2.0 equiv.) And DIEA (3.66 ml, 21.0 mmol, 1.5 equiv.). The reaction was completed after 1 hour, as indicated by HPLC. The reaction mixture was concentrated to give an oil which was precipitated with EtOAc and then triturated with EtOAc (approx. 25 ml). Then, the dissolved substance was again precipitated with ether (approx. 200 ml) and triturated for 15 minutes. The solid was filtered and dried under high vacuum, whereby compound AB was obtained, which was 93% pure, as indicated by HPLC, and used in the next step without further purification. Yield: 9.7 g (94%).

Example 2-Preparation of Compound 1

The HCl salt of phenylalanine tert-butyl ester (868 mg, 3 mmol), N-Boc-dolaproin (668 mg, 1 equiv.), DEPC (820 μl, 1.5 equiv.) And DIEA (1.2 ml) were diluted dichloromethane (3 ml). After 2 hours (h) at room temperature (approximately 28 ° C), the reaction mixture was diluted with dichloromethane (20 ml) and washed successively with saturated aqueous (aq.) NaHCO ₃ (2 × 10 ml) and saturated aqueous NaCl (2 × 10 ml). The organic layer was separated and concentrated. The resulting residue was resuspended in ethyl acetate and purified by flash chromatography in ethyl acetate. The appropriate fractions were combined and concentrated to give the dipeptide as a white solid: 684 mg (64%). ES-MS m / z 491.3 [M + H] ⁺ .

To selectively cleave Boc in the presence of tert-butyl ether, the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 ml). Then 4M HCl / dioxane (960 μl, 3 equiv.) Was added and the reaction mixture was stirred overnight at room temperature. RP-HPLC showed almost complete removal of the Boc protecting group with a minimal level of tert-butyl ether cleavage. The resulting mixture was cooled in an ice bath and triethylamine (500 μl) was added. After 10 minutes, the mixture was removed from the cooling bath, diluted with dichloromethane (20 ml) and washed successively with saturated aqueous NaHCO ₃ (2 × 10 ml) and saturated aqueous NaCl (2 × 10 ml). The organic layer was concentrated to give a yellow foam: 287 mg (57%). The intermediate was used without further purification.

The tripeptide Fmoc-Meval-val-dil-Ot-Bu (prepared as described in 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 was concentrated to dryness, and the residue was evaporated with toluene (3 × 20 ml) and dried in vacuo overnight. The residue was diluted with dichloromethane (5 ml) and added to the dipeptide with removed protecting groups (287 mg, 0.73 mmol), and then DIEA (550 μl, 4 equiv.) And DEPC (201 μl, 1.1 equiv.) Were added. After 2 hours at room temperature, the reaction mixture was diluted with ethyl acetate (50 ml) and washed successively with 10% aqueous citric acid (2 × 20 ml), saturated aqueous NaHCO ₃ (2 × 10 ml) and saturated aqueous NaCl (10 ml) . The organic layer was separated and concentrated. The resulting residue was resuspended in ethyl acetate and purified by flash chromatography in ethyl acetate. The appropriate fractions were combined and concentrated to give Fmoc-Meval-val-dil-dap-phe-Ot-Bu as a white solid: 533 mg (71%). R _f 0.4 (EtOAc). ES-MS m / z 1010.6 [M + H] ⁺ .

The resulting product (200 mg, 0.2 mmol) was diluted with dichloromethane (3 ml) and diethylamine (1 ml). The reaction mixture was stirred overnight at room temperature. Then, the solvents were removed and an oil was obtained which was purified by flash chromatography on silica gel in a stepwise gradient of 0-10% MeOH in dichloromethane, whereby compound 1 was obtained as a white solid: 137 mg (87%). R _f 0.3 (10% MeOH / CH ₂ Cl ₂ ). ES-MS m / z 788.6 [M + H] ⁺ .

Example 3 Preparation of Compound 2

Compound 2 was obtained from compound 1 (30 mg, 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 vacuo overnight to give compound 2 as a hydroscopic white solid: 35 mg (120%, calculated for the HCl salt). ES-MS m / z 732.56 [M + H] ⁺ .

Example 4-Preparation of 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 vacuo overnight to give 50 mg of the intermediate as a hydroscopic white solid.

The white solid intermediate (20 mg, 0.02 mmol) was diluted with dichloromethane (1 ml), DEPC (5 μl, 0.03 mmol, 1.5 eq.) Was added, and then DIEA (11 μl, 0.06 mmol) was added. , 3 equiv.) And tert-butylamine (3.2 μl, 0.03 mmol, 1.5 equiv.). After 2 hours at room temperature, the reaction was not completed, as indicated by RP-HPLC. Then, DEPC (10 μl) and tert-butylamine (5 μl) were added again and the reaction mixture was stirred for another 4 hours. Then the reaction mixture was diluted with dichloromethane (15 ml) and washed successively with water (5 ml), 0.1 M water. HCl (10 ml) and saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The resulting residue was diluted with dichloromethane and purified by flash chromatography in a stepwise gradient of 0-5% MeOH in dichloromethane. The appropriate fractions were combined and concentrated to give an Fmoc-protected intermediate as a white solid: 7.3 mg (36%). R _f 0.75 (10% MeOH / CH ₂ Cl ₂ ).

The Fmoc-protected intermediate 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% MeOH in dichloromethane, whereby compound 3 was obtained as a white solid: 4 mg (70%). R _f 0.2 (10% MeOH / CH ₂ Cl ₂ ). ES-MS m / z 787 [M + H] ⁺ , 809 [M + Na] ⁺ .

Example 5-Preparation of compound 4

Boc-L-phenylalanine (265 mg, 1 mmol, 1 equiv.) And triethylene glycol monomethyl ether (164 μl, 1 mmol, 1 equiv.) Was diluted with dichloromethane (5 ml). After this, DCC (412 mg, 2 mmol, 2 equiv.) Was added, followed by DMAP (10 mg). The reaction mixture was stirred at room temperature overnight. The precipitate was filtered off. The solvent was removed in vacuo and the residue was diluted with ethyl acetate, and then purified by flash chromatography on silica gel in ethyl acetate. Fractions containing the desired product were combined, concentrated and dried in vacuo to give a white solid: 377 mg (91%). R _f 0.5 (EtOAc). ES-MS m / z 434 [M + Na] ⁺ .

The removal of the Boc protecting group was carried out by treating the above product in dioxane (10 ml) with 4 M HCl / dioxane (6 ml) for 6 hours at room temperature. The solvent was removed in vacuo and the residue dried in vacuo to give a white solid.

The HCl salt of triethylene glycol phenylalanine monomethylethyl ether (236 mg, 0.458 mmol, 1 equiv.) And N-Boc-dolaproin (158 mg, 0.55 mmol, 1.2 equiv.) Were diluted with dichloromethane (3 ml). DEPC (125 μl, 1.5 equiv.) Was added to the mixture, followed by DIEA (250 μl, 3 equiv.). After 2 hours at room temperature, the reaction mixture was diluted with ethyl acetate (30 ml) and washed successively with saturated aqueous NaHCO ₃ (2 × 10 ml), 10% aqueous citric acid (2 × 10 ml) and saturated aqueous NaCl (10 ml) . The organic layer was separated and concentrated. The resulting residue was resuspended in ethyl acetate and purified by flash chromatography on silica gel in ethyl acetate. The appropriate fractions were combined and concentrated to give the intermediate compound as a white foam: 131 mg (50%). R _f 0.25 (EtOAc). ES-MS m / z 581.3 [M + H] ⁺ .

The Boc protecting group was removed in dichloromethane (2 ml) and TFA (0.5 ml) at room temperature for 2 hours. Then the solvent was removed in vacuo and the residue was evaporated with toluene (3 × 25 ml), and then dried in vacuum with obtaining 138 mg of the TFA salt of the dipeptide.

Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.) And the TFA salt of the dipeptide (138 mg) were diluted with dichloromethane (2 ml). DEPC (63 μl, 1.5 equiv.) Was added to the mixture, followed by DIEA (160 μl, 4 equiv.). After 2 hours at room temperature, the reaction mixture was diluted with dichloromethane (30 ml) and washed successively with 10% aqueous citric acid (2 × 20 ml) and saturated aqueous NaCl (20 ml). The organic layer was separated and concentrated. The resulting residue was resuspended in dichloromethane and purified by flash chromatography on silica gel in a stepwise gradient of 0-5% MeOH in dichloromethane. The appropriate fractions were combined and concentrated to give a white foam: 205 mg (81%). R _f 0.4 (10% MeOH / CH ₂ Cl ₂ ). ES-MS m / z 1100.6 [M + H] ⁺ , 1122.4 [M + Na] ⁺ .

The Fmoc protecting group was removed by treatment with diethylamine (2 ml) in dichloromethane (6 ml). After keeping at room temperature for 6 hours, the solvent was removed in vacuo and the resulting product was isolated by flash chromatography on silica gel in a stepwise gradient of 0-10% MeOH in dichloromethane. The appropriate fractions were combined and concentrated. After evaporation from a dichloromethane / hexane mixture 1: 1, compound 4 was obtained as a white foam: 133 mg (80%). R _f 0.15 (10% MeOH / CH ₂ Cl ₂ ). ES-MS m / z 878.6 [M + H] ⁺ .

Example 6 Preparation of 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, prepared as described by Pettit GR et al ., Anti-Cancer Drug Design 1998, 13, 243-277) was dissolved in CH ₂ Cl ₂ (10 ml), and then diisopropylethylamine (0.30 ml, 1.71 mmol, 2.2 eq.) Was added. Then, DEPC (0.20 ml, 1.17, 1.5 equiv.) Was added and the mixture was kept under argon. The reaction was completed after 1 hour, as indicated by HPLC. The mixture was concentrated to give an oil and purified by chromatography on SiO ₂ (on a 300 × 25 mm column), eluting with 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 λ _max 215, 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 kept at room temperature for 2 hours. After this, the reaction was completed, as indicated by HPLC, and the reaction mixture was concentrated to obtain an oil, dissolved in 2 ml of DMSO and injected into a preparative HPLC column (C ₁₂ -RP column, 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 product containing fractions were evaporated to give the trifluoroacetic acid salt as a white powder. Yield: 0.126 g (98%). R _f 0.28 (100% EtOAc); ES-MS m / z 746.59 [M + H] ⁺ , 768.51 [M + Na] ⁺ ; UV λ _max 215 nm.

Example 7 Preparation of Compound 6

Trifluoroacetate 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 diisopropylethylamine (22.5 μl, 0.13 mmol, 1.0 eq.) Was added and the yellow solution was stirred under argon. After 3 hours, another 1.0 eq. DIEA. After another 24 hours, 0.5 equiv. activated linker. The reaction was completed after 40 hours. The contents of the vessel were evaporated, dissolved in DMSO and injected into a preparative HPLC column (C ₁₂ -RP column, 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 were evaporated to give the product as a yellow oil. Methylene chloride (approx. 2 ml) and an excess of ether were then added, resulting in compound 6 as 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 λ _max 215, 248 nm.

Example 8-Preparation of compound 7

Compound 4 (133 mg, 0.13 mmol, 1 eq.), Compound AB (123 mg, 0.167 mmol, 1.1 eq.) And HOBt (4 mg, 0.2 eq.) Was diluted with DMF (1.5 ml ) After 2 minutes, pyridine (5 ml) was added and the reaction was monitored by RP-HPLC. It was found that the reaction was completed after 18 hours. The reaction mixture was diluted with dichloromethane (20 ml) and washed successively 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 resulting residue was resuspended in dichloromethane and purified by flash chromatography on silica gel in a stepwise gradient of 0-10% MeOH in dichloromethane. The appropriate fractions were combined and concentrated to give compound 7 as a white foam: 46 mg (21%). R _f 0.15 (10% MeOH / CH ₂ Cl ₂ ). ES-MS m / z 1476.94 [M + H] ⁺ .

Example 9-Preparation of tert-butyl ester MC-val-cit-PAB-MMAF 8

Compound 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.) Were 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 indicated by HPLC. Then the mixture was evaporated, dissolved in a minimal amount of DMSO and purified by preparative HPLC (column C ₁₂ -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 λ _max 205, 248 nm.

Example 10-Preparation of MC-val-cit-PAB-MMAF 9

Compound 8 (45 mg, 32 μmol) was suspended in methylene chloride (6 ml), and then TFA (3 ml) was added. The resulting solution was kept for 2 hours. The reaction mixture was concentrated in vacuo and purified by preparative HPLC (column C ₁₂ -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 to give maleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF (MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MS m / z 1330.29 [M + H] ⁺ , 1352.24 [M + Na] ⁺ ; UV λ _max 205, 248 nm.

Example 11-Preparation of tert-butylamide MS-val-cit-PAB-MMAF 10

Compound 3 (217 mg, 0.276 mmol, 1.0 equiv.), Compound AB (204 mg, 0.276 mmol, 1.0 equiv.) And HOBt (11 mg, 0.0828 mmol, 0.3 equiv.) Were diluted with the mixture. pyridine / DMF (6 ml). DIEA (0.048 ml) was added to this mixture, and the mixture was stirred for about 16 hours. Volatile organic substances were evaporated in vacuo. The crude residue was purified using Chromatotron® (radial thin layer chromatography) with a stepwise gradient of 0-5-10% methanol in DCM to give tert-butylamide MS-val-cit-PAB-MMAF 10. Yield: 172 mg (45%). ES-MS m / z 1386.33 [M + H] ⁺ , 1408.36 [M + Na] ⁺ ; UV λ _max 215, 248 nm.

Example 12 Preparation of AC10-MS-MMAE by Conjugation of AC10 with MS-MMAE

AC10 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 with Sephadex G25 resin and PBS was eluted with 1 mM DTPA. The thiol / Ab value was estimated by determining the concentration of the reduced antibody at an optical density of 280 nm, determining the thiol concentration by reacting with DTNB (Aldrich, Milwaukee, WI) and determining the concentration at an optical density of 412 nm. The reconstituted antibody dissolved in PBS was cooled on ice.

The drug-linker reagent, maleimidocaproyl-monomethylauristatin E, i.e. MS-MMAE dissolved in DMSO was diluted in acetonitrile and water at a known concentration and added to chilled reduced AC10 antibody in PBS. After about one hour, an excess of maleimide was added to quench the reaction and to “cap” any unreacted thiol groups of the antibody. The reaction mixture was concentrated by ultrafiltration in a centrifuge and AC10-MS-MMAE was purified and desalted by elution with G25 resin in PBS, then filtered through 0.2 μm filters under sterile conditions and frozen for subsequent storage.

Example 13 Preparation of AC10-MS-MMAF by Conjugation of AC10 with MS-MMAF

AC10-MS-MMAF was obtained by conjugation of AC10 with MS-MMAF in accordance with the procedure described in example 12.

Example 14 - Preparation of AC10-MS-val-cit-PAB-MMAE by conjugation of AC10 with MS-val-cit-PAB-MMAE

AC10-MS-val-cit-PAB-MMAE was prepared by conjugating AC10 with MS-val-cit-PAB-MMAE in accordance with the procedure described in Example 12.

Example 15-Preparation of AC10-MS-val-cit-PAB-MMAF by conjugation of AC10 with MS-val-cit-PAB-MMAF (9)

AC10-MS-val-cit-PAB-MMAF was prepared by conjugating AC10 with MS-val-cit-PAB-MMAF (9) in accordance with the procedure described in Example 12.

Example 16-Determination of cytotoxicity of selected compounds

The cytotoxic activity of MMAF and compounds 1-5 can be evaluated on Lewis Y-positive OVCAR-3 cell lines and on Lewis Y-positive cell lines of breast carcinoma H3396, lung carcinoma L2987 and colon carcinoma LS174t. 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. Cytotoxicity assays are usually performed within 96 hours after the addition of test compounds. After cultivation, in the last 4-6 hours of incubation, 50 μl of resazurine dye can be added to each well to assess cell viability. Dye reduction can be determined using fluorescence spectrometry at an excitation wavelength and an emission wavelength of 535 nm and 590 nm, respectively. For analysis, the degree of recovery of resazurin by the treated cells can be compared with the degree of recovery of resazurin by the untreated control cells.

In 1-hour cytotoxicity assays, the analyzed cells were treated with the drug for 1 hour and then washed; whereby the cytotoxic effect can be determined after incubation for 96 hours.

Example 17 In vitro Cytotoxicity Assay of Selected Compounds

Table 10 illustrates the cytotoxic effect of cAC10 conjugates and compounds 7-10 on the Karpas 299 CD30 ⁺ cell line, analyzed as described in General Procedure 1. Data are presented for two separate experiments. It was found that the CAC10 conjugates and compounds 7-9 have a slightly higher activity than the conjugate CAC10-val-cit-MMAE.

Table 10 Conjugate IC ₅₀ (ng / ml) cAC10-val-cit-MMAE 6 CAC10-7 1,0 CAC10-8 fifteen CAC10-9 0.5 CAC10-10 twenty

In other experiments, the BR96-val-cit-MMAE conjugate was at least 250 times more potent than free MMAF.

General Procedure I-Determination of cytotoxicity . To assess the cytotoxicity of representative conjugates, 7-10 cells were 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 were treated with appropriate doses of representative conjugates 7-10 with four repetitions. Cytotoxicity assays were performed within 96 hours after the addition of test compounds. At the end of cultivation, in the last 4-6 hours of incubation, 50 μl of resazurin dye can be added to each well to assess cell viability. Dye reduction was determined using fluorescence spectrometry at an excitation wavelength and an emission wavelength of 535 nm and 590 nm, respectively. For analysis, the degree of recovery of resazurin by the treated cells was compared with the degree of recovery of resazurin by the untreated control cells.

Example 18 In Vitro Cell Proliferation Assay

The effectiveness of 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 the cell culture containing approximately 10 ⁴ cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in the medium was besieged in each well of a 96-well plate with opaque walls.

2. Received control wells containing medium and not containing cells.

3. ADC was added to the experimental wells and incubated for 3-5 days.

4. The plates were equilibrated to room temperature over about 30 minutes.

5. A volume of CellTiter-Glo reagent equal to the volume of the cell culture medium present was added to each well.

6. The contents of the wells were mixed for 2 minutes on an orbital shaker to induce cell lysis.

7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal.

8. Luminescence was recorded and plotted as RLU = relative luminescence units.

Example 19 - Removal of conjugates and antibodies from rat plasma

The pharmacokinetics of excretion of antibody-drug conjugates and total antibodies from plasma was studied in Sprague-Dawley rats (Charles River Laboratories, the body weight of each rat was 250-275 g). Animals were given a booster dose by injection into the tail vein (injection IV). Approximately 300 μl of whole blood was collected via cannula through the jugular vein or by tail puncture into vessels with lithium / anticoagulant heparin at the following time intervals: 0 (before dose), after 10 and 30 minutes; after 1, 2, 4, 8, 24 and 36 hours; and 2, 3, 4, 7, 14, 21, and 28 days after dosing. Total antibody titer was measured by ELISA using ECD / GxhuFc-HRP. Antibody drug conjugate levels were measured by ELISA using MMAE / MMAF / ECD-Bio / SA-HRP.

Example 20 - Removal of conjugates and antibodies from plasma of monkeys

The pharmacokinetics of excretion of the antibody-drug conjugates and the total amount of antibody from plasma can be studied in dog-like monkeys. Figure 12 illustrates a two-stage study of the removal of plasma concentrations after administration of H-MS-vc-MMAE to dog-like monkeys in various doses: 0.5; 1.5; 2.5; and 3.0 mg / kg on days 1 and 21. Concentrations of total antibody and ADC were measured versus time (H = trastuzumab).

Example 21-Determination of tumor volume in vivo in transgenic explant mice

Animals suitable for transgenic experiments can be obtained from standard commercially available sources such as Taconic (Germantown, NY). Animals of many species are suitable for these experiments, but female FVB mice are preferred because they are most susceptible to tumor formation. FVB males can be used for mating, and the deduced population of vasectomized CD.1 mice can be used to stimulate pseudopregnancy. Vasoectomized mice can be obtained from any commercially available source. Parent animals can be crossed with either FVB mice or 129 / BL6 x FVB mice heterozygous for p53. Mice heterozygous for the p53 allele can be used as potential animals with increased ability to form tumors. Some F1 tumors come from mixed species. Tumors of the parent mice can only be of type FVB.

Animals having tumors (an allograft cultured in transgenic Ft5 mmtv mice) can be treated with one or more doses of ADC by intravenous (i.v.) injection. Tumor volume can be determined at various time periods after injection.

Example 22 Synthesis of MS-MMAF via tert-butyl ether

Synthesis 1:

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163 mmol) was suspended in CH ₂ Cl ₂ (0.500 ml). After that, 6-maleimidocaproic acid (68.9 mg, 0.326 mmol) and 1,3-diisopropylcarbodiimide (0.0505 ml, 0.326 mmol) were added, followed by pyridine (0.500 ml). The reaction was allowed to stir for 1 hour. HPLC analysis indicated complete consumption of starting compound 1. Volatile organic matter was evaporated under reduced pressure. The product was isolated by flash column chromatography in a stepwise gradient of 0-5% methanol in CH ₂ Cl ₂ . A total of 96 mg of pure MS-MeVal-Val-Dil-Dap-Phe-OtBu (12) was obtained (60% yield). ES-MS m / z 981.26 [M + H] ⁺ ; 1003.47 [M + Na] ⁺ ; 979.65 [MH] ^- .

MS-MeVal-Val-Dil-Dap-Phe-OtBu (compound 12, 74 mg, 0.0754 mmol) was suspended in CH ₂ Cl ₂ (2.0 ml) and TFA (1 ml) at room temperature. After 2.5 hours, HPLC analysis indicated complete consumption of the starting compound. Volatile organic substances were evaporated under reduced pressure and the product was isolated by preparative RP-HPLC on a Phenomenex C ₁₂ Synergi Max-RP 80A column (250 × 21.20 mm). Eluent: a linear gradient of 10% -90% MeCN / 0.05% TFA (aq) for 30 minutes, and then an isorate mixture of 90% MeCN / 0.05% TFA (aq) for another 20 minutes. ES-MS m / z 925.33 [M + H] ⁺ ; 947.30 [M + Na] ⁺ ; 923.45 [MH] ^- .

Example 23a-Synthesis of MS-MMAF (11) through dimethoxybenzyl ether

Synthesis 2:

Obtaining Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ether (Fmoc-Phe-ODMB)

Fmoc-L-phenylalanine (200 g, 516 mmol, Bachem), 2,4-dimethoxybenzyl alcohol (95.4 g, 567 mmol, Aldrich) and CH ₂ Cl ₂ (2.0 L) were charged into a three-necked 5-liter round bottom flask. ) To the resulting suspension, tert-butyl acetal of N, N-dimethylformamide (155 ml, 586 mmol, Fluka) was added over 20 minutes under N ₂ atmosphere, resulting in a clear solution. Then the reaction mixture was stirred at room temperature overnight, after which TLC analysis (0.42, heptane / EtOAc = 2: 1) indicated the completion of the reaction. The reaction mixture was concentrated under reduced pressure to give a light yellow oil, which was redissolved in CH ₂ Cl ₂ (200 ml) and purified on a short silica gel column (25 cm × 25 cm, CH ₂ Cl ₂ ) to give a colorless foam (250 d). To the resulting foam was added MeCN (1 L), which completely dissolved this batch of the reaction mixture. The mixture was then concentrated to dryness and again dissolved in MeCN (1 L) and the resulting suspension was stirred for 1 hour and filtered, after which the filter cake was washed with MeCN (2 × 200 ml) to obtain Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ether as a white solid (113.58 g, 41%, 95.5% AUC according to HPLC analysis). Data: HPLC.

Obtaining L-phenylalanine-2,4-dimethoxybenzyl ether (Phe-ODMB)

Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ether (26.00 g, 48.3 mmol), CH ₂ Cl ₂ (150 ml) and diethylamine (75 ml, Acros) were charged into a 500 ml round bottom flask. The mixture was stirred at room temperature and the progress of the reaction was monitored by HPLC. After 4 hours, the mixture was concentrated (bath temperature <30 ° C). The residue was resuspended in CH ₂ Cl ₂ (200 ml) and concentrated. This procedure was repeated one more time. MeOH (20 ml) was added to the residue, which led to the formation of a gel. This residue was diluted with CH ₂ Cl ₂ (200 ml), concentrated and the turbid oil was kept in vacuo overnight. The residue was suspended in CH ₂ Cl ₂ (100 ml), and then toluene (120 ml) was added. The mixture was concentrated and the residue was kept in vacuo overnight. Data: HPLC, ¹ H-NMR.

Obtaining Fmoc-dolaproin (Fmoc-Dap)

Boc-dolaproin (58.8 g, 0.205 mol) was suspended in 4 N. HCl in 1,4-dioxane (256 ml, 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC analysis indicated the completion of the reaction (10% MeOH / CH ₂ Cl ₂ ), and the mixture was concentrated almost to dryness. An additional 50 ml of 1,4-dioxane was then charged and the mixture was concentrated to dryness and dried in vacuo overnight. The resulting white solid was dissolved in H ₂ O (400 ml) and transferred to a three-liter, three-necked round-bottom flask equipped with a mechanical stirrer and a temperature sensor. Then, N, N-diisopropylethylamine (214.3 ml, 1.23 mol, Acros) was added over 1 minute, which led to an increase in temperature from 20.5 to 28.2 ° C (internal). The mixture was cooled in an ice bath and 1,4-dioxane (400 ml) was added. After that, a solution of Fmoc-OSu (89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 ml) was added over a dropping funnel over a period of 15 minutes, keeping the temperature of the reaction mixture below 9 ° C. The mixture was allowed to warm to room temperature and stirred for 19 hours, after which the mixture was concentrated on a rotary evaporator to give an aqueous suspension (390 g). This suspension was diluted with H ₂ O (750 ml) and Et ₂ O (750 ml), which led to an abundant precipitation of a white precipitate. The layers were separated, keeping solids together with the organic layer. The aqueous layer was acidified with concentrated HCl (30 ml) and extracted with EtOAc (3 × 500 ml). The combined extracts were dried over MgSO ₄ , filtered and concentrated to give 59.25 g of yellow oil A. The Et ₂ O extract was extracted once with saturated NaHCO ₃ (200 ml), keeping the solids together with the aqueous layer. The aqueous suspension was acidified with concentrated HCl (50 ml) and extracted with Et ₂ O (50 ml), keeping the solids together with the organic layer. The organic layer was filtered and concentrated to give 32.33 g of yellow oil B. The two oil substances (A and B) were combined and purified by flash chromatography on silica gel eluting with CH ₂ Cl ₂ (3.5 L) and then 3% MeOH / CH ₂ Cl ₂ (9 L), whereby 68.23 g of Fmoc-dolaproin were obtained as a white foam (81%, purity 97.5%, HPLC (AUC)).

Obtaining Fmoc-Dap-Phe-ODMB

The crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 ml, Acros) for 5 minutes, and then Fmoc- Dap (19.80 g, 48.3 mmol) was added. The mixture was cooled in an ice bath and TBTU (17.08 g, 53.20 mmol, Matrix Innovations) was added. Then, N, N-diisopropylethylamine (25.3 ml, 145.0 mmol, Acros) was added via syringe over 3 minutes. After 1 hour, the ice bath was removed and the mixture was left for 30 minutes to heat. The mixture was then 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 brine (150 ml), dried (MgSO ₄ ) and filtered (through filter paper) to remove insoluble materials (inorganic substances and some dibenzofulven). After concentration, the residue (41 g) was adsorbed onto silica (41 g) and purified by chromatography (22 cm × 8 cm column; 65% heptane / EtOAc (2.5 L); 33% heptane / EtOAc (3.8 L) as a result, 29.4 g of product were obtained in the form of a white foam (86%, purity 92%, HPLC).

Data: HPLC, ¹ H-NMR, TLC (1: 1 EtOAc / heptane, R _f = 0.33, vanillin red).

Getting Dap-Phe-ODMB

Fmoc- Dap- Phe-ODMB (27.66 g), CH ₂ Cl ₂ (122 ml) and diethylamine (61 ml, Acros) were charged into a 1 liter round bottom flask. The solution was stirred at room temperature and the progress of the reaction was monitored by HPLC. After 7 hours, the mixture was concentrated (bath temperature <30 ° C). The residue was resuspended in CH ₂ Cl ₂ (300 ml) and concentrated. This procedure was repeated two times. MeOH (20 ml) and CH ₂ Cl ₂ (300 ml) were added to the residue, and the solution was concentrated. The residue was suspended in CH ₂ Cl ₂ (100 ml) and toluene (400 ml), then concentrated and the residue was kept in vacuo overnight to give a creamy residue.

Data: HPLC, ¹ H-NMR, MS.

Obtaining Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB

The crude Dap -Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135 ml, Acros) for 5 minutes, and then Fmoc-MeVal-Val-Dil-OH (24.94 g, 39.1 mmol, see obtaining the compounds of example 2). The mixture was cooled in an ice bath and TBTU (13.81 g, 43.0 mmol, Matrix Innovations) was added. Then, N, N-diisopropylethylamine (20.5 ml, 117.3 mmol, Acros) was added via syringe over 2 minutes. After 1 hour, the ice bath was removed and the mixture was left for 30 minutes to heat. The mixture was then poured into water (1.5 L) and 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 brine (200 ml), dried (MgSO ₄ ) and filtered (through filter paper) to remove insoluble materials (inorganic substances and some dibenzofulvene). After concentration, the residue (49 g) was scraped off the walls of the flask and adsorbed onto silica (49 g) and then purified by chromatography (15 cm × 10 cm column; 2: 1 EtOAc / heptane (3 L); EtOAc (5 L) ; 250 ml fractions), resulting in 31.84 g of Fmoc-MeVal-Val-Dil- Dap- Phe-ODMB as a white foam (73%, purity 93%, HPLC (AUC)).

Data: HPLC, TLC (2: 1 EtOAc / heptane, R _f = 0.21, vanillin red).

Obtaining MeVal-Val-Dil-Dap-Phe-ODMB

Fmoc-MeVal-Val-Dil- Dap- Phe-ODMB (28.66 g), CH ₂ Cl ₂ (80 ml) and diethylamine (40 ml) were charged into a 1 liter round bottom flask. The solution was stirred at room temperature overnight, and then concentrated under reduced pressure. The residue was adsorbed onto silica (30 g), and then purified by flash chromatography (15 cm × 8 cm column; 2% MeOH / DCM (2 L), 3% MeOH / DCM (1 L), 6% MeOH / DCM (4 L); 250 ml fractions), resulting in 15.88 g of MeVal-Val-Dil- Dap- Phe-ODMB as a white foam (69%, 96% purity, HPLC (AUC)).

Data: HPLC, TLC (6% MeOH / DXM, R _f = 0.24, vanillin red).

Obtaining MS-MeVal-Val-Dil-Dap-Phe-ODMB

MeVal-Val-Dil-Dap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4 ml), maleimidocaproic acid (180 mg, 0.85 mmol) and TBTU (300) were charged into a 50 ml round bottom flask. mg, 0.93 mmol, Matrix Innovations) at room temperature. Then N, N-diisopropylethylamine (450 μl, 2.57 mmol) was added via syringe. After 1.5 hours, the mixture was poured into water (50 ml) and diluted with ethyl acetate (30 ml). NaCl was added to facilitate separation. After separation of the layers, the aqueous layer was extracted with ethyl acetate (25 ml). The combined organic layers were dried (MgSO ₄ ), filtered and concentrated. The resulting oil (1 g) was purified by flash chromatography (100 ml silica; 25% heptane / EtOAc (100 ml), 10% heptane / EtOAc (200 ml), EtOAc (1.5 L)), whereby MS was obtained MeVal-Val-Dil- Dap -Phe-ODMB (13) as a white foam (521 mg, 57%, 94% purity, HPLC (AUC)).

Data: ¹ H-NMR, HPLC.

Preparation of MS-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF) (11)

MC-MeVal-Val-Dil-Dap-Phe-ODMB (compound 13, 428 mg, 0.39 mmol) was charged into a 50 ml round bottom flask and dissolved in 2.5% TFA / CH ₂ Cl ₂ (20 ml). After 2 minutes, the solution turned pinkish-purple. The reaction was monitored by HPLC and TLC (6% MeOH / DCM, KMnO ₄ staining). After 40 minutes, three drops of water were added and the cloudy pinkish-purple mixture was concentrated to give 521 mg of a pink residue. After purification by chromatography (15% IPA / DCM), 270 mg of MS-MMAF (73%, purity 92%, HPLC) was obtained as a white solid.

Example 23b Synthesis of the mc-MMAF Analog

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol) was suspended in DMF (0.250 ml). After that, 4- (2,5-dioxo-2,5-dihydropyrrol-1-yl) benzoic acid (11 mg, 0.049 mmol) and HATU (17 mg, 0.044 mmol) were added, followed by DIEA (0.031 ml, 0, 17 mmol). This reaction mixture was allowed to stir for 2 hours. HPLC analysis showed complete consumption of starting compound 1.

The product was isolated by preparative RP-HPLC on a Phenomenex C ₁₂ Synergi Max-RP 80A column (250 × 21.20 mm). Eluent: a linear gradient of 10% -80% MeCN / 0.05% TFA (aq.) For 8 minutes, then an isorate mixture of 80% MeCN / 0.05% TFA (aq) for another 12 minutes. Only 20 mg of pure product (14) was isolated (0.02 mmol, 46% yield). ES-MS m / z 987.85 [M + H] ⁺ ; 1019.41 [M + Na] ⁺ ; 985.54 [MH] ^- .

MB-MeVal-Val-Dil-Dap-Phe-OtBu (compound 14, 38 mg, 0.0385 mmol) was suspended in CH ₂ Cl ₂ (2.0 ml) and TFA (1 ml). The mixture was stirred for 2 hours, and then the volatile organic substances were evaporated under reduced pressure. The product was purified by preparative RP-HPLC on a Phenomenex C ₁₂ Synergi Max-RP 80A column (250 × 21.20 mm). Eluent: a linear gradient of 10% -80% MeCN / 0.05% TFA (aq) for 8 minutes, and then an isorate mixture of 90% MeCN / 0.05% TFA (aq) for another 12 minutes. A total of 14.4 mg of MV-MMAF product was isolated (0.015 mmol, 40% yield). ES-MS m / z 930.96 [M + H] ⁺ , 952.98 [M + Na] ⁺ ; 929.37 [MH] ^- .

Example 23c — Preparation of MS-MeVal-Cit-PAB-MMAF (16)

To a suspension of Fmoc-MeVal-OH (3.03 g, 8.57 mmol) and N, N'-disuccimidyl carbonate (3.29 g, 12.86 mmol) in CH ₂ Cl ₂ (80 ml) was added DIEA at room temperature (4.48 ml, 25.71 mmol). This reaction mixture was allowed to stir for 3 hours and then poured into a separatory funnel and the organic mixture was extracted with 0.1 M HCl (aq). The crude organic residue was concentrated under reduced pressure, and the product was isolated by flash column chromatography on silica gel using a linear gradient of 20-100% ethyl acetate / hexane. A total of 2.18 g of pure Fmoc-MeVal-OSu (4.80 mmol, 56% yield) was obtained.

To a suspension of Fmoc-MeVal-OSu (2.18 g, 4.84 mmol) in DME (13 ml) and THF (6.5 ml), a solution of L-citrulline (0.85 g, 4.84 mmol) was added at room temperature ) and NaHCO ₃ (0.41 g, 4.84 mmol) in H ₂ O (13 ml). The suspension was left for 16 hours at room temperature for stirring, and then was extracted with a mixture of tert-BuOH / CHCl ₃ / H ₂ O and acidified with 1M HCl to pH 2.3. The organic phase was separated, dried and concentrated under reduced pressure. The residue was triturated with diethyl ether to give 2.01 g of Fmoc-MeVal-Cit-COOH, which was used without further purification.

The crude Fmoc-MeVal-Cit-COOH was suspended in 2: 1 CH ₂ Cl ₂ / MeOH (100 ml) and p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g) were added to this suspension. 7.9 mmol). This suspension was allowed to stir for 125 hours, and then the volatiles were removed under reduced pressure and the residue was purified by flash column chromatography on silica gel using 10% MeOH / CH ₂ Cl ₂ . As a result, pure Fmoc-MeVal-Cit-RAB-OH (0.55 g, 0.896 mmol, 18.5% yield), ES-MS m / z 616.48 [M + H] ^{+ were} obtained.

To a suspension of Fmoc-MeVal-Cit-RAB-OH (0.55 g, 0.896 mmol) in CH ₂ Cl ₂ (40 ml) was added STRATOSPHERES ^™ (bound to piperazine-resin) (> 5 mmol / g, 150 mg). After stirring at room temperature for 16 hours, the mixture was filtered through celite (pre-washed with MeOH) and concentrated under reduced pressure. The residue was triturated with diethyl ether and hexane. The resulting solid, MeVal-Cit-RAB-OH, was suspended in CH ₂ Cl ₂ (20 ml), and then MS-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, however, the HPLC analysis of the reaction mixture indicated an incomplete reaction, and therefore, the suspension was concentrated under reduced pressure to a volume of 6 ml, and then 10% NaHCO ₃ solution (aq) was added and the suspension was stirred for another 16 hours. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel in a gradient of 0-10% MeOH / CH ₂ Cl ₂ to give 42 mg (0.072 mmol, 8% yield) MS-MeVal-Cit-RAV-OH .

To a suspension of MS-MeVal-Cit-RAB-OH (2.37 g, 4.04 mmol) and bis (nitrophenyl) carbonate (2.59 g, 8.52 mmol) in CH ₂ Cl ₂ (10 ml) was added (1.06 ml, 6.06 mmol). This suspension was stirred for 5.5 hours, concentrated under reduced pressure and purified by trituration with diethyl ether. MS-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a pyridine / DMF solution (1: 5) (3 ml), and 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 purified by preparative RP-HPLC (× 3) on a Phenomenex C ₁₂ Synergi Max-RP 80A column (250 × 21.20 mm). Eluent: a linear gradient of 10% -90% MeCN / 0.05% TFA (aq) for 30 minutes, then an isorate mixture of 90% MeCN / 0.05% TFA (aq) for another 20 minutes. MS-MeVal-Cit-PAB-MMAF (16) was obtained as a yellowish solid (24.5 mg, 0.0182, 0.45% yield). ES-MS m / z 1344.95 [M + H] ⁺ ; 1366.94 [M + Na] ⁺ .

Example 23d — Preparation of Suberyl-Val-Cit-PAB-MMAF Succinimidoester (17)

Compound I (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol, 1.5 eq.) Were suspended in anhydrous pyridine, and then 5 ml of HOBt (10 mg) was added. 0.076 mmol, 0.2 eq.) And DIEA (199 μl, 1.14 mmol, 3 eq.). The reaction mixture was sonicated for 10 minutes and then stirred at room temperature overnight. Pyridine was removed under reduced pressure and the residue was resuspended in CH ₂ Cl ₂ . The mixture was separated by flash chromatography on silica gel in a stepwise gradient of MeOH, from 0 to 10%, in CH ₂ Cl ₂ . The product containing fractions were combined, concentrated, dried in vacuo overnight to give 317 mg (59% yield) 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 / CH ₂ Cl ₂ (10 ml) for 2 hours. The mixture was diluted with CH ₂ Cl ₂ (50 ml). The organic layer was washed successively with water (2 × 30 ml) and brine (1 × 30 ml). The organic phase was concentrated and loaded onto a silica gel layer in 10% MeOH / CH ₂ Cl ₂ . The product was eluted with 30% MeOH / CH ₂ Cl ₂ . After drying in vacuo overnight, Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid, 38 mg, 40% yield. ES-MS m / z 1357.7 [MH] ^- .

Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH ₂ Cl ₂ (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 was evaporated with pyridine (2 ml) and then with toluene (2 × 5 ml) and dried in vacuum. Val-Cit-PAB-MMAF was obtained as a brownish oil and used without further purification.

All Val-Cit-PAB-MMAF obtained from 67 mg of Fmoc-Val-Cit-PAB-MMAF was suspended in pyridine (2 ml) and added to a solution of disuccinimidyl suberate (74 mg, 0.2 mmol, 4 eq.) In pyridine (1 ml). The reaction mixture was stirred at room temperature. After 3 hours, ether (20 ml) was added. The precipitate was collected and washed with additional ether. The reddish solid was suspended in 30% MeOH / CH ₂ Cl ₂ and filtered through a pad of silica gel using 30% MeOH / CH ₂ Cl ₂ as eluent. Compound 17 was obtained as a white solid (20 mg, 29% yield). ES-MS m / z 1388.5 [MH] ^- .

Example 24- In vivo efficacy of mcMMAF antibody-drug conjugates

Efficacy of cAC10-mcMMAF in Karpas-299 ALCL Xenografts . To evaluate in vivo efficacy of the cAC10-mcMMAF conjugate containing, on average, 4 drug molecules per antibody (cAC10-mcF4), human Karpas-299 ALCL cells were implanted subcutaneously in C. B-17 SCID immunodeficient mice (5 × 10 ⁶ cells on the mouse). Tumor volume was calculated by the formula (0.5 × L × W ² ), where, of the two bidirectional measurements, one of L and W is longer and the other is shorter. When the average tumor volume of the studied animals reached approximately 100 mm ³ (ranging from 48 to 162), the mice were divided into 3 groups (5 mice per group), and these mice were either not treated or they received one intravenous injection into the tail vein 1 mg / kg or 2 mg / kg cAC10-mcF4 (Fig. 1). Tumors in untreated mice grew rapidly, and 7 days after the start of the experiment, their average volume reached> 1000 mm ³ . In contrast, all cAC10-mcF4-treated mice showed a rapid decrease in tumor size, with 3 of 5 group mice receiving a dose of 1 mg / kg and 5 of 5 group mice receiving a dose of 2 mg / kg showed a complete antitumor answer. One of the mice with absolute responsiveness in the group receiving a dose of 2 mg / kg nevertheless had a relapse of the tumor approximately 4 weeks after the start of therapy, however, the remaining mice of this group, i.e. in 4 out of 5 mice, no detectable tumors were detected, and in 3 mice with absolute response in the group receiving a dose of 1 mg / kg, no tumors were observed 10 weeks after the start of therapy.

Efficacy of cBR96- mcMMAF in L2987 NSCLC Xenografts. cBR96 is a chimeric antibody that recognizes the Le ^Y antigen. To evaluate the in vivo efficacy of the cBR96-mcMMAF conjugate containing 4 drug molecules per antibody (cBR96-mcF4), tumor-free (naked) mice were implanted with tumor fragments of non-small cell lung cancer (NSCLC) L2987. When the average tumor volume reached approximately 100 mm ³ , the mice were divided into 3 groups: one group that was not subjected to treatment, and 2 treatment groups. For therapy, as shown in FIG. 3a, mice were injected with cBR96-mcF4 every 4 days at a dose of 3 mg / kg or 10 mg / kg / injection, a total of 4 injections (q4d × 4). As shown in FIG. 3b, mice were injected every 4 days with cBR96-mF4 or the non-binding control conjugate CAC10-mcF4 at a dose of 10 mg / kg / injection, a total of 4 injections (q4d × 4). As shown in figa and 3b, BR96-mcF4 provided a significant slowdown in tumor growth compared with the control.

Figure 2 illustrates an in vivo assay for the effectiveness of a single dose of cAC10-mcMMAF in subcutaneous L540CY. These studies were performed on 4 mice of the untreated group and 10 mice in each of the treatment groups.

Example 25- In Vitro Efficacy of Antibody Medicament MC-MMAF Conjugates

The activity of conjugates "CAC10-antibody-drug" against CD30 ⁺ cell lines . Figures 4a and 16b show dose-response curves constructed from a representative experiment in which Karpas-299 (anaplastic large cell lymphoma) and L428 (Hodgkin lymphoma) cell cultures were incubated with serially diluted CAC10-mcMMAF (Fig. 4a) or cAC10-vcMMAF (Fig. 4b) for 96 hours. The cultures were labeled with 50 μM resazurin [10-oxide 7-hydroxy-3H-phenoxazin-3-one] for 4 hours and the fluorescence intensity was measured. These data were converted using GraphPad Prism version 4.00 in accordance with the procedure for constructing a 4-parameter dose-response curve. IC ₅₀ values were defined as the concentration at which cell growth was reduced by 50% compared with the untreated control culture. Each concentration was tested with four repetitions.

The activity of conjugates “ cBR96- antibody-drug” against Le ^{Y +} cell lines . Figures 5a and 5b show dose-response curves constructed from a representative experiment in which cultures of H3396 (breast carcinoma) and L2987 (non-small cell lung carcinoma) cultures were incubated with serially diluted cBR96-mcMMAF (Fig. 5a) ) or cBR96-vcMMAF (Fig. 5b) for 96 hours. Cultures were labeled with 50 μM resazurin for 4 hours and the fluorescence intensity was measured. These data were converted using GraphPad Prism version 4.00 in accordance with the procedure for constructing a 4-parameter dose-response curve. IC ₅₀ values were defined as the concentration at which cell growth was reduced by 50% compared with the untreated control culture. Each concentration was tested with four repetitions.

Antibody c1F6-drug conjugate activity against CD70 ⁺ cell lines of renal carcinoma . Figures 6a and 6b show a closed dose-response plotted from a representative experiment in which Caki-1 and 786-O cell cultures were incubated with serially diluted c1F6-mcMMAF (Fig. 6a) or c1F6-vcMMAF (Fig. 6b) within 96 hours. Cultures were labeled with 50 μM resazurin for 4 hours and the fluorescence intensity was measured. These data were converted using GraphPad Prism version 4.00 in accordance with the procedure for constructing a 4-parameter dose-response curve. IC ₅₀ values were defined as the concentration at which cell growth was reduced by 50% compared with the untreated control culture. Each concentration was tested with four repetitions.

Example 26-Purification of trastuzumab

The contents of one vessel, comprising 440 mg of Herceptin® antibody (huMAb4D5-8, rhuMAb HER2, US patent No. 5821337), were dissolved in 50 ml of MES buffer (25 mm MES, 50 mm NaCl, pH 5.6) and loaded onto a cation exchange column ( Sepharose S, 15 cm × 1.7 cm), which was then balanced in the same buffer. Then the column was washed with the same buffer (5 column volumes). Trastuzumab was eluted by adjusting the concentration of NaCl buffer to 200 mM. Antibody-containing fractions were combined, diluted to 10 mg / ml and dialyzed into a buffer containing 50 mM potassium phosphate, 500 mM NaCl, 2 mM EDTA, pH 6.5.

Example 27-Preparation of Trastuzumab-MS-MMAE Conjugate by Conjugation of Trastuzumab with MS-MMAE

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 37 ° C for 30 minutes, the buffer was replaced by elution with Sephadex G25 resin, and then PBS was eluted with 1 mM DTPA. The thiol / Ab value was evaluated by determining the concentration of the reduced antibody at an optical density of 280 nm and the thiol concentration by reacting with DTNB (Aldrich, Milwaukee, WI) and determining the concentration at an optical density of 412 nm. The reconstituted antibody dissolved in PBS was cooled on ice.

The drug-linker reagent, maleimidocaproyl-monomethylauristatin E (MMAE), i.e. MS-MMAE, dissolved in DMSO, was diluted in acetonitrile and in water at a known concentration and added to the chilled reduced antibody trastuzumab in PBS. After about one hour, an excess of maleimide was added to quench the reaction and to “cap” any unreacted thiol groups of the antibody. The reaction mixture was concentrated by ultrafiltration in a centrifuge and trastuzumab-MS-MMAE was purified and desalted by elution with G25 resin in PBS, and then filtered through 0.2 μm filters under sterile conditions and frozen for subsequent storage.

Example 28-Preparation of Trastuzumab-MS-MMAF Conjugate by Conjugation of Trastuzumab with MS-MMAF

The trastuzumab-MS-MMAF conjugate was prepared by conjugating trastuzumab with MS-MMAF in accordance with the procedure of Example 27.

Example 29 — Preparation of trastuzumab-MS-val-cit-RAV-MMAE conjugate by conjugation of trastuzumab with MS-val-cit-RAV-MMAE

The trastuzumab-MS-val-cit-RAV-MMAE conjugate was prepared by conjugating trastuzumab with MS-val-cit-RAV-MMAE in accordance with the procedure of Example 27.

Example 30 — Preparation of trastuzumab-MS-val-cit-RAB-MMAF conjugate by conjugation of trastuzumab with MS-val-cit-RAV-MMAF9

The trastuzumab-MS-val-cit-RAB-MMAF conjugate was prepared by conjugating trastuzumab with MS-val-cit-RAB- MMAF9 in accordance with the procedure of Example 27.

Example 31 — Toxicity to Rat

The acute toxicity profile of free drugs and ADC was evaluated in young Sprague-Dawley rats (each weighing 75-125 grams, Charles River Laboratories (Hollister, CA). The animals were injected on day 1, and then on day 0, day 3 and day 5 complete chemical and hematological analyzes were performed, and a complete autopsy was performed on day 5. All the animals measured the levels of enzymes in the liver, and for three randomly taken animals of each group a routine histological analysis of the following tissues was performed: chest, liver, kidney, thymus, spleen, thick and thin to . Shki experimental groups were the following groups:

For conjugates trastuzumab-MS-val-cit-MMAF, trastuzumab-MS (Me) -val-cit-RAV-MMAF, trastuzumab-MS-MMAF and trastuzumab-MS-val-cit-RAV-MMAF μg MMAF / m ² calculated using a mass (Mw) of MMAF of 731.5 and a mass (Mw) of herceptin of 145167.

Body surface area was calculated using the following formula: [{(body weight in grams to the power of 0.667) × 11.8} / 10000] (Guidance for Industry and Reviewers, 2002).

Doses of the solution were administered intravenously into the tail vein as a single loading dose on the 1st day of the study at a dose of 10 ml / kg. Before the dose was administered on the 1st day of the study and daily after its administration, the animal body weight was measured. Whole blood was collected in EDTA-containing tubes for hematological analysis. Whole blood was collected in serum separator tubes for clinical biochemical analyzes. Blood samples were taken before dosing on study day -4, day 3 and day 5. At autopsy, whole blood was also collected in tubes containing sodium heparin, and the plasma was frozen at -70 ° C for possible further analysis. At autopsy, tissues of the following organs were taken and placed in a neutral buffered formalin: liver, kidney, heart, thymus, spleen, brain, chest, and sections of the gastrointestinal tract, including the stomach and large and small intestines. A study was also conducted of the chest, small intestine, large intestine, liver, thymus, spleen and kidneys.

Levels of liver serum enzymes at each time period were within the range comparable to their levels in normal female Sprague-Dawley rats (5- and 95-percentiles). The number of leukocytes and platelets in each time period was within the range comparable with their number in normal female Sprague-Dawley rats (5- and 95-percentiles).

High dose studies were performed on normal female Sprague-Dawley rats for the following groups:

Group 1: Carrier Group 2: trastuzumab-MS-MMAF, 52.24 mg / kg, 4210 μg / m ² Group 3: trastuzumab-MS-MMAF, 68.25 mg / kg, 5500 μg / m ² Group 4: trastuzumab-MS-MMAF, 86.00 mg / kg, 6930 μg / m ²

Eleven animals underwent routine histological analysis of tissues. These animals participated in the studies on the limits of acute dose-dependent toxicity of the trastuzumab-MS-MMAF immunoconjugate. Animals were observed for 12 days after dosing.

Example 32 - Toxicity / Safety for Dog-like Monkeys

Three groups of four dog-like Macaca fascicularic monkeys (2 males and 2 females) not previously participating in the experiments were studied for the action of the trastuzumab-MS-vc-RAV-MMAE and trastuzumab-MS-vc-RAV-MMAF conjugates. Intravenous administration was performed on the 1st and 22nd day of the study.

Sample Group Dose Carrier one
1M / 1F Day 1
Day 22 H-MS-vc-RAV-MMAE 2
2M / 2F 180 mcg / m ² (0.5 mg / kg) on day 1
1100 mcg / m ² (3.0 mg / kg) on day 22 H-MS-vc-RAV-MMAE 3
2M / 2F 550 mcg / m ² (1.5 mg / kg) on day 8
550 mcg / m ² (1.5 mg / kg) on day 29 H-MS-vc-RAV-MMAE four
2M / 2F 880 mcg / m ² (2.5 mg / kg) on day 15
880 mcg / m ² (2.5 mg / kg) on day 36

Sample Group Dose Carrier one
1M / 1F Day 1
Day 22 H-MS-vc-RAV-MMAF 2
2M / 2F 180 mcg / m ² (0.5 mg / kg) on day 1
1100 mcg / m ² (3.0 mg / kg) on day 22 H-MS-vc-RAV-MMAF 3
2M / 2F 550 mcg / m ² (1.5 mg / kg) on day 1
550 mcg / m ² (1.5 mg / kg) on day 22 H-MS-vc-RAV-MMAF four
2M / 2F 880 mcg / m ² (2.5 mg / kg) on day 1
880 mcg / m ² (2.5 mg / kg) on day 22

H = trastuzumab

The dose was expressed as the surface area of the animal’s body so that it corresponded to the doses administered by animals of a different species, since the dose expressed in μg / m ² does not depend on the type of animal, and therefore the doses for different animals can be compared. ADC preparations contained PBS, 5.4 mM 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 1, 3, 5, 7, 14 and 21 days after the introduction of each dose. The number of red blood cells (RBC) and platelets (PLT) was measured by light scattering. White blood cell count (WBC) was measured using the peroxidase / basophil method. The number of reticulocytes was measured by light scattering using a cationic dye. The number of cells was measured on an Advia 120 apparatus. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were measured in units / l using UV / NADH, a technique developed by the International Federation of Clinical Chemistry (IFCC) and implemented on an Olympus AU400 apparatus, and the total antibody titer was measured by ELISA using ECD / GxhuFc-HRP. Ab-conjugate levels were measured using ELISA-MMAE / MMAF / ECD-Bio / SA-HRP tests.

Example 33-Production, Characterization and Humanization of Monoclonal Antibody 4D5 against ErbB2

The murine monoclonal antibody 4D5, which specifically binds to the extracellular domain of ErbB2, was produced as described by Fendly et al. (1990) Cancer Research 50: 1550-1558. Briefly, cells were NIH-3T3 / HER2-3 ₄₀₀ (expressing approximately 1 × ¹⁰ May ErbB2 / cell molecules) produced as described in Hudziak et al. Proc. Natl. Acad. Sci. (USA) 84: 7159-7163 (1987)) were collected using phosphate buffered saline (PBS) containing 25 mM EDTA and used to immunize BALB / c mice. These ip mice were injected with 10 ⁷ cells in 0.5 ml PBS at 0-, 2-, 5- and 7th week. For mice with antiserum that contained ³² P-labeled ErbB2 as immunoprecipitate, ip injections of ErbB2 membrane extract purified using wheat germ agglutinin (WGA) - sepharose were administered ip 9 and 13 weeks. And then iv injection was injected with 0.1 ml of ErbB2 preparation and the splenocytes were fused to the mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for binding to ErbB2 by ELISA and radioimmunoprecipitation.

Epitope mapping and characterization

The ErbB2 epitope bound to the 4D5 monoclonal antibody was characterized by a competitive binding assay (Fendly et al., Cancer Research 50: 1550-1558 (1990)). Cross-blocking studies were performed by directly measuring fluorescence on intact cells using a PANDEX ^™ screening device to quantify fluorescence. The monoclonal antibody was conjugated to fluorescein isothiocyanate (FITC) in accordance with well-known procedures (Wofsy et al. Selected Methods in Cellular Immunology, p. 287, Mishel & Schiigi (eds.) San Francisco: WJ Freeman Co. (1980)). The confluent monolayers of NIH-3T3 / HER2-3 ₄₀₀ cells were trypsinized, washed once and resuspended at 1.75 × 10 ⁶ cells / ml in cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN ₃ . To avoid clogging the membranes of PANDEX ^™ plates, a final concentration of 1% latex particles was added (IDC, Portland, OR). Cells in suspension, 20 μl, and 20 μl of purified monoclonal antibodies (100 μg / ml-0.1 μg / ml) were added to the wells of the PANDEX ^™ plate and the mixture was incubated on ice for 30 minutes. A predefined dilution of an FITC-labeled monoclonal antibody in 20 μl was added to each well, incubated for 30 minutes, washed and the fluorescence intensity was quantified on PANDEX ^™ . Monoclonal antibodies were considered to have a common epitope if each antibody blocked the binding of another antibody by 50% or more compared to an irrelevant control monoclonal antibody. In this experiment, epitope I was assigned to a 4D5 monoclonal antibody (amino acid residues from about 529 to 625, including residues of the extracellular domain of ErbB2).

The tumor-inhibitory properties of 4D5 monoclonal antibody were evaluated on a mammary tumor cell line, SK-BR-3 (see Hudziak et al. (1989) Molec. Cell. Biol. 9 (3): 1165-1172). Briefly, SK-BR-3 cells were separated using 0.25% (v / v) trypsin and suspended in complete medium at a density of 4 × 10 ⁵ cells per ml. Aliquots of 100 μl (4 × 10 ⁴ cells) were seeded in 96-well micro-dilution plates, then the cells were left for adhesion, after which 100 μl of one medium or medium containing a monoclonal antibody was added (at a final concentration of 5 μg / ml). After 72 hours, the plates were washed twice with PBS (pH 7.5), stained with crystal violet (0.5% in methanol) and analyzed for relative cell proliferation as described by Sugarman et al., (1985) Science 230: 943-945 . Monoclonal antibody 4D5 inhibited relative proliferation of SK-BR-3 cells by approximately 56%.

Monoclonal antibody 4D5 was also evaluated for its ability to inhibit HRG-stimulated tyrosine phosphorylation of proteins having M _{r of the} order of 180,000 in lysates of whole MCF7 cells (Lewis et al. (1996) Cancer Research 56: 1457-1465). MCF7 cells have been reported to express all known ErbB receptors, but at relatively low levels. Since ErbB2, ErbB3, and ErbB4 have almost the same molecular weight, it is impossible to determine which of these proteins is tyrosine phosphorylated by Western blot analysis of cell lysates. However, these cells are ideal for testing for HRG-stimulated tyrosine phosphorylation, since under the used analytical conditions, in the absence of exogenously added HRG, these cells produce low or undetectable levels of tyrazine phosphorylated proteins with M _{r of the} order of 180,000.

MCF7 cells were seeded in 24-well plates and monoclonal anti-ErbB2 antibodies were added to each well, followed by incubation for 30 minutes at room temperature, and then rHRGβ1 _{177-244 was} added to each well to a final concentration of 0.2 nM and incubation continued 8 minutes Then, medium was carefully aspirated from each well and the reaction was stopped by adding 100 μl SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl) was subjected to electrophoresis in a 4-12% gradient gel (Novex), and then was electrophoretically transferred onto a polyvinylidene difluoride membrane. Immunoblots of antibodies against phosphotyrosine (4G10, from UBI, used at a concentration of 1 μg / ml) showed and the intensity of the predominant reactive band at M _r = 180,000 was quantified using reflectance 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)).

The 4D5 monoclonal antibody showed a significant inhibition of the generation of an HRG-induced tyrosine phosphorylation signal at M _r 180,000. In the absence of HRG, no tyrosine phosphorylation of M _r proteins of the order of 180,000 was stimulated. In addition, this antibody did not cross-react with EGFR (Fendly et al., Cancer Research 50: 1550-1558 (1990)), ErbB3 or ErbB4. Monoclonal antibody 4D5 was able to block HRG stimulation of tyrosine phosphorylation by 50%.

The tumor inhibitory properties of 4D5 monoclonal antibody were evaluated on MDA-MB-175 and SK-BR-3 cells in the presence or absence of exogenous rHRGβ1 (see Schaefer et al. Oncogene 15: 1385-1394 (1997)). The levels of ErbB2 in MDA-MB-175 cells were 4-6 times higher than the levels found in normal mammary epithelial cells, and the ErbB2-ErbB4 receptor in MDA-MB-175 cells is constitutively phosphorylated for tyrosine. The 4D5 monoclonal antibody is capable of inhibiting the proliferation of MDA-MB-175 cells both in the presence and absence of exogenous HRG. Inhibition of cell proliferation by 4D5 depends on the level of expression of ErbB2 (Lewis et al. Cancer Immunol. Immunother. 37: 255-263 (1993)). The maximum detectable inhibition of SK-BR-3 cells was 66%. However, a greater effect can be achieved using exogenous HRG.

The 4D5 murine monoclonal antibody was humanized using the “gene-converting mutagenesis” strategy described in US Pat. No. 5,821,337, which is incorporated herein by reference in its entirety. The humanized 4D5 monoclonal antibody used in the following experiments was designated huMAb4D5-8. This antibody has an IgG1 isotype.

Cited Works

The scope of the present invention is not limited to the specific embodiments described in the examples, which are presented only to illustrate several aspects of the present invention, and any functionally equivalent variants are included in its scope. Indeed, in addition to the modifications that have been illustrated and described in this application, various other modifications obvious to those skilled in the art can be made to the present invention, and such modifications are also included in the scope of the attached claims.

All works cited here in their entirety and for all purposes of practical application are introduced into the present description by reference, since if each individual publication, patent or patent application were specifically and separately introduced in its entirety and for all purposes of practical use by reference .

Claims (118)

1. The compound having the formula D _F :

or a pharmaceutically acceptable salt of compound D _F ,
where independently in each position:
R ^{2 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{5 is} selected from the group consisting of —H and methyl;
R ^{6 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ⁸ represents —O— (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents O or NH;
R ^{11 is} selected from the group consisting of —H, —C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents —C ₂ -C ₈ alkyl;
R ¹⁴ represents —H or —C ₁ -C ₈ alkyl. 2. The compound according to claim 1, having the formula:

or a pharmaceutically acceptable salt thereof. 3. The compound or its pharmaceutically acceptable salt according to any one of claims 1 to 2 in isolated and purified form. 4. The conjugate having the formula:
L- (LU-D _F ) _P
or a pharmaceutically acceptable salt thereof;
where L is a ligand component;
linker component (LU) has the formula:
-A _a -W _w -Y _y -,
where -A- is an extension component,
a represents 0 or 1,
each of -W- independently represents an amino acid component,
w is an integer from 0 to 12,
-Y- is a spacer component,
y is 0, 1 or 2; and p ranges from 1 to about 20;
D _F has the formula:

or a pharmaceutically acceptable salt thereof,
where the dashed line in D _F indicates the site of covalent binding to the ligand component L, selected from the group including protein, polypeptide and peptide, through the linker component LU;
independently in each position:
R ^{2 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{5 is} selected from the group consisting of H and methyl;
R ^{6 is} selected from H and C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ⁸ represents O- (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents O or NH;
R ^{11 is} selected from the group consisting of H, C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents C ₂ -C ₈ alkyl;
R ¹⁴ represents H or C ₁ -C ₈ alkyl. 5. The conjugate having the formula:
LU-D _F
or a pharmaceutically acceptable salt thereof,
where -LU- is a linker component;
where the linker component (LU) contains;
-A _a -W _w -Y _y -,
where -A- is an extension component,
equal to 0 or 1,
each -W- independently represents an amino acid component,
w is an integer from 0 to 12,
-Y- is a spacer component, and
y is 0, 1 or 2;
D _F has the formula:

where the dashed line in D _F indicates the site of covalent binding to the linker component of LU, and
independently in each position:
R ^{2 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{5 is} selected from the group consisting of H and methyl;
R ^{6 is} selected from H and C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ⁸ represents O- (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents O or NH;
R ^{11 is} selected from the group consisting of H, C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents C ₂ -C ₈ alkyl;
R ¹⁴ represents H or C ₁ -C ₈ alkyl. 6. The conjugate according to claim 4, where the conjugate is an antibody-drug conjugate having the formula Ia ':

or a pharmaceutically acceptable salt thereof,
where Ab is an antibody,
-A _a -W _w -Y _y - is a linker component,
A is an extension component,
equal to 0 or 1,
each W independently represents an amino acid component,
w is an integer from 0 to 12,
Y is a spacer component
y is 0, 1 or 2, and
p ranges from 1 to about 20. 7. The conjugate "antibody-drug" according to claim 6, where each of a, w and y is present, or a is 1 and each of w and y is 0,
or a pharmaceutically acceptable salt thereof. 8. The conjugate "antibody-drug" according to claim 6, having the formula:

or a pharmaceutically acceptable salt thereof,
where L is an antibody and R ¹⁷ is C ₁ -C ₁₀ alkylene-, -C ₃ -C ₈ carbocyclo-, -O- (C ₁ -C ₈ alkyl), -arylene, -C ₁ -C ₁₀ alkylene arylene, arylene-C ₁ -C ₁₀ alkylene-, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ carbocyclo) -, - (C ₃ -C ₈ carbocyclo) -C ₁ -C ₁₀ alkylene- , -C ₃ -C ₈ heterocyclo-, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ heterocyclo) -, - (C ₃ -C ₈ heterocyclo) -C ₁ -C ₁₀ alkylene-, - (CH ₂ CH ₂ O) _r - and - (CH ₂ CH ₂ O) _r -CH ₂ -; and r is an integer from 1 to 10. 9. The antibody-drug conjugate of claim 6 or a pharmaceutically acceptable salt thereof, wherein the antibody is coupled to a linker component or to compound D _F via a cysteine residue of the antibody. 10. The conjugate "antibody-drug" according to claim 9 or its pharmaceutically acceptable salt, in which p is from 2 to 5. 11. The conjugate "antibody-drug" according to claim 9, having the formula:

or a pharmaceutically acceptable salt thereof,
where L is an antibody and R ¹⁷ is C ₁ -C ₁₀ alkylene-, -C ₃ -C ₈ carbocyclo-, -O- (C ₁ -C ₈ alkyl), -arylene, -C ₁ -C ₁₀ alkylene arylene, arylene-C ₁ -C ₁₀ alkylene-, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ carbocyclo) -, - (C ₃ -C ₈ carbocyclo) -C ₁ -C ₁₀ alkylene- , -C ₃ -C ₈ heterocyclo-, -C ₁ -C ₁₀ alkylene- (C ₃ -C ₈ heterocyclo) -, - (C ₃ -C ₈ heterocyclo) -C ₁ -C ₁₀ alkylene-, - (CH ₂ CH ₂ O) _r - and - (CH ₂ CH ₂ O) _r -CH ₂ -; and r is an integer from 1 to 10. 12. The conjugate "antibody-drug" according to claim 8, having the formula:

or a pharmaceutically acceptable salt thereof. 13. The conjugate "antibody-drug" according to claim 11, having the formula:

or a pharmaceutically acceptable salt thereof,
where each of w and y is 0. 14. The conjugate "antibody-drug" according to claim 6 or its pharmaceutically acceptable salt, where w is an integer from 2 to 12. 15. The conjugate "antibody-drug" according to 14 or its pharmaceutically acceptable salt, where w is equal to 2. 16. The antibody-drug conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein W _w is -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-. 17. The antibody-drug conjugate of claim 8 or a pharmaceutically acceptable salt thereof, wherein W _w is 5-aminovalerianic acid, homophenylalanine lysine, tetraisoquinoline carboxylate lysine, cyclohexylalanine lysine, isonipecotinic acid lysine, beta-alanine lysine, valine glycine glutamine or isonipecotinic acid. 18. The conjugate "antibody-drug" according to clause 16, having the formula:

or a pharmaceutically acceptable salt thereof. 19. The conjugate "antibody-drug" according to clause 16, having the formula:

or a pharmaceutically acceptable salt thereof. 20. The conjugate "antibody-drug" according to clause 16, having the formula:

or a pharmaceutically acceptable salt thereof. 21. The conjugate "antibody-drug" according to any one of claims 6 to 8 and 12-20, where D _F has the formula:

or a pharmaceutically acceptable salt thereof. 22. The antibody-drug conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody;
a bispecific antibody, a chimeric antibody, a humanized antibody, a diabody, and an antibody fragment. 23. The conjugate "antibody-drug" according to claim 6, having the formula:

or

or a pharmaceutically acceptable salt thereof,
where Ab is an antibody, Val is valine and Cit is citrulline. 24. The antibody-drug conjugate of claim 23 or a pharmaceutically acceptable salt thereof, wherein the antibody binds to CD30, CD20, CD33, or the Lewis Y antigen. 25. A pharmaceutical composition for treating cancer, comprising an effective amount of a conjugate according to any one of claims 4 and 6-24, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. 26. The use of the conjugate according to any one of claims 4 and 6-24, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. 27. The use of claim 26, wherein the cancer is lymphoma. 28. The application of claim 26, wherein the cancer is breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, colon, thyroid, pancreas, prostate, or bladder cancer. 29. A method of treating cancer, comprising administering an amount of an antibody-drug conjugate according to any one of claims 4 and 6-24, or a pharmaceutically acceptable salt thereof, wherein said amount is effective for treating cancer, the antibody-drug drug conjugate antibody binds to antigen expressed by a cancer cell. 30. The use of claim 26, wherein the cancer is breast cancer. 31. The use of claim 26, wherein the cancer is kidney cancer. 32. The conjugate or its pharmaceutically acceptable salt according to any one of claims 4-24 in isolated and purified form. 33. The antibody-drug conjugate comprising an antibody covalently attached to one or more drug molecules, wherein the antibody-drug conjugate has the formula Ic:

or a pharmaceutically acceptable salt thereof,
where Ab is an antibody that binds to one or more tumor associated antigens (1) to (35):
(1) BMPR1B (type I bone morphogenesis protein receptor);
(2) E16 (LAT1, SLC7A5);
(3) STEAP1 (six-domain transmembrane prostate epithelial antigen);
(4) 0772P (CA125, MUC16);
(5) MPF (MPF, MSLN, SMR, megakaryocytic potentiating factor, mesothelin);
(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 of the family of soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transporting protein 3b type II);
(7) Sema 5b (FLJ10372, KIAA1445; Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorine) 5 B);
(8) PSCA nlg (2700050C12Rik, C530008016Rik, cDNA RIKEN 2700050C12, cDNA of the RIKEN gene 2700050C12);
(9) ETBR (type B endothelin receptor);
(10) MSG783 (RNF124, hypothetical protein FLJ20315);
(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein prostate gland);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of subfamily M);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor derived from teratocarcinoma);
(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792);
(15) CD79b (IGb (immunoglobulin-linked beta protein), B29);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a containing the 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 linked to immunoglobulin alpha protein);
(29) CXCR5 (Burkitt's lymphoma receptor 1);
(30) HLA-DOB (beta subunit of the MHC class II molecule (antigen Ia), which binds to peptides and presents them to CD4 ⁺ T lymphocytes);
(31) P2X5 (ion channel 5 opened by the ligand of the P2X purinergic receptor);
(32) CD72 (antigen CD72 line B-cell differentiation, Lyb-2);
(33) LY64 (lymphocytic antigen 64 (RP105), a membrane protein belonging to the leucine-rich repeating protein family (LRR), type I);
(34) FCRH1 (Fc receptor-like protein: 1); and
(35) IRTA2 (translocation-associated immunoglobulin 2 superfamily receptor),
A is an extension component,
equal to 0 or 1,
each W independently represents an amino acid component,
w is an integer from 0 to 12,
Y is a spacer component
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a drug molecule selected from the group consisting of formulas D _E and D _F :

where the wavy line in D _E and D _F denotes sites of covalent attachment to A, W or Y, and independently in each position:
R ^{2 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{5 is} selected from the group consisting of H and methyl;
R ^{6 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ^{8 is} independently selected from the group consisting of H, OH, C ₁ -C ₈ alkyl and O- (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of H and C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents O or NH;
R ^{11 is} selected from the group consisting of H, C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents C ₂ -C ₈ alkyl;
R ¹⁴ represents H or C ₁ -C ₈ alkyl; and
R ¹⁸ represents —C (R ⁸ ) ₂ —C (R ⁸ ) ₂ —C ₆ —C ₁₀ aryl. 34. The conjugate "antibody-drug" according to clause 33, in which D has the formula D _E :

or a pharmaceutically acceptable salt thereof. 35. The conjugate "antibody-drug" according to clause 33, in which D has the formula D _F :

or a pharmaceutically acceptable salt thereof. 36. The antibody-drug conjugate of claim 33, or a pharmaceutically acceptable salt thereof, wherein the antibody is coupled to a drug molecule via the cysteine residue of the antibody. 37. The conjugate "antibody-drug" according to clause 36 or its pharmaceutically acceptable salt, where p is from 1 to 4. 38. The conjugate "antibody-drug" according to p. 33 or its pharmaceutically acceptable salt, where p is from 2 to 8. 39. The conjugate "antibody-drug" according to p. 33 or its pharmaceutically acceptable salt, where p is from 2 to 5. 40. The conjugate "antibody-drug" according to clause 36, having the formula:

or a pharmaceutically acceptable salt thereof. 41. The conjugate "antibody-drug" according to paragraph 40, where each of w and y is 0, having the formula:

or a pharmaceutically acceptable salt thereof. 42. The conjugate "antibody-drug" according to paragraph 41 or its pharmaceutically acceptable salt, where D has the formula D _E. 43. The conjugate "antibody-drug" according to § 42, in which the formula D _E has the formula:

or a pharmaceutically acceptable salt thereof. 44. The conjugate "antibody-drug" according to paragraph 41 or its pharmaceutically acceptable salt, where D has the formula D _F. 45. The conjugate "antibody-drug" according to item 44, in which the formula D _F has the formula:

or a pharmaceutically acceptable salt thereof. 46. The conjugate "antibody-drug" according to clause 33, having the formula:

or a pharmaceutically acceptable salt thereof. 47. The conjugate "antibody-drug" according to item 46, having the formula:

or a pharmaceutically acceptable salt thereof. 48. The conjugate "antibody-drug" according to item 47, having the formula:

or a pharmaceutically acceptable salt thereof. 49. The conjugate "antibody-drug" according to § 48 or its pharmaceutically acceptable salt, where D has the formula D _E. 50. The conjugate "antibody-drug" according to § 49, in which the formula D _E has the formula:

or a pharmaceutically acceptable salt thereof. 51. The conjugate "antibody-drug" according to item 47 or its pharmaceutically acceptable salt, in which D has the formula D _F. 52. The conjugate "antibody-drug" according to § 51, in which the formula D _F has the formula:

or a pharmaceutically acceptable salt thereof. 53. The conjugate "antibody-drug" according to p. 33 or its pharmaceutically acceptable salt, where w is an integer from 2 to 12. 54. The conjugate "antibody-drug" according to p. 33 or its pharmaceutically acceptable salt, where w is equal to 2. 55. The conjugate "antibody-drug" according to clause 35 or its pharmaceutically acceptable salt, where W _w is a valine-citrulline-. 56. The conjugate "antibody-drug" according to p, in which D is selected from the group consisting of formulas:

and

or a pharmaceutically acceptable salt thereof. 57. The antibody-drug conjugate of claim 33, or a pharmaceutically acceptable salt thereof, wherein the antibody specifically binds to the HER2 receptor. 58. The antibody-drug conjugate of claim 57, or a pharmaceutically acceptable salt thereof, which specifically binds to the extracellular domain of the HER2 receptor and inhibits the growth of tumor cells that overexpress the HER2 receptor. 59. The antibody-drug conjugate according to claim 33 or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, and an antibody fragment. 60. The conjugate "antibody-drug" according to § 59 or its pharmaceutically acceptable salt, where the antibody fragment is a Fab fragment. 61. The conjugate "antibody-drug" according to § 59 or a pharmaceutically acceptable salt thereof, wherein the humanized antibody is 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. The antibody-drug conjugate of claim 61 or a pharmaceutically acceptable salt thereof, wherein the antibody is huMAb4D5-8 (trastuzumab). 63. The conjugate "antibody-drug" according to p. 33, having the formula:

or a pharmaceutically acceptable salt thereof,
where Val is valine and Cit is citrulline. 64. The conjugate "antibody-drug" according to item 63 or its pharmaceutically acceptable salt, where the antibody is huMAb4D5-8 (trastuzumab). 65. A pharmaceutical composition for treating cancer, comprising an effective amount of an antibody-drug conjugate according to any one of claims 33-64, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. 66. A method of lysing or inhibiting the proliferation of tumor or cancer cells, comprising treating the tumor or cancer cells with an amount of an antibody-drug conjugate according to any one of claims 33-64, wherein said amount is effective for lysing or inhibiting the proliferation of tumor or cancer cells. 67. A method of treating cancer, comprising administering an amount of an antibody-drug conjugate according to any one of claims 33-64, wherein said amount is effective for treating cancer. 68. The method according to p, where the cancer is selected from the group consisting of cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, colon, thyroid gland, pancreas, prostate and bladder . 69. The method of claim 67, wherein the cancer is lymphoma. 70. The method according to p, where the lymphoma is a non-Hodgkin's lymphoma. 71. A method of inhibiting cell proliferation, comprising:
treating mammalian cells in the cell culture medium with an antibody-drug conjugate according to any one of claims 33-64 or a pharmaceutically acceptable salt thereof, and
measuring the cytotoxic activity of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 72. A method of treating cancer, comprising administering an effective amount of an antibody-drug conjugate composition according to any one of claims 33-64 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, carrier or excipient. 73. The method of claim 72, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof specifically binds to a receptor encoded by the EbrB2 gene. 74. The method of claim 72, wherein the amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof administered to a patient is in the range of about 0.1 to about 10 mg / kg of the patient's body weight. 75. The method of claim 72, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is administered at intervals of about three weeks. 76. The method of claim 72, wherein said antibody-drug conjugate or a pharmaceutically acceptable salt thereof is prepared together with a pharmaceutically acceptable parenteral carrier and administered parenterally. 77. The method of claim 72, wherein said antibody-drug conjugate or a pharmaceutically acceptable salt thereof is formulated as an injectable drug. 78. The method of claim 72, wherein said antibody-drug conjugate or a pharmaceutically acceptable salt thereof is administered intravenously. 79. The method of claim 72, further comprising administering a second antibody that binds to a tumor-associated antigen selected from (1) to (35):
(1) BMPR1B (type I bone morphogenesis protein receptor);
(2) E16 (LAT1, SLC7A5);
(3) STEAP1 (six-domain transmembrane prostate epithelial antigen);
(4) 0772P (CA125, MUC16);
(5) MPF (MPF, MSLN, SMR, megakaryocytic potentiating factor, mesothelin);
(6) Napi3b (NAPI-3B, NpTIIb, SLC34A2, member 2 of the family of soluble carriers 34 (sodium phosphate), sodium-dependent phosphate-transporting protein 3b type II);
(7) Sema 5b (FLJ 10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, seme domain, seven domain thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain (semaphorin ) 5B);
(8) PSCA hlg (2700050C12Rik, C530008016Rik, cDNA RIKEN 2700050C12, cDNA of the RIKEN gene 2700050C12);
(9) ETBR (type B endothelin receptor);
(10) MSG783 (RNF124, hypothetical protein FLJ20315);
(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, six-domain transmembrane epithelial antigen of the prostate 2, six-domain transmembrane protein prostate gland);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, potential-dependent cation channel of the transient receptor, member 4 of subfamily M);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor derived from teratocarcinoma);
(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792);
(15) CD79b (IGb (immunoglobulin-linked beta protein), B29);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a containing the 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 linked to immunoglobulin alpha protein);
(29) CXCR5 (Burkitt's lymphoma receptor 1);
(30) HLA-DOB (beta subunit of the MHC class II molecule (antigen Ia), which binds to peptides and presents them to CD4 ⁺ T lymphocytes);
(31) P2X5 (ion channel 5 opened by the ligand of the P2X purinergic receptor);
(32) CD72 (antigen CD72 line B-cell differentiation, Lyb-2);
(33) LY64 (lymphocytic antigen 64 (RP105), a membrane protein belonging to the leucine-rich repeating protein family (LRR), type I);
(34) FCRH1 (Fc receptor-like protein, 1); and
(35) IRTA2 (translocation-associated immunoglobulin 2 superfamily receptor). 80. A method of inhibiting the growth of tumor cells that overexpresses a tumor-associated antigen, comprising administering an antibody-drug conjugate according to any one of claims 33-64 or a pharmaceutically acceptable salt thereof that specifically binds to said tumor-associated antigen, and a chemotherapeutic an agent wherein each of said antibody-drug conjugate or a pharmaceutically acceptable salt thereof and a chemotherapeutic agent are administered in amounts effective for ngibirovaniya the growth of tumor cells. 81. The method of claim 80, wherein said antibody-drug conjugate or a pharmaceutically acceptable salt thereof provides tumor cells with a sensitivity to said chemotherapeutic agent. 82. A method of treating a person susceptible to a disease or a person who has been diagnosed with a disease characterized by overexpression of an ErbB2 receptor, wherein said method comprises administering an effective amount of an antibody-drug conjugate combination or a pharmaceutically acceptable salt thereof according to any one of claims. 33-64 and a chemotherapeutic agent. 83. Analysis for the detection of cancer cells, including:
treating the cells with an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 33-64 and
determining the degree of binding of the antibody-drug conjugate or its pharmaceutically acceptable salt to the cells. 84. The analysis of claim 83, wherein the cells are breast tumor cells. 85. The assay of Claim 83, wherein the degree of binding is determined by measuring the levels of a nucleic acid encoding a tumor-associated antigen by fluorescence in situ hybridization (FISH). 86. The analysis of claim 83, wherein the degree of binding is determined by the immunohistochemical method (IHC). 87. An article containing:
an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 33-64;
container; and
a package insert or label on the package indicating that the antibody-drug conjugate or a pharmaceutically acceptable salt thereof can be used to treat cancer. 88. The product of claim 87, wherein said package insert or label indicates that the antibody-drug conjugate or a pharmaceutically acceptable salt thereof can be used to treat cancer characterized by overexpression of the ErbB2 receptor. 89. The product according to p, where the cancer is breast cancer. 90. A method of treating cancer in a mammal, wherein the cancer is overexpressed by an ErbB2 receptor (HER2) and is immune or poorly susceptible to treatment with an anti-ErbB2 antibody, and wherein said method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate according to any one of claims .33-64 or a pharmaceutically acceptable salt thereof. 91. The use of the antibody-drug conjugate according to any one of claims 33-64 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the lysis or inhibition of proliferation of tumor cells or cancer cells. 92. Use of the antibody-drug conjugate according to any one of claims 33-64 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of hyperproliferative disorder. 93. The application of paragraph 92, where the hyperproliferative disorder is a cancer selected from the group consisting of cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, colon, thyroid gland, pancreas, prostate gland and bladder. 94. The use of claim 92, wherein the cancer is breast cancer. 95. The use of claim 92, wherein the cancer is lymphoma. 96. The compound having the formula:

where R ² selected from the group including -H and C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
where R ⁵ selected from the group comprising H and methyl;
R ^{6 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
R ⁸ represents O- (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents —NH—;
R ^{11 is} selected from the group consisting of —H, C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents —C ₂ -C ₈ alkyl;
R ¹⁴ represents —H or C ₁ -C ₈ alkyl;
or a pharmaceutically acceptable salt thereof. 97. The compound having the formula:

where independently in each position:
R ^{2 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of —H and ═C ₁ -C ₈ alkyl;
where R ⁵ selected from the group comprising H and methyl;
R ^{6 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ⁸ represents —O— (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
or a pharmaceutically acceptable salt thereof. 98. The compound having the formula:

where R ² selected from the group including -H and C ₁ -C ₈ alkyl;
R ^{3 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
R ^{4 is} selected from the group consisting of —H and C ₁ -C ₈ alkyl;
wherein R ^{5 is} selected from the group consisting of —H and methyl;
R ^{6 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ^{7 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ⁸ represents —O— (C ₁ -C ₈ alkyl);
R ^{9 is} selected from the group consisting of —H and —C ₁ -C ₈ alkyl;
R ¹⁰ represents C ₆ -C ₁₀ aryl;
Z represents —O—;
R ^{11 is} selected from the group consisting of —H, C ₁ -C ₂₀ alkyl, C ₆ -C ₁₀ aryl, and - (R ¹³ O) _m —R ¹⁴ ;
m is an integer from 1 to 1000;
R ¹³ represents —C ₂ -C ₈ alkyl;
R ¹⁴ represents —H or C ₁ -C ₈ alkyl;
or a pharmaceutically acceptable salt thereof. 99. The compound according to any one of claims 96-98, or a pharmaceutically acceptable salt thereof, in isolated and purified form. 100. The conjugate "antibody-drug" according to clause 33, having the formula:

or a pharmaceutically acceptable salt thereof, wherein Val is valine and Cit is citrulline. 101. The conjugate "antibody-drug" according to p. 33, having the formula:

or a pharmaceutically acceptable salt thereof. 102. The conjugate "antibody-drug" according to p. 33, having the formula:

or a pharmaceutically acceptable salt thereof. 103. The method according to p, where the cancer is a cancer selected from the group comprising cancer of the breast, ovaries, kidneys, prostate and liver. 104. The method of claim 67, wherein the cancer is hematopoietic cancer. 105. The method of claim 104, wherein the hematopoietic cancer is non-Hodgkin's lymphoma. 106. The conjugate "antibody-drug" according to item 23, where the antibody is associated with CD70. 107. The compound according to any one of claims 1 and 96-98, or a pharmaceutically acceptable salt thereof, wherein each R ² and R ⁶ is methyl and R ⁹ is —H. 108. The conjugate according to any one of claims 4 to 20 and 22, or a pharmaceutically acceptable salt thereof, wherein each R ² and R ⁶ is methyl and R ⁹ is —H. 109. The compound according to any one of claims 1 and 96-98, or a pharmaceutically acceptable salt thereof, wherein each R ² and R ⁶ is methyl, R ⁵ is —H, R ⁷ is sec-butyl, in each case R ⁸ represents —OCH ₃ , and R ⁹ represents —H. 110. The conjugate according to any one of claims 4 to 20, 22 and 24, or a pharmaceutically acceptable salt thereof, wherein each R ² and R ⁶ is methyl, R ⁵ is —H, R ⁷ is sec-butyl, in each case R ⁸ represents —OCH ₃ , and R ⁹ represents —H. 111. The conjugate "antibody-drug" according to claim 6, having the formula:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody, Val is valine, and Cit is citrulline. 112. The conjugate "antibody-drug" according to claim 6, having the formula:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody, Val is valine, and Cit is citrulline. 113. The conjugate "antibody-drug" according to claim 9 or its pharmaceutically acceptable salt, where p is from 1 to 4. 114. The conjugate "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 claims 33-42, 44, 46-49, 51, 53-55, 57-62 and 64, or a pharmaceutically acceptable salt thereof, wherein R ² and R ^{6 are} each methyl and R ⁹ is by yourself -N. 116. The conjugate according to any one of claims 33-42, 44, 46-49, 51, 53-55, 57-62 and 64, or a pharmaceutically acceptable salt thereof. 117. The compound according to any one of claims 1, 96 and 98, or a pharmaceutically acceptable salt thereof, wherein R ^{11 is} selected from the group consisting of H, C ₁ -C ₂₀ alkyl and (R ¹³ O) _m -R ¹⁴ . 118. The conjugate according to any one of claims 4 to 20, 22 and 24, or a pharmaceutically acceptable salt thereof, wherein R ^{11 is} selected from the group consisting of H, C ₁ -C ₂₀ alkyl and (R ¹³ O) _m —R ¹⁴ .

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