CN1993146A - Antibody-drug conjugates and methods - Google Patents

Abstract

The present invention relates to antibody-drug conjugate compounds of Formula I: Ab-(L-D) _p , wherein one or more maytansinoid drug moieties (D) are covalently linked to an antibody (Ab) via L, and the antibody (Ab) binds to an ErbB receptor or one or more tumor-associated antigens or cell surface receptors. These compounds can be used in the diagnosis or treatment of cancer and other diseases and disorders.

Description

Antibody-drug conjugates and methods

This non-provisional application filed under 37 CFR §1.53(b) requires US provisional application 60/576,517 filed June 1, 2004, US provisional application filed October 5, 2004, under 35 USC §119(e) Benefits of application 60/616,098, which are incorporated by reference in their entirety.

Field of invention

The present invention relates generally to compounds having anticancer activity, and more particularly to antibodies conjugated to chemotherapeutic maytansinoid drugs or toxins. The present invention also relates to methods of in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or related pathological conditions, using antibody-drug conjugate compounds.

Background of the invention

Monoclonal antibody therapies have been established for targeted therapy in patients with cancer, immunological and angiogenic disorders. Antibody-drug conjugates (ADCs) or immunoconjugates are used to locally deliver cytotoxic or cytostatic agents, drugs that kill or inhibit tumor cells in cancer therapy (Payne, G. (2003) Cancer Cell 3:207-212 ; Trail et al (2003) Cancer Immunol.Immunother.52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv.Drug Del.Rev.26:151- 172; US4975278) theoretically allow the targeted delivery of drug moieties to tumors and accumulate intracellularly in tumors, where systemic administration of these unconjugated agents may result in unacceptable levels of toxicity to both normal cells and the tumor cells to be removed (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview, "in Monoclonal Antibodies' 84: Biological And Clinical Applications , A. Pinchera et al. (eds.), pp.475-506). Thus what is sought is maximum efficacy with minimum toxicity. Efforts to design and improve ADCs have focused on the selectivity and drug-attachment and drug-release properties of monoclonal antibodies (mAbs). Both polyclonal and monoclonal antibodies have been reported for use in these strategies (Rowland et al. (1986) Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunorubicin, doxorubicin, methotrexate, mitomycin, neocarcinstatin (Takahashi et al. (1988) Cancer 61:881-888) and vindesine (Rowland et al. , (1986) ibid). Toxins for antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin (US4753894; US 5629197; US 4958009; US 4956453), small molecule toxins such as geldanamycin (Mandler etc. (2000) J.of the Nat.Cancer Inst.92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med.Chem.Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem.13: 786-791), maytansinoids (maytansinoids) (EP1391213; Liu et al., (1996) Proc.Natl.Acad.Sci.USA 93:8618-8623), and calicheamicin (calicheamicin) (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). The toxin may affect its cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or have reduced activity when conjugated to large antibodies or protein receptor ligands.

The antibody-radioisotope conjugate ZEVALIN_ (ibritumomab tiuxetan, Biogen/Idec), consisting of a murine IgG1 kappa monoclonal antibody against the CD20 antigen and ¹¹¹ In or ⁹⁰ y Radioisotope composition (Wiseman et al. (2000) Eur.J.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). In 2000, the antibody-drug conjugate MYLOTARG ^™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), consisting of the hu CD33 antibody linked to calicheamicin, was approved for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000 )25(7):686; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001). Antibody-drug conjugate Cantuzumab mertansine (Immunogen, Inc.) consisting of the huC242 antibody linked to the maytansinoid drug moiety DM1 via a disulfide linker SPP (Xie et al. (2004) J.of Pharm.and Exp .Ther.308(3): 1073-1082; Tolcher et al. (2003) J.Clin.Oncology 21(2): 211-222; US 5208020) have treated cancers expressing CanAg such as colon cancer, pancreatic cancer, gastric cancer and others Phase I trials in cancer. MLN-2704 (Millennium Pharm., BZLBiologics, Immunogen Inc.), an antibody-drug conjugate consisting of an anti-prostate-specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety DM1, is under investigation Developed for effective treatment of prostate tumors. The same maytansinoid drug moiety DM1 was linked to mouse monoclonal antibody TA.1 via a non-disulfide linker SMCC (Chari et al. (1992) Cancer Research 52:127-131) . This conjugate is reported to be 200-fold less potent than the corresponding disulfide linker conjugate. The SMCC linker is considered "non-cleavable" in such conjugates (see also: US 4981979). HERCEPTIN® (trastuzumab) linked to DM1 via SMCC has been reported (WO 2005/037992).

In an effort to find effective cellular targets for cancer diagnosis and therapy, investigators have sought to identify transmembrane or tumor-associated polypeptides that are specifically expressed on a on the surface of one or more specific types of cancer cells. Typically, these tumor-associated polypeptides are more expressed on the surface of cancer cells than on the surface of non-cancer cells. The identification of these tumor-associated cell surface antigen polypeptides, tumor-associated antigens (TAAs), has yielded the ability to specifically target cancer cells for destruction by antibody-based therapies.

Monoclonal antibody therapy has been established for the targeted treatment of patients with cancer, immune disorders, and angiogenesis disorders. An example of successful antibody therapy is the recombinant DNA-derived humanized monoclonal antibody HERCEPTIN® (trastuzumab), which selectively binds with high affinity (Kd = 5 nM) to Extracellular domain of human epidermal growth factor receptor 2 protein HER2 (ErbB2) (US 5821337; US 6054297; US 6407213; US 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 containing human framework regions with a murine antibody complementarity-determining region (4D5) that binds HER2. Trastuzumab binds to the HER2 antigen, thus inhibiting the growth of cancer cells. Because trastuzumab is a humanized antibody, it minimizes any HAMA response in patients. The anti-HER2 humanized antibody is produced by suspension culture of mammalian cells (Chinese Hamster Ovary (CHO)). The HER2 (or c-erbB2) proto-oncogene encodes a 185 kDa transmembrane receptor protein that is structurally related to the epidermal growth factor receptor. HER2 protein overexpression is observed in 25%-30% of primary breast cancers and can be determined using immunohistochemistry-based assessment of fixed tumor blockade (Press MF, et al. (1993) Cancer Res 53:4960 -70). Trastuzumab has been shown to inhibit the proliferation of human tumor cells overexpressing HER2 in in vitro tests and in animals (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 cytotoxicity, ADCC (HotalingTE, 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; Sliwkowski et al. (1999) Seminars in Oncology 26(4), Suppl 12:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews: Molecular Cell Biology, Macmillan Magazines, Ltd., Vol. 2:127-137). Clinically, HERCEPTIN® is active in ErbB2-overexpressing metastatic breast cancer patients who have previously received extensive anticancer therapy (Baselga et al., (1996) J. Clin. Oncol. 14:737- 744). Although HERCEPTIN is a breakthrough in the treatment of patients with ErbB2-overexpressing breast cancer who have previously received extensive anticancer therapy, the majority of patients in this population do not respond or respond poorly to HERCEPTIN therapy. Therefore, there is a clear clinical need to develop additional HER2-directed cancer therapies for the treatment of patients with HER2-overexpressing tumors or other diseases associated with HER2 expression who do not respond or respond poorly to HERCEPTIN therapy. In addition to HER2, targeted therapies also have the potential to exploit other tumor-associated antigens.

overview

The present invention provides novel compounds with biological activity against cancer cells. The compounds can inhibit the growth of tumors in mammals and are useful in the treatment of human cancer patients.

The present invention relates to the delivery, transport, accumulation or maintenance of therapeutic antibody-drug conjugate (ADC) compounds within cells. The present invention more particularly relates to obtaining high concentrations of active metabolite molecules in cancer cells. Intracellular targeting can be achieved by methods and compounds that allow the accumulation or maintenance of biologically active agents within the cell. Such effective targeting is applicable to a variety of therapeutic formulations and methods.

The surprising discovery has been made that antibody-drug conjugates having a stable non-active maytansinoid drug moiety attached to the antibody lead to enhanced potency in vitro and efficacy in vivo. Disulfide linker group. Furthermore, the antibody-drug conjugates showed unexpectedly safer results in vivo relative to certain disulfide linker conjugates.

Antibody-drug conjugate (ADC) compounds comprise an antibody covalently linked by a linker to one or more maytansinoid drug moieties. ADC can be represented by formula I:

Ab-(LD) _p I

wherein one or more maytansinoid drug moieties (D) are covalently linked via L to the antibody (Ab). Abs are antibodies that bind ErbB receptors, or that bind one or more tumor-associated antigens or cell surface receptors. The linker L may be stable extracellularly, ie extracellularly. Linker L, maytansinoid drug moiety D, or linker and maytansinoid drug moiety together (L-D) do not contain a disulfide group.

In one embodiment, a substantial amount of the drug moiety is not cleaved by the antibody until the antibody-drug conjugate enters a cell having a cell surface receptor specific for the antibody-drug conjugated antibody, and the drug moiety is cleaved by the antibody when the antibody-drug conjugate does enter the cell.

In another embodiment, the ADC specifically binds receptors encoded by the ErbB gene, such as EGFR, HER2, HER3, and HER4. ADC can specifically bind the extracellular domain of HER2 receptor. ADCs can inhibit the growth of tumor cells that overexpress the HER2 receptor.

In another embodiment, the antibody (Ab) of formula I is a humanized antibody such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab).

Another aspect of the present invention is a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

Another aspect provides a pharmaceutical combination comprising a compound of formula I and a second compound having anticancer properties or other therapeutic effect.

Another aspect includes diagnostic and therapeutic uses of the compounds and compositions disclosed herein.

Another aspect is a method for killing or inhibiting the proliferation of tumor cells or cancer cells, which includes using a certain amount of antibody-drug conjugate effective in killing or inhibiting the proliferation of tumor cells or cancer cells, or a pharmaceutically acceptable salt or solvent thereof Chemicals to treat the cells.

Another aspect is a method of treating cancer comprising administering to a patient a formulation of a compound of formula I. A method is for treating cancer in a mammal, wherein the cancer is characterized by overexpression of ErbB receptors. The mammal is optionally non-responsive, or minimally responsive, to treatment with a non-conjugated anti-ErbB antibody. The method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound.

Another aspect is a method for inhibiting the growth of tumor cells overexpressing a growth factor receptor selected from HER2 receptors and EGF receptors, the method comprising administering to a patient a drug that specifically binds to the growth factor receptor An antibody-drug conjugate compound and a chemotherapeutic agent, wherein the antibody-drug conjugate and the chemotherapeutic agent are respectively administered in an amount effective to inhibit the growth of tumor cells in a patient.

Another aspect is a method of treating a human patient susceptible to or diagnosed with a condition characterized by ErbB2 receptor overexpression, the method comprising administering a formula I Combinations of antibody-drug conjugate compounds and chemotherapeutic agents.

Another aspect is a testing method for detecting cancer cells, comprising: contacting the cells with an antibody-drug conjugate compound, and measuring the degree of binding of the antibody-drug conjugate compound to the cells.

Another aspect relates to methods of screening ADC drug candidates for the treatment of a disease or disorder characterized by overexpression of HER2.

Another aspect includes an article of manufacture, ie a kit, comprising an antibody-drug conjugate, a container, and a package insert or label directing the treatment.

Another aspect includes a method of treating a disease or disorder characterized by HER2 overexpression in a patient with the antibody-drug conjugate compound.

Another aspect includes the production method, preparation method, synthesis method, coupling method, and purification method of the antibody-drug conjugate compound and intermediates used in the preparation, synthesis, and coupling of the antibody-drug conjugate compound.

Brief description of the drawings

Figure 1 shows in vitro cell proliferation assays performed on SK-BR-3 cells treated with antibody-drug conjugates: - △-Trastuzumab-SPP-DM1, -△-Trastuzumab-SPDP-DM1, and -○-trastuzumab-SMCC-DM1.

Figure 2 shows in vitro cell proliferation assays performed on BT-474 cells treated with antibody-drug conjugates: - -Trastuzumab-SPP-DM1, -△-Trastuzumab-SPDP-DM1, and -○ - Trastuzumab-SMCC-DM1.

Figure 3 shows in vitro cell proliferation assays performed on MCF7 cells treated with antibody-drug conjugates: - □-Trastuzumab-SPP-DM1, -△-Trastuzumab-SPDP-DM1, and -○-Trastuzumab Tuzumab-SMCC-DM1.

Figure 4 shows the in vitro cell proliferation assay performed on MDA-MB-468 cells treated with antibody-drug conjugates: - -Trastuzumab-SPP-DM1, -△-Trastuzumab-SPDP-DM1, and -○-trastuzumab-SMCC-DM1.

Figure 5 shows the serum clearance of trastuzumab-SMCC-DM1 relative to trastuzumab-SPP-DM1 in tumor-free beige nude mice, at six time points in 7 days (5 minutes, 1 hour, 6 minutes after dosing). hours, 24 hours, 72 hours, and 168 hours) to measure conjugate and total antibody serum concentrations.

Figure 6 shows the stability over time of the following conjugates in tumor-free nude mice: Trastuzumab-SPDP-DM1, Trastuzumab-SPP-DM1, Trastuzumab-SPP-DM3, Trastuzumab Serum concentrations of anti-SPP-DM4, and trastuzumab-SMCC-DM1 were measured at six time points (5 minutes, 1 hour, 6 hours, 24 hours, 72 hours, 168 hours after administration) in 7 days.

Figure 7 shows serum concentration measurements of total trastuzumab/trastuzumab-SMCC-DM1 and total trastuzumab/trastuzumab-SPP-DM1 in mice with and without tumors after 7 days of treatment.

Figure 8 shows the plasma concentration clearance study after administration of 10 mg/kg Trastuzumab-SPP-DM1 to 4 test rats. Total antibody and Trastuzumab-SPP-DM1 concentrations were determined. (tr = trastuzumab)

Figure 9 shows the plasma concentration clearance study after administration of 10 mg/kg Trastuzumab-SMCC-DM1 to 4 test rats. Total antibody and trastuzumab-SMCC-DM1 concentrations were determined.

Figure 10 shows the mean tumor volume change over time in mice dosed with: Vehicle (PBS pH 6.5), Trastuzumab-SPP-DM1 (370 μg DM1/m ² ), and Trastuzumab- SMCC-DM1 (330 μg DM1/m ² ), wherein the dose refers to the dose of DM1 administered.

Figure 11 shows the mean tumor volume change over time in athymic nude mice with Fo5 tumor allografts dosed on day 0 with: vehicle (PBS pH 6.5), 10 mg/kg trastuzumab-SIAB -DM1 (3.4DM1/Ab; 168 μg DM1/kg), and 10 mg/kg trastuzumab-SMCC-DM1 (3.2DM1/Ab; 158 μg DM1/kg), where dose refers to the antibody-drug conjugate administered dosage.

Figure 12 shows over time, a single injection of vehicle (PBS pH 6.5), 10 mg/kg trastuzumab-SPP-DM1, 10 mg/kg trastuzumab-SPP-DM4, 10 mg/kg trastuzumab-SPP-DM4, 10 mg/kg trastuzumab- The average tumor volume change of SPP-DM3 and MMTV-Her2 Fo5 beige nude mice (7 in each group, all tumors, Ti=7) with 10 mg/kg trastuzumab-SMCC-DM1.

Figure 13 shows that in HER2-Fo5 tumors, vehicle (PBS pH 6.5), trastuzumab-SPP-DM1, trastuzumab-SPP-DM4, trastuzumab-SPP-DM3, and trastuzumab- Time to tumor volume doubling and logarithmic cell killing analysis of SMCC-DM1.

Figure 14 shows the change in body weight over time in rats dosed with: vehicle (10 mM sodium succinate, 100 mg/mL sucrose, 0.1% Tween 20, pH 5.0), trastuzumab-SPP-DM1 (1860 μg DM1/m ² ), Trastuzumab-SMCC-DM1 (1860 μg DM1/m ² ), Trastuzumab-SMCC-DM1 (3260 μg DM1/m ² ), and No DM1 (650 μg/m ² ).

Figure 15 shows the liver function test values measured in units of AST per liter over time in a rat model dosed with: Vehicle (10 mM sodium succinate, 100 mg/mL sucrose, 0.1% Tween 20, pH 5.0 ), Trastuzumab-SPP-DM1(22.3mg/kg), Trastuzumab-SMCC-DM1(10mg/kg), Trastuzumab-SMCC-DM1(25mg/kg), Trastuzumab- SMCC-DM1 (50 mg/kg), and no DM1.

Figure 16 shows the safety profile of PLT units per liter of cells measured over time in a rat model dosed with: vehicle (10 mM sodium succinate, 100 mg/mL sucrose, 0.1% Tween 20, pH 5.0 ), Trastuzumab-SPP-DM1(22.3mg/kg), Trastuzumab-SMCC-DM1(10mg/kg), Trastuzumab-SMCC-DM1(25mg/kg), Trastuzumab- SMCC-DM1 (50 mg/kg), and no DM1.

Figure 17 shows in vitro cell proliferation assays with HT1080EphB2(C8) cells treated with antibody-drug conjugates: -▲-anti-EphB2R 2H9-SPP-DM1, and -_-anti-EphB2R2H9-SMCC-DM1.

Detailed Description of Exemplary Embodiments

Reference will now be made in detail to specific embodiments of the invention, examples of which are set forth in the accompanying structures and formulae. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are defined in Singleton et al., (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York.

definition

Unless otherwise stated, the following terms and phrases used herein shall have the following meanings:

Where a trade name is used herein, Applicants intend to include independently the preparation of the trade name product, the drug class of the trade name product, and the active pharmaceutical ingredient.

The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired The desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or antibodies derived from other species. Antibodies are proteins produced by the immune system that recognize and bind specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5thEd., Garland Publishing, New York). The target antigen generally has many binding sites recognized by the CDRs of various antibodies, Also called an epitope. Each antibody that specifically binds a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site or a portion thereof that immunospecifically binds an antigen of a target of interest, including but not limited to cancer Cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins may be of any species origin. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.

An "antibody fragment" comprises a portion of a full-length antibody, usually its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; fragments produced from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions) , and any of the above epitope-binding fragments, single-chain antibody molecules that immunospecifically bind cancer cell antigens, viral antigens, or microbial antigens; and multispecific antibodies formed from antibody fragments.

The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, each monoclonal antibody is directed against a single determinant on an antigen, in contrast to a polyclonal antibody preparation that includes different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by recombinant DNA methods (see, US 4816567). For example, monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described by Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597.

Monoclonal antibodies herein specifically include "chimeric" antibodies, as well as fragments of these antibodies, in which a portion of the heavy and/or light chain is identical to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class or subclass of antibodies or homologous, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to other antibody classes or subclasses, provided they exhibit the desired biological activity (US4816567; and Morrison et al (1984 ) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable region antigen-binding sequences derived from a non-human primate (e.g., old world Monkey or Ape) and human constant region sequences.

A "whole antibody" here is an antibody comprising the VL and VH domains, as well as the light chain constant region (CL) and the heavy chain constant regions CH1, CH2 and CH3. The constant region may be a native sequence constant region (eg, a human native sequence constant region) or an amino acid sequence variant thereof. An intact antibody may possess one or more "effector functions," ie, those biological activities attributable to the antibody Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Negative regulation.

Depending on the amino acid sequence of the constant region of their heavy chains, intact antibodies can be assigned to different classes. There are five major intact antibody classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant regions that correspond to the different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

"ErbB receptors" are receptor protein tyrosine kinases that belong to the ErbB receptor family, which are important mediators of cell growth, differentiation and survival. The ErbB receptor family includes four distinct members, including epidermal growth factor receptor (EGFR, ErbB1, HER1 ), HER2 (ErbB2 or p185 ^neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies has been identified with the human breast tumor cell line SKBR3 (Hudziak et al (1989) Mol. Cell. Biol. 9(3): 1165-1172. The greatest inhibition was obtained with an antibody called 4D5, which inhibited The percentage of cell proliferation was 56%. In this test, other antibodies in this group reduced cell proliferation to a lower degree. It was further found that antibody 4D5 sensitized ErbB2 overexpressing breast tumor cell lines to the cytotoxic effects of TNF-α (US5677171). 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; Sliwkowski 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 further identified the anti-ErbB2 antibodies discussed in Hudziak et al.

Franklin et al (2004) Cancer Cell 5: 317-328; Tagliabue et al (1991) Int.J. Cancer 47: 933-937; McKenzie et al (1989) Oncogene 4: 543-548; Maier et al (1991) Cancer Res.51: 5361-5369; Bacus et al (1990) Molecular Carcinogenesis 3: 350-362; Stancovski et al (1991) PNAS (USA) 88: 8691-8695; Bacus et al (1992) Cancer Research 52: 2580-2589 ; Xu et al (1993) Int. J. Cancer 53: 401-408; WO94/00136; Kasprzyk et al (1992) Cancer Research 52: 2771-2776; Hancock 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; US 5783186; Other anti-ErbB2 antibodies with different properties are described in and Klapper et al (1997) Oncogene 14:2099-2109.

Sequence identity screens have identified two other ErbB receptor family members; ErbB3 (US 5,183,884; US 5,480,968; Kraus et al (1989) PNAS (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 receptors show enhanced expression on at least some breast cancer cell lines.

ErbB receptors will typically include an extracellular domain that binds ErbB ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a receptor containing several phosphorylated tyrosine residues. Carboxy-terminal signaling domain. The ErbB receptor may be a "native sequence" ErbB receptor or an "amino acid sequence variant" thereof. The ErbB receptor can be a native sequence human ErbB receptor. Thus, an "ErbB receptor family member" is EGFR (ErbBl), ErbB2, ErbB3, ErbB4 or any other ErbB receptor currently known or to be identified in the future.

The terms "ErbB1", "epidermal growth factor receptor", "EGFR" and "HER1" are used interchangeably herein to refer to EGFR as disclosed, for example, in Carpenter et al (1987) Ann. Rev. Biochem. 56:881-914 , including its naturally occurring mutant forms (eg, the deletion mutant EGFR in Humphrey et al., (1990) PNAS (USA), 87:4207-4211). The term erbB1 refers to the gene encoding the EGFR protein product. For example, Murthy et al (1987) Arch. Biochem. Biophys., 252:549-560 and WO 95/25167 describe antibodies against HER1.

The terms "ERRP", "EGF-receptor-related protein", "EGFR-related protein" and "epidermal growth factor receptor-related protein" are used interchangeably herein to refer to ERRP as disclosed, for example, in US 6399743 and US2003/0096373.

The terms "ErbB2" and "HER2" are used interchangeably herein to refer to, for example, Semba et al (1985) PNAS (USA), 82: 6497-6501 and Yamamoto et al (1986) Nature, 319: 230-234 (Genbank Access No. X03363) human HER2 protein described in. The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to the gene encoding rat pl85neu.

"ErbB3" and "HER3" refer to the receptor polypeptides disclosed in, eg, US 5183884; US 5480968; Kraus et al (1989) PNAS (USA) 86:9193-9197. Antibodies against ErbB3 are known in the art (US 5183884; US 5480968; WO 97/35885).

The terms "ErbB4" and "HER4" here refer to, for example, EP Patent Application 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366: 473-475 (1993), including isoforms thereof, disclosed in, for example, WO 99/19488. Antibodies against HER4 are described, for example, in WO 02/18444.

Antibodies to the ErbB receptor are commercially available from a number of sources including, for example, Santa Cruz Biotechnology, Inc., California, USA.

"ErbB ligand" means a polypeptide that binds and/or activates an ErbB receptor. The ErbB ligand can be a native sequence human ErbB ligand 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 keratinocyte autocrine growth factor (Shoyab et al (1989) Science 243 : 1074-1076; Kimura et al (1990) Nature348: 257-260; Cook et al (1991) Mol.Cell.Biol., 11: 2547-2557); Betacellulin (betacellulin) (Shing et al (1993) Science 259: 1604-1607; Sasada et al (1993) Biochem. Biophys. Res. Commun. 190: 1173); Heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al (1991) Science 251: 936-939) ; epiregulin (Toyoda et al (1995) J.Biol.Chem.270: 7495-7500; Komurasaki et al (1997) Oncogene 15: 2841-2848); heregulin (see below); neuregulin-2 ( NRG-2) (Carraway et al., Nature, 387:512-516 (1997)); Neuregulin-3 (NRG-3) (Zhang et al (1997) Proc.Natl.Acad.Sci., 94: 9562-9567); neuregulin-4 (NRG-4) (Harari et al (1999) Oncogene, 18: 2681-89) or cripto (CR-1) (Kannan et al (1997) J.Biol.Chem. , 272(6):3330-3335). ErbB ligands that bind EGFR include EGF, TGF-α, amphiregulin, β-cytokinin, HB-EGF, and epiregulin. ErbB ligands that bind ErbB3 include heregulin. ErbB ligands capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4, and heregulin. The ErbB ligand may also be a synthetic ErbB ligand. Synthetic ligands can be specific for a particular ErbB receptor, or can recognize a particular ErbB receptor complex. An example of a synthetic ligand is the synthetic heregulin/EGF chimera biregulin (see, e.g., Jones et al (1999) FEBS Letters, 447:227-231, which is incorporated by reference).

"Heregulin" (HRG) refers to the polypeptide encoded by the heregulin gene product disclosed in US 5641869 or Marchionni et al (1993) Nature 362:312-318. Examples of heregulins include heregusin-α, heregusin-β1, heregusin-β2, and heregusin-β3 (Holmes et al (1992) Science 256:1205-1210; and US 5641869); neu differentiation factor (NDF) (Peles et al (1992) Cell 69: 205-216); Acetylcholine receptor-inducing activity (ARIA) (Falls et al (1993) Cell 72: 801-815); Glial growth factors (GGFs) (Marchionni et al (1993 ) Nature, 362:312-318); sensory and motor neuron-derived factor (SMDF) (Ho et al (1995) J.Biol.Chem.270: 14523-14532); γ-heregulin (Schaefer et al (1997 ) Oncogene, 15:1385-1394). The term includes biologically active fragments and/or amino acid sequence variants of native sequence HRG polypeptides, such as EGF-like domain fragments thereof (eg, HRGβ1177-244).

An "ErbB hetero-oligomer" is a non-covalently associated oligomer comprising at least two different ErbB receptors. An "ErbB dimer" is a non-covalently bound oligomer comprising two different ErbB receptors. Cells expressing two or more ErbB receptors can form this complex when exposed to ErbB ligands. For example ErbB oligomers, such as ErbB dimers, can be isolated by immunoprecipitation and SDS-PAGE analysis as described in Sliwkowski et al (1994) J. Biol. Chem., 269(20):14661-14665. Examples of such ErbB hetero-oligomers include EGFR-ErbB2 (also known as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes. In addition, ErbB hetero-oligomers may comprise two or more ErbB2 receptors in combination with different ErbB receptors such as ErbB3, ErbB4 or EGFR (ErbB1). Other proteins such as cytokine receptor subunits (eg, gp130) can be included in the hetero-oligomer.

"Ligand activation of an ErbB receptor" means signal transduction mediated by the binding of an ErbB ligand to an ErbB hetero-oligomer comprising an ErbB receptor of interest (e.g., by an ErbB receptor or a substrate polypeptide caused by phosphorylation of the intracellular kinase domain of tyrosine residues in ErbB receptors). This usually involves the binding of an ErbB ligand to an ErbB hetero-oligomer, which activates the kinase domain of one or more ErbB receptors in the hetero-oligomer, thereby resulting in Phosphorylation of tyrosine residues and/or phosphorylation of tyrosine residues in other substrate polypeptides. ErbB receptor activation can be quantified with a variety of tyrosine phosphorylation assays.

A "native sequence" polypeptide is one that has the same amino acid sequence as a polypeptide derived from nature (eg, ErbB receptor or ErbB ligand). Such native sequence polypeptides can be isolated from nature, or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide may have the amino acid sequence of a naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.

The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs in some way from a native sequence polypeptide. Typically, the amino acid sequence variant will have at least about 70% sequence identity to at least one receptor binding domain of a native ErbB ligand or to at least one ligand binding domain of a native ErbB receptor, or to such receptor Or the ligand binding domains are at least about 80%, or at least about 90% homologous. The amino acid sequence variants have substitutions, deletions, and/or insertions at positions within the amino acid sequence of the native amino acid sequence.

"Sequence identity" is defined as the percentage of residues in amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to maximize the percentage sequence identity. Methods and computer programs for such alignments are well known in the art. One such computer program is "Align 2," designed by Genentech, Inc., which was filed with the United States Copyright Office, Washington, DC 20559 on December 10, 1991 with user documentation.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to cell-mediated responses in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g. natural killer (NK) cells, neutrophils , and macrophages) recognize bound antibodies on target cells, which subsequently leads to lysis of the target cells. NK cells, the main cells that mediate ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol, 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed (US 55003621; US 5821337). Useful effector cells for this test include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al (1998) PNAS (USA), 95:652-656.

"Maytansinoid drug moiety" means a substructure of an antibody-drug conjugate having the structure of a maytansinoid compound. Maytansine was first isolated from the East African shrub Maytenus serrata (US 3896111). It was later found that certain microorganisms also produce maytansinoids such as maytansinol and C-3 maytansinol esters (US 4151042). Synthetic maytansinol and maytansinol analogs have been reported.参见美国专利4137230；4248870；4256746；4260608；4265814；4294757；4307016；4308268；4308269；4309428；4313946；4315929；4317821；4322348；433 1598；4361650；4364866；4424219；4450254；4362663；和4371533，以及Kawai et al (1984) Chem. Pharm. Bull. 3441-3451), both expressly incorporated by reference.

The term "Fc receptor" or "FcR" is used to describe a receptor, such as a native sequence human FcR, that binds the Fc region of an antibody. FcRs can bind IgG antibodies (gamma receptors), including receptors FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants of these receptors and differently spliced forms of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review M. in Da_ron, Annu. Rev. Immunol., 15:203-234 (1997)). Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991); Capel et al (1994) Immunomethods, 4: 25-34; and de Haas et al (1995) J. Lab. Clin. Med. FcRs are reviewed in , 126:330-41. Herein the term "FcR" includes other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al (1976) J. Immunol. 117:587, and Kim et al (1994) J. Immunol. 24:249).

"Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to molecules (eg, antibodies) complexed with the relevant antigen. To assess complement activation, a CDC assay, such as that described by Gazzano-Santoro et al (1996) J. Immunol. Methods, 202:163, can be performed.

"Native antibodies" are typically heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, however the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by a number of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end. The constant region of the light chain is juxtaposed with the first constant region of the heavy chain, and the variable region of the light chain is juxtaposed with the variable region of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable regions.

The term "variable" refers to certain portions of the variable regions of antibodies that vary widely in sequence for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable regions of all antibodies. In both the light and heavy chain variable regions, attention has been focused on three segments called hypervariable regions. The more conserved portions of the variable domains are called the framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, largely adopting a β-sheet conformation, connected by three hypervariable regions, which form loops linking and sometimes forming β-sheet structures a part of. The hypervariable regions in each chain are very tightly bound together by FRs and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). The constant regions are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions typically comprise amino acid residues from "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 in the light chain variable region (L3) and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region; Kabat et al supra) and/or those residues from the "hypervariable loop" (e.g. residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable region and 26-32(H1), 53-55 in the heavy chain variable region (H2) and 96-101 (H3); Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). "Framework region" or "FR" residues are those variable region residues other than the hypervariable region residues defined herein.

Papain digestion of antibodies yields two identical antigen-binding fragments, termed "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of conjugating antigen.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of tightly, non-covalently associated variable regions of one heavy chain and one light chain. It is this conformation in which the three hypervariable domains of each variable domain interact defines the antigen-binding site on the surface of the VH-VL dimer. Together, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable domains specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the full binding site.

The Fab fragment also contains the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the CH1 region of the heavy chain, including one or more cysteines from the antibody hinge region. Fab' having at least one free thiol group in the cysteine residue of the constant region is referred to herein as Fab'-SH. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between the Fab' fragments. Other chemical conjugates of antibody fragments are also known.

Based on the amino acid sequence of its constant region, the "light chain" of an antibody from any vertebrate species can be assigned to one of two clearly defined types called kappa (κ) and lambda (λ).

"Single-chain Fv" or "scFv" means a single-chain variable region antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form a structure required for antigen binding (Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer- Verlag, New York, pp.269-315 (1994). Anti-ErbB2 antibody scFv fragments are described in WO 93/16185; US 5571894; US 5587458.

The term "diabodies" refers to small antibody fragments with two antigen binding sites comprising a heavy chain variable region (VH-VL) linked to a light chain variable region (VL) in the same polypeptide chain (VH-VL). VH). By using a linker sequence that is too short to allow pairing of the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain, creating two antigen-binding sites. Diabodies are described in more detail in eg EP404,097; WO 93/11161; and Hollinger et al (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448.

"Humanized" forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are, for the most part, human immunoglobulins (recipient antibody) in which residues from hypervariable regions of the recipient are replaced by residues from hypervariable regions of a non-human species (donor antibody), The non-human species is, for example, a mouse, rat, rabbit, or non-human primate having the desired specificity, avidity, and capabilities. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications were constructed to further improve antibody performance. In general, humanized antibodies will comprise substantially all, or at least one, and typically two variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin. loop, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al (1986) Nature, 321: 522-525; Riechmann et al (1988) Nature 332: 323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2: 593-596 .

Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 described in Table 3 of US 5821337 (herein expressly incorporated by reference) and huMAb4D5-8 (HERCEPTIN®, trastuzumab); humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies.

An "isolated" antibody refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to materials that would interfere with the diagnostic or therapeutic use of an antibody, and may include enzymes, hormones, and other protein or nonprotein solutes. The antibody can be purified to (1) greater than 95% by weight of the antibody as determined by the Lowry method, (2) to at least 15% of the N-terminal or internal amino acid sequence sufficient to obtain an N-terminal or internal amino acid sequence as determined by a spinning cup protein sequencer. residues, or (3) perform SDS-PAGE under reducing or non-reducing conditions and stain with Coomassie blue or silver to achieve homogeneity. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

An antibody "which binds" an antigen of interest, eg, the ErbB2 antigen, is one that is capable of binding that antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen. Wherein said antibody refers to an antibody that binds ErbB2, as opposed to other ErbB receptors, it will generally bind ErbB2 preferentially, and also refers to an antibody that does not significantly cross-react with other proteins such as EGFR, ErbB3 or ErbB4. In such embodiments, the antibody binds to these non-ErbB2 proteins (e.g., cell surface proteins that bind endogenous receptors) as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). The degree will be less than 10%. Occasionally, anti-ErbB2 antibodies do not significantly cross-react with rat neu protein, as described, for example, in Schecter et al (1984) Nature 312:513, and Drebin et al (1984) Nature, 312:545-548.

An antibody that "blocks" ligand activation of the ErbB receptor reduces or prevents such activation, wherein the antibody is substantially more effective at blocking ligand activation of the ErbB receptor than monoclonal antibody 4D5, for example, About as potent as monoclonal antibodies 7F3 or 2C4 or their Fab fragments. For example, an antibody that blocks ligand activation of the ErbB receptor may be an antibody that is about 50-100% more effective at blocking ErbB hetero-oligomer formation than 4D5. Blocking of ligand activation of ErbB receptors can occur in any way, for example, by interfering with: ligand binding to ErbB receptors, ErbB complex formation, tyrosine kinase activity of ErbB receptors in ErbB complexes and/or ErbB Phosphorylation of tyrosine kinase residues in or by ErbB receptors.

An antibody having the "biological characteristics" of a given antibody, such as monoclonal antibody 2C4 (Omnitarg, Genentech, Inc.), refers to an antibody that has one or more biological characteristics of the antibody that distinguish it from Other antibodies that bind the same antigen (eg, ErbB2) are distinguished. For example, antibodies with biological characteristics of 2C4 can block HRG activation of ErbB hetero-oligomers including ErbB2 and ErbB3, ErbB1 or ErbB4; block EGF, TGF-α, HB receptors of ErbB receptors including EGFR and ErbB2; - EGF, epiregulin, and/or amphiregulin activation; block EGF, TGF-α, and/or HRG-mediated activation of MAPK; and/or bind to the same epitope in the extracellular domain of ErbB2 as 2C4 binds (eg, an antibody that blocks the binding of monoclonal antibody 2C4 to ErbB2).

Unless otherwise indicated, the expression "monoclonal antibody 2C4" refers to an antibody having antigen-binding residues of, or derived from, the murine 2C4 antibody as exemplified below. For example, monoclonal antibody 2C4 can be murine monoclonal antibody 2C4 or a variant thereof, such as humanized antibody 2C4, which has the antigen-binding amino acid residues of murine monoclonal antibody 2C4 (WO 01/00245). Unless otherwise indicated, when the expression "rhuMAb 2C4" is used herein, it refers to an antibody comprising the light chain variable region (VL) and heavy chain variable region (VH) sequences of SEQ ID Nos. 3 and 4, respectively, said Light chain variable region (VL) and heavy chain variable region (VH) sequences fused to human light and heavy chain IgG1 (non-A isotype) optionally expressed by Chinese hamster ovary (CHO) cells (WO 01/00245) isotype) constant region sequence.

Unless otherwise indicated, the term "monoclonal antibody 4D5" refers to an antibody having antigen-binding residues of, or derived from, the murine 4D5 antibody (ATCC CRL10463). For example, monoclonal antibody 4D5 can be murine monoclonal antibody 4D5 or a variant thereof, such as humanized 4D5, which has the antigen-binding residues of murine monoclonal antibody 4D5. Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (HERCEPTIN®) in US 5821337.

"Growth inhibitory agent"refers to a compound or composition that inhibits the growth of a cell, eg, an ErbB-expressing cancer cell, in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of ErbB expressing cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (in a non-S phase position), such as agents that induce G1 arrest and M-phase arrest (The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13). Examples of "growth inhibitory" antibodies are those antibodies that bind ErbB2 and inhibit the growth of ErbB2-overexpressing cancer cells. At an antibody concentration of about 0.5 to 30 μg/ml, the growth inhibitory anti-ErbB2 antibody can inhibit the growth of Sk-Br-3 breast tumor cells in cell culture by more than 20%, or more than 50% (for example, from about 50% to about 100%), where growth inhibition was determined six days after Sk-Br-3 cells were exposed to the antibody (US5677171).

An antibody that "induces cell death" refers to an antibody that causes a living cell to become non-viable. The cells are typically cells expressing ErbB2 receptors, especially cells overexpressing ErbB2 receptors. The cells may be cancer cells, eg, breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cells. In vitro, the cells may be Sk-Br-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cells. Cell death can be measured in vitro in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, cell death assays can be performed with heat-inactivated serum (ie, in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody is capable of inducing cell death, loss of membrane integrity relative to untreated cells can be assessed by means of propidium iodide (PI), trypan blue (see Moore et al. 1995) Cytotechnology, 17:1-11) or 7AAD uptake assessment. Antibodies that induce cell death refer to those antibodies that induce PI uptake in the PI uptake assay of BT474 cells (see below).

An antibody that "induces apoptosis" refers to an antibody that induces programmed cell death through annexin V binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum swelling, cell fragmentation, and/or membrane vesicle formation (called Apoptotic body) assay. The cells are typically cells that overexpress the ErbB2 receptor, including tumor cells, eg, breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cells. In vitro, the cells may be Sk-Br-3, BT474, Calu 3 cells, MDA-MB-453, MDA-MB-361 or SKOV3 cells.

The terms "treat" or "treatment" both refer to therapeutic treatment and preventive or preventive measures, wherein the object is to arrest or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in extent of disease, stabilization of disease status (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease status , and symptom relief (whether partial or total), whether detectable or undetectable. "Treatment" can also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those susceptible to having the condition or disorder, or those in which the condition or disorder is to be prevented.

"Disorder" refers to any condition that would benefit from treatment of the present invention. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose mammals to such disorders. Non-limiting examples of disorders to be treated herein include benign and malignant tumors; leukemia and lymphoid malignancies, especially breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreas, prostate or bladder carcinoma; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunological disorders. Exemplary disorders according to the invention to be treated are solid malignancies.

The term "therapeutically effective amount" refers to an amount of drug effective to treat a disease or disorder in a mammal. As far as cancer is concerned, the therapeutically effective dose of the drug can: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, block, and slow down the infiltration of cancer cells into peripheral organs to a certain extent and preferably terminate; (iv) inhibit (ie slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; and/or (vi) alleviate to some extent one or more symptoms associated with cancer. To the extent a drug is able to prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. In animal models, efficacy can be assessed by physically measuring tumors during the course of disease after ADC administration, and by measuring partial and complete regression of tumors. For cancer therapy, efficacy can be determined, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The term "bioavailability" refers to the systemic availability (ie, blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term referring to a measure of the time (rate) and total amount (extent) of a drug reaching the systemic circulation from an administered dosage form.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals typically characterized by unrestrained cell growth. A "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 include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma ( squamous carcinoma), peritoneal carcinoma, hepatocellular carcinoma, gastric or gastric cancer including gastrointestinal cancer, gastrointestinal stromal tumor (GIST), pancreatic cancer, glioblastoma ), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer , kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer (hepatic carcinoma), anal cancer, penile cancer, and head and neck cancer.

An "ErbB-expressing cancer" refers to a cancer comprising cells that have ErbB protein present on their cell surface. An "ErbB2-expressing cancer" refers to a cancer that produces sufficient levels of ErbB2 on its cell surface so that an anti-ErbB2 antibody can bind to it and have a therapeutic effect against the cancer.

A cancer that "overexpresses" a receptor, such as an ErbB receptor, refers to a cancer that has significantly higher levels of the receptor on its cell surface compared to non-cancerous cells of the same tissue type. Such overexpression can result from gene proliferation or enhanced transcription or translation. ErbB receptor overexpression can be determined in diagnostic or prognostic tests by assessing elevated ErbB protein levels on the cell surface (eg, by immunohistochemical tests; IHC). Alternatively, or in addition, the concentration of β in cells can be determined by, for example, fluorescence in situ hybridization (FISH; see WO 98/45479), southern blotting, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR). Levels of nucleic acid encoding ErbB. Overexpression of an ErbB ligand can be determined diagnostically by assessing the level of said ligand (or nucleic acid encoding it) in a patient, for example, in a tumor biopsy or by various diagnostic tests such as IHC, FISH, southern blot, PCR or in vivo testing. ErbB receptor overexpression can also be studied by assaying shed antigen (e.g., ErbB extracellular domain) in biological fluids such as serum (see, e.g., US4933294; WO 91/05264; US 5401638; and Sias et al (1990) J. Immunol. Methods 132:73-80). In addition to the tests described above, a variety of other in vivo tests are available to the skilled artisan. For example, cells in a patient can be contacted with an antibody, optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to the patient's cells can be assessed, e.g., by radioactive external scanning, or by assaying live A tissue biopsy taken from a patient who has been previously exposed to the antibody.

Tumors overexpressing HER2 are graded by immunohistochemical scoring corresponding to the number of copies of the HER2 molecule expressed per cell, and biochemical assays can be performed: 0 = 0-10,000 copies/cell, 1+ = at least approx. 200,000 copies/cell, 2+ = at least about 500,000 copies/cell, 3+ = about 1-2 x ¹⁰⁶ copies/cell. Overexpression of HER2 at 3+ levels results in ligand-independent activation of tyrosine kinase (Hudziak et al., (1987) Proc. Natl. Acad. Sci. USA 84:7159-7163), which occurs in approximately 30% of In breast cancer, and in these patients, relapse-free survival and overall survival are reduced (Slamon et al (1989) Science 244:707-712; Slamon et al (1987) Science, 235 : 177-182). In contrast, a cancer "not characterized by overexpression of ErbB2 receptors" is a cancer that does not express higher than normal levels of ErbB2 receptors in diagnostic testing compared to non-cancerous cells of the same tissue type. Murine monoclonal anti-HER2 antibody inhibits growth of breast cancer cell lines that overexpress HER2 at 2+ and 3+ (1-2 x ¹⁰⁶ HER2 receptors per cell), but less Cells with low levels of HER2 are inactive (Lewis et al (1993) Cancer Immunol. Immunother. 37:255-263). Based on this observation, the antibody 4D5 was humanized (huMAb4D5-8, rhuMAb HER2, US 5821337; Carter et al (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289), and tested in breast cancer patients tested, the breast cancer patient's tumor overexpressed HER2, but it progressed after conventional chemotherapy (Cobleigh et al (1999) J. Clin. Oncol. 17:2639-2648). In this trial, most patient tumors expressed HER2 at 3+ levels, while a fraction were 2+.

A "hormone-independent" cancer is one whose proliferation does not depend on the presence of hormones that bind receptors expressed by cancer cells. Such cancers do not undergo clinical regression when pharmacological or surgical strategies are implemented to attenuate hormone concentrations in or near the tumor. Examples of hormone-independent cancers include androgen-independent prostate cancer, estrogen-independent breast cancer, endometrial cancer, and ovarian cancer. Such cancers can start as hormone-dependent tumors and progress from hormone-sensitive to hormone-resistant tumors after antihormonal therapy.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular functions and/or causes cellular destruction. The term will include radioisotopes (such as ²¹¹ At, ^{131 I} , ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, 188 Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ⁶⁰ C, and Lu radioisotopes), chemotherapeutic agents, and Toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

A "chemotherapeutic agent" is a compound used in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA_, Genentech/OSI Pharm.), Bortezomib (VELCADE_, Millenium Pharm.), Fulvestrant (FASLODEX_, Astrazeneca), Sutent (SU11248, Pfizer), letrozole ) (FEMARA_, Novartis), Imatinib mesylate (GLEEVEC_, Novartis), PTK787/ZK222584 (Novartis), Oxaliplatin (Eloxatin_, Sanofi), 5-FU (5-fluorouracil), formyl tetra Leucovorin, Rapamycin (Sirolimus, RAPAMUNE_, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA_, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN_cyclophosphamide; alkyl sulfonates such as busulfan ), improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uridine uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, Triethiylenethiophosphoramide and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (camptothecin) (including the synthetic analogs topotecan; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin) (bizelesin) synthetic analogues); cryptophycin (especially cryptophyllin 1 and cryptophyllin 8); dolastatin (dolastatin); duocarmycin (including synthetic analogues, KW-2189 and CB1 -TM1); eleutherobin; pancratisatin; sarcodictyn; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide , estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, Phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosurea such as carmustine , chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediynes (enediyne) antibiotics (such as calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see e.g. Agnew, Chem.Intl.Ed.Engl.33:183-186, 1994 )); anthracyclines, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin Chromophores and related chromophores (enediyne antibiotics (chromophores), aclacinomysin, actinomycin, athramycin, azaserine, blem Bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, soft red Daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN_ doxorubicin (including morpholine doxorubicin, cyanomorph Adriamycin, 2-pyrroline doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin , mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, Puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubene ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as di Denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiametidine Purine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azuridine, carmofur (carmofur), cytarabine ( cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, hydroxyandrosterone propionate (dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trolosteine (trilostane); folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine); bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eifornithine; eiiiptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and maytansinol Ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin ( pirarubicin); losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK_polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane ( urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C");cyclophosphamide;thiotepa; taxoids such as TAXOL_paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ^™ , paclitaxel without cremophor (Cremophor) albumin modified nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE_docetaxel (doxetaxel) (Rhéne-Poulenc Rorer, Antony, France); Chloranbucil; GEMZAR_gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etopol Glycoside (etoposide, VP-16); Ifsfamide; Mitoxantrone; Vincristine; NAVELBINE_vinorelbine; Novantrone; Tenipo teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor The agent RFS 2000; Difluoromethylornithine (DMFO); Retinoids (retinoids), such as retinoic acid; Capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the above agents thing.

The definition "chemotherapeutic agent" also includes (i) antihormonal agents that modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including for example tamoxifen (including NOLVADEX_ tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene; (ii) an aromatase inhibitor that inhibits the aromatase that regulates estrogen production in the adrenal gland, such as, for example, 4( 5)-Imidazole, aminoglutethimide, MEGASE_megestrol acetate, AROMASIN_exemestane, formestanie, fadrozole, RIVISOR _vorozole, FEMARA_letrozole, and ARIMIDEX_anastrozole; (iii) antiandrogens such as flutamide, nilutamide ), bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog ); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, especially those that inhibit signaling pathways involved in abnormal cell proliferation Antisense oligonucleotides for gene expression, such as, for example, PKC-alpha, Raf and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (such as ANGIOZYME_ribozyme) and HER2 expression inhibitors; (ix) vaccines , such as gene therapy vaccines, such as ALLOVECTIN_vaccine, LEUVECTIN_vaccine, and VAXID_vaccine; PROLEUKIN_rIL-2; LURTOTECAN_topoisomerase 1 inhibitor; ABARELIX_rmRH; (x) antiangiogenic agents such as bevacizumab (bevacizumab ) (AVASTIN®, Genentech); and (xi) a pharmaceutically acceptable salt, acid or derivative of any of the above.

Protein kinase inhibitors include tyrosine kinase inhibitors, which inhibit, to some extent, the tyrosine kinase activity of tyrosine kinases such as ErbB receptors. Examples of tyrosine kinase inhibitors include EGFR-targeted drugs such as: (i) antibodies that bind EGFR, including MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCCCRL 8509) (see, US 4943533, Mendelsohn et al.) and variants thereof, such as chimeric 225 (C225 or Cetuximab; ERBITUX_, Imclone) and reshaped human 225 (H225) ( WO 96/40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (US 5212290); humanized and chimeric antibodies that bind EGFR (US 5891996); and human antibodies that bind EGFR, such as ABX-EGF ( WO 98/50433); (ii) anti-EGFR antibodies conjugated to cytotoxic agents (EP 659439A2); and small molecules that bind EGFR include ZD1839 or Gefitinib (IRESSA ^™ ; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA ^TM ; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen), quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline (4-(3-chloroanilino)quinazoline), pyridopyrimidine ( pyridopyrimidines), pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidine, 4-(anilino)-7H-pyrrolo[2,3-d]pyrimidine (4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines), curcumin (diferuloyl methane), 4,5-bis(4-fluoroaniline) phthalimide ) (4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Wamer-Lambert); antisense molecules (eg, those that bind to ErbB-encoding nucleic acids) ; quinoxalines (quinoxalines) (US 5804396); tryphostins (US 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); 3521; Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib ( Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as US 5804396; WO 99/09016 (American Cyanamid); WO98/43960 (American Cyanamid); 38983 (Warner Lambert); WO99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); /33980 (Zeneca).

"Anti-angiogenic agent" refers to a compound that in some way blocks, or interferes with, the development of blood vessels. Anti-angiogenic factors can be, for example, small molecules or antibodies that bind growth factors or growth factor receptors involved in promoting angiogenesis. Exemplary anti-angiogenic agents are antibodies that bind vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®, Genentech).

The term "cytokine" is a generic term for proteins released by one population of cells that act as intercellular mediators in another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included in the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone (N-methionyl human growth hormone), and bovine growth hormone; parathyroid hormone; thyroxine; insulin; insulin Pro; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; gonadotropin-associated peptide); inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; Transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); osteoinductive factors (osteoinductive factors); -β, and -γ; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); Interleukins (ILs) 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). The term cytokine as used herein includes proteins of natural origin or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or transformed into a more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchard et al., (ed.), pp.247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate (ester)-containing prodrugs, thiophosphate (ester)-containing prodrugs, sulfate (ester)-containing prodrugs, peptide-containing prodrugs, and D-amino acid modified prodrugs , glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluoro Cytosine and other 5-fluorouridine prodrugs that can be converted to more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized in prodrug form for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.

"Liposomes" are vesicles composed of various types of lipids, phospholipids, and/or surfactants that can be used to deliver drugs, such as the anti-ErbB2 antibodies and optionally chemotherapeutic agents disclosed herein, to mammals. things. The components of liposomes are usually arranged in a bilayer structure, similar to the lipid arrangement of biological membranes.

The term "package insert" is used to mean the instructions customarily included in commercial packages of therapeutic products which contain information on the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

"Cardioprotectant" refers to a compound or composition that prevents or attenuates myocardial dysfunction (cardiomyopathy and/or congestive heart failure) that is associated with the administration of drugs such as anthracycline antibiotics and/or Anti-ErbB2 antibodies are associated. The cardioprotective agent may eg block or attenuate free radical mediated cardiotoxic effects and/or prevent or attenuate oxidative stress damage. Examples of cardioprotective agents encompassed by this definition include the iron chelator dexrazoxane (ICRF-187) (Seifert et al., The Annals of Pharmacotherapy, 28:1063-1072 (1994)); lipid-lowering agents And/or antioxidants such as probucol (probucol) (Singalet al., J.Mol.Cell Cardiol., 27:1055-1063 (1995)); Amifostine (aminothiol 2-[(3-aminopropyl Amifostine (aminothiol2-[(3-aminopropyl)amino]ethanethiol-dihydrogen phosphate ester), also known as WR-2721 and its dephosphorylated cellular uptake known as WR-1065 form) and S-3-(3-methylaminopropylamino)propylphosphorothioic acid (S-3-(3-methylaminopropylamino)propylphosphorothioic acid), (WR-151327), see Green et al., (1994) Cancer Research, 54:738-741; digoxin (digoxin) (Bristow, M.R.ed. (1980) Drug-Induced Heart Disease. NewYork: Elsevier 191-215); beta-blockers such as metoprolol (metoprolol ) (Hjalmarson et al (1994) Drugs 47: Suppl 4: 31-9; and Shaddy et al (1995) Am. Heart J., 129: 197-9); vitamin E; ascorbic acid (vitamin C); free radical scavengers Such as oleanolic acid (oleanolic acid), ursolic acid (ursolic acid) and N-acetyl cysteine (NAC); spin trapping (spin trapping) compounds such as α-phenyl-tert-butylnitrone (alpha- phenyl-tert-butyl nitrogen) (PBN) (Paracchini et al (1993) Anticancer Res., 13: 1607-1612); selenized organic compounds (selenoorganic compounds) such as P251 (Elbesen); and the like.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is commonly associated in antibody nucleic acids of natural origin. An isolated nucleic acid molecule refers to a nucleic acid molecule in a form or composition other than that found in nature. Isolated nucleic acid molecule thus refers to a nucleic acid molecule other than that found in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express an antibody, eg, where the nucleic acid molecule is at a different chromosomal location than in natural cells.

"Alkyl"refers to a _C1 - _C18 hydrocarbon containing normal, secondary, tertiary or ring carbon atoms. Examples of alkyl groups include C1-C8 hydrocarbon moieties such as but not limited to: 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, i-propyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, s-butyl, -CH(CH ₃ ) CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH ₃ ) ₃ ), 1-pentyl ( n-pentyl, -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-hexyl (-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" refers to a C ₂ -C ₁₈ hydrocarbon, containing normal, secondary, tertiary or ring carbon atoms, having at least one site of unsaturation, ie, a carbon-carbon sp ² double bond. Examples of alkenyl groups include _C2 - _C8 hydrocarbon moieties such as but not limited to: ethylene or vinyl (-CH= _CH2 ), allyl ( _-CH2CH = _CH2 ), cyclopentenyl ( -C ₅ H ₇ ), and 5-hexenyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH=CH ₂ ).

"Alkynyl" refers to a _C2 - _C18 hydrocarbon, containing normal, secondary, tertiary or ring carbon atoms, with at least one site of unsaturation, ie, a carbon-carbon sp triple bond. Examples of alkynyl groups include C2-C8 hydrocarbon moieties such as, but not limited to, acetylene (-C≡CH) and propargyl ( _-CH2C≡CH ).

"Alkylene" means a saturated, branched or straight chain or cyclic hydrocarbon group of 1 to 18 carbon atoms and having two monovalent Alkanes derived from the same or two different carbon atoms by removing two hydrogen atoms. Typical alkylene groups include C ₁ -C ₈ hydrocarbon moieties such as but 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 -), and the like.

"Alkenylene" means an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms and having two monovalent radical centers which pass through Derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkenes. Typical alkenylene groups include _C2 - _C8 hydrocarbon moieties such as, but not limited to, 1,2-ethene (-CH(CH-).

"Alkynylene" means an unsaturated, branched or straight chain or cyclic hydrocarbon group of 2 to 18 carbon atoms and having two monovalent Derived from the same or two different carbon atoms of the parent alkyne by removing two hydrogen atoms. Typical alkynylene groups include C ₂ -C ₈ hydrocarbon moieties such as, but not limited to: acetylene (-C≡C-), propargyl (-CH ₂ C≡C-), and 4-pentynyl (-CH _{2CH2CH2C≡C- ) .}

"Aryl", alone or in combination, refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms, derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl groups may contain one, two or three rings, where such rings may be attached together in a pendent manner, such as biphenyl, or may be fused, such as naphthalene or anthracene. Some arene groups are represented by the exemplary structure of "Ar". Typical arene groups include _C6 - _C12 hydrocarbon moieties such as, but not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, and the like.

"Arylalkyl" refers to an acyclic alkyl group in which one hydrogen atom bonded to a carbon atom, typically a terminal or sp ³ carbon atom, is replaced by an aryl group. Typical aralkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-styryl-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthyneethynyl -1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl (2-naphthophenylethan-1-yl) and the like. An aralkyl group containing 6 to 20 carbon atoms, for example an alkanoyl, alkenyl or alkynyl group having 1 to 6 carbon atoms in the alkyl portion of the aralkyl group and 5 to 14 carbon atoms in the aryl portion .

"Heteroarylalkyl" refers to an acyclic alkyl group in which one hydrogen atom bonded to a carbon atom, typically a terminal or sp ³ carbon atom, is replaced by an aromatic heterocycle. Typical aromatic heterocycloalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl and the like. Arylheterocycloalkyl containing 6 to 20 carbon atoms, for example, the alkyl part of aromatic heterocycloalkyl includes alkanyl, alkenyl or alkynyl of 1 to 6 carbon atoms, and the aromatic heterocyclic part is 5 to 14 carbons atoms, and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaromatic ring portion of the heteroaryl cycloalkyl group can be monocyclic (2 to 6 carbon atoms) having 3 to 7 ring members or bicyclic (4 to 9 carbon atoms) having 7 to 10 ring members (4 to 9 carbon atoms, and 1 to 3 heteroatoms selected from N, O, P, and S), for example: bicyclic [4,5], [5,5], [5,6], or [6,6] systems.

Alkyl, alkenyl, aryl, aralkyl, and heteroarylcycloalkyl groups may be substituted, wherein one or more hydrogen atoms are each independently replaced by a substituent. Typical substituents include but are not limited to -X, -R, -O ^- , -OR, -SR, -S ^- , -NR ₂ , -NR ₃ , =NR, -CX ₃ , -CN, -OCN, - SCN, -N=C=O, -NCS, -NO, -NO ₂ , =N ₂ , -N ₃ , -NC(=O)R, -C(=O)R, -C(=O)NR ₂ , -SO ₃ ^- , -SO ₃ H, -S(=O) ₂ R, -OS(=O) ₂ OR, -S(=O) ₂ NR, -S(=O)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 , wherein each X is independently halogen: F, Cl, Br, or I; each R is independently H, C ₁ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₄ heterocyclic protecting group or prodrug moiety. The aforementioned alkenyl, alkylene, and alkynylene groups may also be substituted.

"Heteroaromatic ring" also known as heterocycle or heterocyclyl refers to a ring system group in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur. The aromatic heterocyclic group contains 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaromatic ring portion of the heteroaryl cycloalkyl group can be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a ring having 7 Bicyclic rings of up to 10 ring members (4 to 9 carbon atoms, and 1 to 3 heteroatoms selected from N, O, P, and S), e.g.: bicyclic [4,5], [5,5 ], [5,6], or [6,6] systems. Aromatic heterocyclic compounds are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A.Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Chemistry" Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950-present), especially volumes 13, 14, 16, 19, and 28; and J.Am.Chem.Soc. (1960) 82:5566 .

"Linker" or "linkage" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently links the antibody to the drug moiety. In many embodiments, the linker is represented by L. Linkers include divalent groups such as alkenyl, arylene, heteroarylene, such as: -(CR ₂ ) _n O(CR ₂ ) _n -, alkoxy (e.g. polyethylene oxide, PEG, repeating units of polymethylene oxide) and alkylamino groups (eg, polyvinylamine, Jeffamine ^™ ); and diacid esters and amides, including succinate, succinamide, diglycolate, malonate, and caproamide.

The term "chiral" molecule refers to a molecule having the property that the mirror image partner is not superimposable, while the term "achiral" refers to a molecule whose mirror image partner is superimposable.

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

"Diastereomers"refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereoisomers can be separated under high resolution analytical methods such as electrophoresis and chromatography.

"Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

Stereochemical definitions and conventions used herein are generally in accordance with S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994 ) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, ie they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule with respect to its chiral centers. The prefixes d and 1 or (+) and (-) are used to indicate the direction of rotation of plane polarized light induced by the compound, (-) or 1 indicating that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers, known as a racemic mixture or racemate, can occur in a chemical reaction or process that has no stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, which lacks optical activity.

As used herein, the phrase "pharmaceutically acceptable salt," refers to a pharmaceutically acceptable organic or inorganic salt of ADC. Exemplary salts include, but are not limited to, sulfuric acid, citric acid, acetic acid, oxalic acid, chloride, bromide, iodide, nitric acid, acid sulfate, phosphoric acid, acid phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, sour lemon acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, gentisinate, fumaric acid, gluconic acid, glucuronic acid, sugar acid, formic acid, benzoic acid, glutamine acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and pamoic acid (ie, 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as acetate, succinate or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Additionally, pharmaceutically acceptable salts can have more than one charged atom in their structure. Examples in which multiply charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

"Pharmaceutically acceptable solvate" refers to the association of one or more solvent molecules with an ADC. 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.

Antibody-Drug Conjugates

Compounds of the present invention include those compounds that have utility in anticancer activity. In particular, the compounds include antibodies conjugated, ie covalently linked via a linker, to a drug moiety, wherein the drug has a cytotoxic or cytostatic effect when not conjugated to the antibody. In this way, the biological activity of the drug moiety is modulated by conjugation to the antibody. The antibody-drug conjugate (ADC) of the present invention can selectively deliver effective doses of cytotoxic agents to tumor tissues, thereby obtaining stronger selectivity, that is, the desired effect can be achieved at a lower dose.

In one embodiment, the ADC or an intracellular metabolite of the ADC has increased bioavailability in a mammal compared to the corresponding maytansinoid compound alone. At the same time, the bioavailability of ADC or intracellular metabolites of ADC is increased in mammals compared to the corresponding antibody alone (antibody of ADC, without drug moiety or linker).

In one embodiment, the maytansinoid drug portion of the ADC is not cleaved from the antibody until the antibody-drug conjugate binds to a cell surface receptor or enters a cell that has the antibody-drug conjugate Antibody-specific cell surface receptors. After the antibody-drug conjugate enters the cell, the drug moiety can be cleaved from the antibody. In mammals, the maytansinoid drug moiety can be cleaved intracellularly from antibodies to the compound or intracellular metabolites of the compound by enzymatic action, hydrolysis, oxidation, or other mechanisms. For example, and in no way meant to limit the invention to a particular mechanism of action, the sulfur atom of the maytansinoid drug moiety of the ADC may be oxidized to a sulfone or sulfoxide group. Intracellularly, the protons bound to the carbons of sulfones and sulfoxides can be removed under general catalysis or enzymatic catalysis and cause beta-elimination fragmentation that cleaves and separates the drug moiety from the antibody of the ADC . Alternatively, other electron-withdrawing groups in the linker such as amides, antibody or drug moieties can induce similar fragmentation/cleavage mechanisms inside the cell.

Antibody-drug conjugates (ADCs) can be represented by Formula I:

Ab-(LD) _p I

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antibody that binds to an ErbB receptor, or one or more tumor-associated antigens or cell surface receptors selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor (bone morphogenetic protein receptor)-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (six transmembrane epithelial antigen, Genbank accession number NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, lysate carrier family (solute carrier family) 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter (sodium-dependent phosphate transporter) 3b, Genbank accession number NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (seven thrombospondin repeats) (type 1 and type 1 ), a transmembrane domain (TM) and a short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 genes, Genbank accession number AY358628);

(9) ETBR (Endothelin (Endothelin) type B receptor, Genbank accession number AY275463);

(10) MSG783 (RNF124, hypothetical protein (hypothetical protein) FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six transmembrane epithelial antigen 2 of the prostate, six transmembrane prostate protein (six transmembrane prostate protein), Genbank accession number AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession number M26004);

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein (phosphatase anchor protein) 1a containing SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20Rα (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activating factor receptor, BlyS receptor 3, BR3, NP_443177.1);

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

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha), covalently interacts with Ig beta (CD79B) and forms a complex with IgM molecules on the surface, transduces genes involved in B cell differentiation Signaling B cell-specific protein, Genbank accession number NP_001774.1);

(29) CXCR5 (Burkitt's) lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, and plays a role in HIV-2 infection and may play a role in AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP_001707.1);

(30) HLA-DOB (Beta subunit (Ia antigen) of MHC class II molecules, which binds peptides and presents them to CD4+ T lymphocytes, Genbank accession number NP_002111.1);

(31) P2X5 (purinergic receptor) P2X ligand-gated ion channel (ligand-gated ion channel) 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its defect may Pathophysiological conditions leading to idiopathic detrusor instability (pathophysilolgy of idiopathic detrusor instability, Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (Lymphocyte Antigen 64 (RP105), Leucine-Rich Repeat Type I Membrane Protein (LRR) Family, Regulates B Cell Activation and Apoptosis, Loss of Function Is Associated with Enhanced Disease Activity in Patients with Systemic Lupus Erythematosus , Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, putative immunoglobulin Fc domain receptor, containing C2-type Ig-like and ITAM domains, may play a role in B lymphocyte differentiation, Genbank accession number NP_443170.1) ;

(35) IRTA2 (translocation-related immunoglobulin superfamily receptor 2, a putative immune receptor that may play a role in B-cell development and lymphomagenesis; gene dysregulation by translocation occurs in some B In cellular malignancies, Genbank accession number NP_112571.1); and

(36) TENB2 (putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, Genbank accession number AF179274;

With the proviso that the antibody is not TA.1.

L is a non-disulfide linker. L includes but is not limited to structures:

Wherein the wavy line represents the covalent connection with Ab and D;

X is:

R is independently H or C ₁ -C ₆ alkyl; and n is 1 to 12;

D is the part of the maytansinoid drug. Maytansinoids include but are not limited to structures:

Wherein the wavy line represents the covalent connection with L;

R is independently H or C ₁ -C ₆ alkyl; and

M is 1, 2, or 3.

The ratio of drug to antibody or drug loading is expressed as p for the compound of formula I. Drug loading values p ranged from 1 to 8. Compounds of Formula I include all mixtures of different loaded and linked antibody-drug conjugates wherein 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently linked to the antibody.

In another embodiment, the Ab is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(16) and (18)- (36), ie does not bind ErbB receptors, including HER2.

Antibody

Within its scope, an antibody unit (Ab-) of formula I includes any antibody unit associated with or complexed with a receptor, antigen or other receptive moiety that binds or is reactive with an administered associated with the target cell population. An antibody can be any protein or protein-like molecule that binds, complexes, or reacts with a desired therapeutically or biologically modified portion of a cell population. In one aspect, the antibody unit functions to deliver the maytansinoid drug moiety to the specific target cell population with which the antibody unit reacts. Such antibodies include, but are not limited to, large molecular weight proteins such as full-length antibodies and antibody fragments.

Antibodies comprising the antibody-drug conjugates of the invention preferably retain the antigen-binding ability of their native, wild-type counterparts. Thus, the antibodies of the invention are capable of binding to an antigen, preferably specifically. Such antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, those associated with tissue development or differentiation (e.g., known or suspected to be functionally contribute to tissue development or differentiation), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (eg, known or suspected to functionally contribute to angiogenesis )molecular. The tumor-associated antigen may be a cluster of differentiation factor (ie, CD protein). An antigen to which an antibody of the invention is capable of binding may be a member of a subgroup of one of the aforementioned classes, wherein other subgroups of said class comprise other molecules/antigens with distinguishing characteristics relative to the antigen of interest.

In one embodiment, the antibody of the antibody-drug conjugate (ADC) specifically binds a receptor encoded by the ErbB gene. The antibody may specifically bind to an ErbB receptor selected from EGFR, HER2, HER3 and HER4. ADCs can specifically bind the extracellular domain (ECD) of HER2 and inhibit the growth of tumor cells overexpressing the HER2 receptor. The antibody of the ADC can be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. The humanized antibody may be huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab). The antibody may be an antibody fragment, such as a Fab fragment.

Antibodies that are in the antibody-drug conjugate (ADC) of Formula I and that can be used in cancer therapy include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAAs). Such tumor-associated antigens are known in the art and can be prepared using methods and information well known in the art for antibody production. In an effort to find effective cellular targets for cancer diagnosis and therapy, investigators have sought to identify transmembrane or tumor-associated polypeptides that are specifically expressed on a on the surface of one or more specific types of cancer cells. Typically, these tumor-associated polypeptides are more expressed on the surface of cancer cells than on the surface of non-cancer cells. The identification of these tumor-associated cell surface antigen polypeptides has yielded the ability to specifically target cancer cells for destruction by antibody-based therapies.

Examples of TAAs include, but are not limited to, the tumor-associated antigens listed below (1)-(36). For convenience, all art-known information for these antigens is listed below, including names, alternate names, Genbank accession numbers and original references, following the National Center for Biotechnology Information (NCBI) conventions for nucleic acid and protein sequence identification. Nucleic acid and protein sequences corresponding to TAA(1)-(36) are available in public databases such as GenBank. Antibody-targeted tumor-associated antigens include all amino acid sequence variants and isoforms that possess at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequence identified in the cited reference, Or all amino acid sequence variants and isoforms that exhibit substantially the same biological properties or characteristics as the TAA having the sequence found in the cited reference. For example, a TAA having a variant sequence that is generally capable of specifically binding an antibody that specifically binds a TAA having the corresponding sequence listed. The sequences and contents of the references specifically cited herein are expressly incorporated by reference.

Tumor-associated antigens (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number 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- A1 (page 38-39); WO2002102235 (claim 13; page 296); WO2003055443 (page 91-92); WO200299122 (Example 2; pages 528-530); WO2003029421 (claim 6); WO2003024392 (claim 2; Figure 112); WO200298358 (claim 1; p. 183); WO200254940 (p. 100-101); WO200259377 (p. 349-350); WO200230268 (claim 27; p. 376); WO200148204 (Example; Figure 4)

NP_001194 bone morphogenetic protein receptor, type IB/pid = NP_001194.1-

Cross references: MIM:603248; NP_001194.1; AY065994

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486)

Biochem.Biophys.Res.Commun.255(2), 283-288(1999), Nature395(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); -129 pages); WO200286443 (claim 27; pages 222, 393); WO2003003906 (claim 10; pages 293); WO200264798 (claim 33; pages 93-95); WO200014228 (claim 5; pages 133 -136 pages); US2003224454 (Fig. 3); WO2003025138 (claim 12; page 150); US 20050107595; US 20050106644;

NP_003477 Lysate carrier family 7 (day ionic amino acid transporter, y+ system), member 5/pid=NP_003477.3-Human (Homo Sapiens) cross reference: MIM:600182; NP_003477.3; NM_015923; NM_003486_1

(3) STEAP1 (six transmembrane epithelial antigens of the prostate, Genbank accession number NM_012449)

Cancer Res.61(15), 5857-5860(2001), Hubert, R.S., et al (1999) Proc.Natl.Acad.Sci.U.S.A.96(25):14523-14528); WO2004065577 (claim 6); WO2004027049 (Fig. 1L); EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); Example 5; pages 618-619); WO2003022995 (Example 9; Figure 13A, Example 53; Page 173, Example 2; Figure 2A);

NP_036581 Prostate six transmembrane epithelial antigen

Cross references: MIM:604415; NP_036581.1; NM_012449_1

(4) 0772P (CA125, MUC16, Genbank accession number AF361486)

J.Biol.Chem.276(29):27371-27375(2001)); WO2004045553 (claim 14); WO200292836 (claim 6; Figure 12); WO200283866 (claim 15; pages 116-121); US2003124140 (Example 16); US2003091580 (claim 6); WO200206317 (claim 6; pages 400-408);

Cross References: GI: 34501467; AAK74120.3; AF361486_1

(5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin, Genbank accession number NM_005823)

Yamaguchi, N., et al Biol.Chem.269(2), 805-808(1994), Proc.Natl.Acad.Sci.U.S.A.96(20):11531-11536(1999), Proc.Natl.Acad. Sci.U.S.A.93(1):136-140(1996), J.Biol.Chem.270(37):21984-21990(1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; the 287th -288 pages); WO2002101075 (claim 4; pages 308-309); WO200271928 (pages 320-321); WO9410312 (pages 52-57);

Cross references: MIM:601051; NP_005814.2; NM_005823_1

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, lysate carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, genbank accession number NM_006424)

J.Biol.Chem.277(22):19665-19672(2002), Genomics62(2):281-284(1999), Feild, J.A., 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; pages 139-140);

Cross references: MIM:604217; NP_006415.1; NM_006424_1

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1), transmembrane structure domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878)

Nagase T., et al (2000) DNA Res.7(2):143-150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; p. 50); WO200188133 (claim claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11);

Accession number: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC: 10737;

(8)PSCAhlg(2700050C12Rik、C530008O16Rik、RIKEN cDNA2700050C12、RIKEN cDNA 2700050C12基因，Genbank登录号AY358628)；Ross et al(2002)Cancer Res.62：2546-2553；US2003129192(权利要求2)；US2004044180(权利要求12 ); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (example 5); WO2003105758 (claim 12); US2003206918 (example 5); EP1347046 (claim 1); ;

Cross References: GI: 37182378; AAQ88991.1; AY358628_1

(9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

Nakamuta M., et al Biochem.Biophys.Res.Commun.177, 34-39, 1991; Ogawa Y., et al Biochem.Biophys.Res.Commun.178, 248-255, 1991; Arai H., et al Jpn.Circ.J.56, 1303-1307, 1992; Arai H., et al J.Biol.Chem.268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys.Res. Commun.178, 656-663, 1991; Elshourbagy N.A., et al J.Biol.Chem.268, 3873-3879, 1993; Haendler B., et al J.Cardiovasc.Pharmacol.20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R.L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C., et al J. Clin. 82, 3116-3123, 1997; Okamoto Y, et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am.J.Med. Genet. 108, 223-225, 2002; Hofstra R.M.W., et al. al Eur.J.Hum.Genet.5, 180-185, 1997; Puffenberger E.G, et al Cell 79, 1257-1266, 1994; Attie T., et al, Hum.Mol.Genet.4, 2407-2409, 1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996; 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.111, 198-206; WO2004045516 (claim 1); WO2004048938 (example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); Claim 1); WO200261087 (Fig. 1); WO2003016494 (Fig. 6); WO2003025138 (claim 12; p. 144); WO200198351 (claim 1; p. 124-125); EP522868 (claim 8; Fig. 2) ; WO200177172 (claim 1; pages 297-299); US2003109676; US6518404 (fig. 3); US5773223 (claim 1a; columns 31-34); WO2004001004;

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

WO2003104275 (claim 1); WO2004046342 (example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; Figure 93); (embodiment 6);

Cross Reference: LocusID: 54894; NP_060233.2; NM_017763_1

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, six transmembrane epithelial antigen 2 of prostate, six transmembrane prostate protein, Genbank login No. AF455138)

Lab.Invest.82(11):1573-1582(2002)); WO2003087306; US2003064397 (claim 1; Figure 1); WO200272596 (claim 13; pages 54-55); WO200172962 (claim 1; Figure 4B ); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; Figure 10); WO200226822 (claim 23; Figure 2); WO200216429 (claim 12; Figure 10);

Cross references: GI:22655488; AAN04080.1; AF455138_1

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636)

Xu, X.Z., et al Proc.Natl.Acad.Sci.U.S.A.98(19):10692-10697(2001), Cell 109(3):397-407(2002), J.Biol.Chem.278(33) : 30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; pages 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); 27; p. 391); US2003219806 (claim 4); WO200162794 (claim 14; Figures 1A-D);

Cross references: MIM:606936; NP_060106.2; NM_017636_1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number 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; pp. 52-53); WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; pp. 94-95, pp. 105) ); WO200222808 (claim 2; Figure 1); US5854399 (Example 2; columns 17-18); US5792616 (Figure 2);

Cross references: MIM:187395; NP_003203.1; NM_003212_1.

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/Epstein-Barr virus receptor) or Hs.73792 Genbank accession number 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.U.S.A.84,9194-9198,1987; Barel M., et al Mol.Immunol.35,1025-1031,1998; Weis J.J., et al Proc.Natl.Acad.Sci.U.S.A.83,5639- 5643,1986; Sinha S.K., et al (1993) J.Immunol.150,5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (Fig. 9.1-9.9); WO2004020595 (claim 1);

Accession numbers: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29, Genbank accession number NM_000626 or 11038674)

Proc. Natl. Acad. Sci. U.S.A. (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, Figure 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (embodiment 2); 15 pages); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Figure 17A/B); WO200055351 (claim 11, pages 1145-1146);

Cross references: MIM:147245; NP_000617.1; NM_000626_1

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase-anchored protein 1a containing SH2 domain), SPAP1B, SPAP1C, Genbank accession numbers NM_030764, AY358130)

Genome Res.13(10):2265-2270(2003), Immunogenetics 54(2):87-95(2002), Blood 99(8):2662-2669(2002), Proc.Natl.Acad.Sci.U.S.A. 98(17):9772-9777 (2001), Xu, M.J., et al (2001) Biochem.Biophys.Res.Commun.280(3):768-775; WO2004016225 (claim 2); WO2003077836; Claim 5; Figures 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25);

Cross references: MIM:606509; NP_110391.2; NM_030764_1

(17) HER2 (ErbB2, Genbank accession number 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. U.S.A. 82 , 6497-6501, 1985; Swiercz J.M., et al J.Cell Biol.165, 869-880, 2004; KuhnsJJ., 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; ); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; Fig. 1A-B); WO2003025228 (claim 37; Fig. 5C); pp); WO200212341 (claim 68; FIG. 7); WO200213847 (page 71-74); WO200214503 (page 114-117); WO200153463 (claim 2; pp. 41-46); WO200141787 (page 15) ; WO200044899 (claim 52; Figure 7); WO200020579 (claim 3; Figure 2); US5869445 (claim 3; columns 1-38); WO9630514 (claim 2; pages 56-61); Claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; Figure 4);

Accession number: P04626; EMBL; M11767; AAA35808.1.EMBL; M11761; AAA35808.1

(18) NCA (CEACAM6, Genbank accession number 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. U.S.A. 99: 16899-16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (example 2); ; WO200260317 (claim 2);

Accession number: P40199; Q14920; EMBL; M29541; AAA59915.1.EMBL; M18728;

(19) MDP (DPEP1, Genbank accession number BC017023)

Proc.Natl.Acad.Sci.U.S.A.99(26):16899-16903(2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 (Figures 6-8); WO9946284 (FIG. 9);

Cross references: MIM:179780; AAH17023.1; BC017023_1

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

Clark H.F., et al Genome Res.13, 2265-2270, 2003; 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) Biochemistry42:12617-12624; Sheikh F., et al (2004) J.Immunol.172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63 ); WO200222153 (page 45-47); US2002042366 (page 20-21); WO200146261 (page 57-59); WO200146232 (page 63-65); WO9837193 (claim 1; page 55-59) ;

Accession numbers: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession number AF229053)

Gary S.C., et al Gene 256, 139-147, 2000; Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; figure 52); US2003119122 (claim 1; figure 52); US2003119126 (claim 1); 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; Figure 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1);

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442)

Chan, J. and 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; p. 41); WO2004065576 (claim 1); WO2004020583 (claim 9); - 132 pages); WO200053216 (claim 1; page 42);

Cross references: MIM:600997; NP_004433.2; NM_004442_1

(23) ASLG659 (B7h, Genbank accession number AX092328)

US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (figure 3); US2003165504 (claim 1); US2003124140 (example 2); US2003065143 (figure 60); ; US2003091580 (Example 2); WO200210187 (claim 6; Figure 10); WO200194641 (claim 12; Figure 7b); WO200202624 (claim 13; Figures 1A-1B); US2002034749 (claim 54; Sections 45-46 pages); WO200206317 (Example 2; pages 320-321, claim 34; pages 321-322); WO200271928 (pages 468-469); WO200202587 (Example 1; Figure 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;

(24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436)

Reiter R.E., et al Proc.Natl.Acad.Sci.U.S.A.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; Figure 17); US2001055751 (Example 1; Figure 1b); WO200032752 (claim 18; Figure 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 97); 94 pages); WO9840403 (claim 2; Figure 1B);

Accession numbers: O43653; EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession number AY260763);

AAP14954 lipoma (lipoma) HMGIC fusion partner-like protein/pid=AAP14954.1-human

Species: Homo sapiens (human)

WO2003054152 (claim 20); WO2003000842 (claim 1);

WO2003023013 (embodiment 3, claim 20); US2003194704 (claim 45);

Cross References: GI: 30102449; AAP14954.1; AY260763_1

(26) BAFF-R (B cell activating factor receptor, BlyS receptor 3, BR3, Genbank accession number: AF116456); BAFF receptor/pid=NP_443177.1-Homo sapiens Thompson, J.S., et al Science 293 (5537 ), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; pp. 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; pp. 615-616); columns 136-137); WO200238766 (claim 3; page 133); WO200224909 (example 3; Figure 3);

Cross references: MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number: AK026467);

Wilson et al (1991) J.Exp.Med.173:137-146; WO2003072036 (claim 1; Figure 1);

Cross references: MIM:107266; NP_001762.1; NM_001771_1

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, covalently interacts with Igbeta (CD79B), forms a complex with IgM molecules on the surface, and transduces B-cell specificity of signals involved in B-cell differentiation Protein) PROTEIN SEQUENCE Fullmpggpgv...dvqlekp(1..226; 226aa), pI: 4.84, MW: 25028TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession number: NP_001774.10)

WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, Figure 16); WO9207574 (Figure 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;

(29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, in HIV-2 infection and perhaps in AIDS, lymph tumor, myeloma, and leukemia) PROTEINSEQUENCE Full mnypltl...atslttf (1..372; 372aa), pI: 8.54MW: 41959TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession number: NP_001707.1)

WO2004040000; WO2004015426; US2003105292 (Example 2); US6555339 (Example 2); WO200261087 (Fig. 1); WO200157188 (claim 20, p. 269); 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;

(30) HLA-DOB (Beta subunit of MHC class II molecules (Ia antigen), which binds peptides and presents them to CD4+ T lymphocytes) PROTEIN SEQUENCE Fullmgsgwvp...vllpqsc (1..273; 273aa, pI : 6.56MW: 30820TM: 1 [P]GeneChromosome: 6p21.3, Genbank accession number: 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, Figure 15); US6153408 (columns 35-38); US5976551 (columns 168-170); US6011146 (Columns 145-146); Kasahara et al (1989) Immunogenetics 30(1):66-68; Larhammar et al (1985) J.Biol.Chem.260(26):14111-14119;

(31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its defect may lead to the pathophysiology of idiopathic detrusor instability Status) PROTEIN SEQUENCE Full mgqagck...lephrst (1..422; 422aa), pI: 7.63, MW: 47206TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession number: 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); WO2003029277 (page 82);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Fullmaeaity...tafrfpd (1..359; 359 aa), pI: 8.66, MW: 40225TM: 1 [P] GeneChromosome: 9p13.3, Genbank accession number: NP_001773.1)

WO2004042346 (claim 65); WO2003026493 (pp. 51-52, 57-58); WO200075655 (pp. 105-106); Von Hoegen et al (1990) J. Immunol.144(12):4870-4877; Strausberg et al(2002) Proc.Natl.Acad.Sci USA99:16899-16903;

(33) LY64 (Lymphocyte Antigen 64 (RP105), a Leucine-Rich Repeat (LRR) Type I Membrane Protein Family, Regulates B Cell Activation and Apoptosis, Loss of Function Is Associated with Enhanced Disease Activity in Patients with Systemic Lupus Erythematosus )PROTEIN SEQUENCE Full mafdvsc...rwkyqhi(1..661; 661aa), pI: 6.20, MW: 74147TM: 1 [P] Gene Chromosome: 5q12, Genbank accession number: NP_005573.1)

US2002193567; WO9707198 (claim 11, page 39-42); Miura et al (1996) Genomics 38 (3): 299-304; Miura et al (1998) Blood 92: 2815-2822; WO2003083047; WO9744452 (claim 8, pp. 57-61); WO200012130 (pp. 24-26);

(34) FcRH1 (Fc receptor-like protein 1, Fc domain putative immunoglobulin receptor, which contains C2-type Ig-like and ITAM domains, may have a role in B lymphocyte differentiation) PROTEINSEQUENCE Full mlprlll... vdyedam (1..429; 429aa), pI: 5.28, MW: 46925TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession number: NP_443170.1)

WO2003077836; WO200138490 (claim 6, Fig. 18E-1-18-E-2); Davis et al (2001) Proc.Natl.Acad.Sci USA 98(17):9772-9777; WO2003089624 (claim 8); (claim 1); WO2003089624 (claim 7);

(35) IRTA2 (translocation-related immunoglobulin superfamily receptor 2, a putative immune receptor that may play a role in B-cell development and lymphomagenesis; it is related to the translocation that occurs in some B-cell malignancies Gene dysregulation caused by) PROTEIN SEQUENCE Fullmllwvil...assaphr(1..977; 977aa), pI: 6.88MW: 106468TM: 1 [P] GeneChromosome: 1q21, Genbank accession number: Human: AF343662, AF343663, AF343664, AF343665 , AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1

WO2003024392 (claim 2, Figure 97); Nakayama et al (2000) Biochem.Biophys.Res.Commun.277(1): 124-127; WO2003077836; WO200138490 (claim 3, Figures 18B-1-18B-2) ;

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, EGF/hergulin family involved in growth factors and follistatin) PROTEINSEQUENCE Full mvlwesp...rastrli(1. .374; 374 aa, NCBI accession numbers: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;

WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944 (page 69-70); )；WO200190304(SEQ IDNO 2706)；US2004249130；US2004022727；WO2004063355；US2004197325；US2003232350；US2004005563；US2003124579；US 6410506；US 66420061；Horie et al(2000)Genomics 67：146-152；Uchida et al(1999)Biochem. Biophys.Res.Commun.266:593-602; Liang et al(2000) Cancer Res.60:4907-12; Glynne-Jones et al(2001) Int J Cancer.Oct 15;94(2):178-84 .

Antibody production

A number of methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to clonal cell lines that produce a single antibody type, uses cells from many species including mouse (rat), hamster, rat, and human. Other methods of making MAbs, including chimeric and humanized antibodies, use genetic engineering, ie, recombinant DNA technology.

Polyclonal antibodies can be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts.

Human myeloma and mouse-human hybridoma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The medium in which the hybridoma cells were cultured was tested for the production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Binding affinities of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al (1980) Anal. Biochem., 107:220.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to the heavy and light chains encoding the murine antibody encoding). The hybridoma cells serve as a source of this DNA. Once the DNA is isolated, it can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce antibody proteins such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells to obtain the synthesis of monoclonal antibodies in recombinant host cells (US 2005/0048572; US 2004/0229310). Reviews on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al (1993) Curr. Opinion in Immunol., 5:256-262 and Plückthun (1992) Immunol. Revs., 130:151-188.

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries using McCafferty et al (1990) Nature 348:552-554; Clackson et al (1991) Nature 352:624- 628; and Marks et al (1991) J. Mol. Biol., 222: 581-597, which describe the isolation of murine and human antibodies, respectively, using phage libraries. Later publications described the production of high-affinity (nM range) human antibodies by strand replacement (Marks et al (1992) Bio/Technology, 10:779-783), and as a combination of strategies for constructing very large phage libraries Infection and in vivo recombination (Waterhouse et al (1993) Nuc. Acids. Res., 21:2265-2266) were performed. Therefore, these techniques are viable alternatives to traditional mAb hybridoma techniques for isolation of mAbs.

DNA can also be modified, for example by substituting human heavy and light chain constant regions for homologous murine sequences (US4816567; and Morrison et al (1984) Proc. The protein polypeptide coding sequence is modified by covalently linking all or part of it to the immunoglobulin coding sequence.

Typically, a non-immunoglobulin polypeptide is substituted for the constant region of the antibody, or for the variable region of one of the antibody's antigen-binding sites, to form a chimeric bivalent antibody comprising an antigen-binding site with antigen-specificity and a Another antigen binding site with a different antigen specificity.

Description According to an exemplary production technique of an antibody (Ab) used in an antibody-drug conjugate (ADC) of the present invention. The production of antibodies will be described with reference to anti-ErbB2 antibodies, but it will be apparent to those skilled in the art that antibodies to other members of the ErbB receptor family, as well as any other receptor or tumor-associated antigen or target, can be similarly produced and modified.

The ErbB2 antigen used for antibody production can be, for example, a soluble form of the ErbB2 extracellular domain or a portion thereof comprising the desired epitope. Alternatively, cells expressing ErbB2 on their cell surface, such as transformed NIH-3T3 cells overexpressing ErbB2; or cancer cell lines such as Sk-Br-3 cells (Stancovski et al (1991) PNAS (USA) 88:8691-8695) Can be used to produce antibodies. Other forms of ErbB2 useful for producing antibodies will be apparent to those skilled in the art.

Example 1 describes the production of exemplary humanized anti-ErbB2 antibodies. A humanized antibody may, for example, comprise non-human hypervariable region residues incorporated into a human variable region heavy chain domain, and may further comprise framework region (FR) substitutions at sites selected from the group consisting of: using Kabat et al ., 69H, 71H and 73H of the variable region numbering system provided in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H and 73H.

As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (eg, mice) capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. (Jakobovits et al (1993) Proc.Natl.Acad.Sci.USA, 90: 2551; Jakobovits et al (1993) Nature 362: 255-258; Bruggermann et al (1993) Year in Immuno. 7: 33; and US 5591669; US 5589369; US 5545807).

Alternatively, phage display technology (McCafferty et al (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro from the repertoire of immunoglobulin variable (V) region genes from non-immunized donors. (Johnson, Kevin S. and Chiswell, David J. (1993) Current Opinion in Structural Biology 3:564-571). A full repertoire of V genes from a non-immune human donor can be constructed and antibodies to essentially a variety of antigens, including self-antigens, can be isolated (Marks et al (1991) J. Mol. Biol. 222:581-597; Griffith et al (1993) EMBO J. 12:725-734; US 5565332; US 5573905). Human antibodies can also be produced by activated B cells in vitro (US 5567610; US5229275). Human anti-ErbB2 antibodies are described (US 5772997 and WO 97/00271).

Various techniques have been developed for producing antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, eg, Morimoto et al (1992) Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al (1985) Science, 229:81). Antibody fragments can also be produced directly from recombinant host cells and the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al (1992) Bio/Technology 10:163-167). According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to the skilled artisan. In other embodiments, the selected antibody is a single chain Fv fragment (scFv). See WO 93/16185; US 5571894; and US 5587458. For example, antibody fragments may also be "linear antibodies", such as described in US 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

Bispecific antibodies with binding specificities for at least two different epitopes (Millstein et al (1983), Nature 305:537-539) can bind two different epitopes of the ErbB2 protein. Other such antibodies may combine ErbB2 binding sites with binding sites for EGFR, ErbB3 and/or ErbB4. Alternatively, the anti-ErbB2 arm can bind to stimulating molecules on leukocytes such as T cell receptor molecules (e.g. CD2 or CD3), or Fc receptors (FcγRs) of IgG, such as FcγRI (CD64), FcγRII (CD32) and FcγRIII ( CD16) to focus the cell's defense mechanisms on ErbB2-expressing cells. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing ErbB2 (WO 96/16673; US 5837234; WO 98/02463; US 5821337). Purification methods for bispecific antibodies have been disclosed (WO 93/08829; Traunecker et al (1991) EMBO J. 10:3655-3659; WO 94/04690; Suresh et al (1986) Methods in Enzymology 121:210; US 5731168 ). Leucine zippers (Kostelny et al (1992) J. Immunol. 148(5): 1547-1553), and single-chain Fv (sFv) dimers (Gruber et al (1994) J. Immunol. 152: 5368) to produce bispecific antibodies.

Techniques for the production of bispecific antibodies from antibody fragments have also been described, for example utilizing the chemical linkages at which intact antibodies are proteolytically cleaved to yield F(ab') ₂ fragments (Brennan et al (1985) Science 229:81). Fab'-SH fragments can be recovered from E. coli and chemically coupled to form bispecific antibodies (Shalaby et al (1992) J. Exp. Med. 175:217-225. "Diabody" technology provides for the preparation of bispecific An alternative approach to antibody fragments (Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).

Antibodies that are more than bivalent can be prepared. Multivalent "octopus"-like antibodies having three or more antigen-binding sites and two or more variable regions can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains (US 2002/0004586; WO 2002/0004586; 01/77342). For example, trispecific antibodies can be prepared (Tutt et al (1991) J. Immunol. 147:60).

Amino acid sequence modifications of antibodies are contemplated. For example, antibody mutants and many isoforms that bind tumor-associated antigens are expected to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody-encoding nucleic acid or by peptide synthesis. Such modifications include, for example, deletion of residues from, and/or insertion of, and/or substitution of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions are made to arrive at the final construct such that the final construct possesses the desired characteristics. Amino acid changes may also alter the post-translational processing of the antibody, for example altering the number or location of glycosylation sites.

A useful method for identifying specific residues or regions of antibodies which are preferred sites for mutagenesis is called "alanine scanning mutagenesis" (Cunningham and Wells (1989) Science, 244: 1081-1085, in which an amino acid residue or group of residues of interest (for example, charged residues such as arg, asp, his, lys, and glu) is identified and neutral or negatively charged amino acids such as alanine or polyalanine Acid substitutions to optimize amino acid interaction with antigen. Amino acid sequence insertions include fusions from one residue to the amino and/or carboxyl termini of polypeptides containing a hundred or more residues, as well as single or multiple amino acid residues Examples of terminal insertions include anti-ErbB2 antibodies with N-terminal methionyl residues or antibodies fused to cytotoxic polypeptides. Other insertional variants of anti-ErbB2 antibody molecules include enzymes (e.g. for ADEPT: Tietze et al( 2003) Current Pharm. Design 9: 2155-2175) or polypeptides and anti-ErbB2 antibody N- or C-terminal fusions, said polypeptides increase the serum half-life of antibodies, such as albumin-binding peptides.

Plasma-protein binding may be an effective means of improving the pharmacokinetic properties of short-lived molecules. Albumin is the most abundant protein in plasma. Serum albumin binding peptides (ABPs) are capable of altering the pharmacodynamics of fused active domain proteins, including alterations in tissue uptake, penetration, and diffusion. These pharmacodynamic parameters can be modulated by specific selection of appropriate serum albumin binding peptide sequences (US20040001827). A series of albumin binding peptides were identified by phage display screening (Dennis et al (2002) "Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins" J Biol Chem. 277:35035-35043; WO 01/45746). Compounds of the present invention include: (i) Tables III and IV in Dennis et al (2002) J Biol Chem.277:35035-35043, page 35038; (ii) [0076] SEQ ID NOS: 9-22 in US 20040001827 and (iii) the ABP sequences taught in WO 01/45746, pages 12-13, SEQ ID NOS: z1-z14, which are incorporated herein by reference.

Amino acid sequence is usually altered by altering the basic nucleic acid sequence. Nucleic acid molecules encoding antibody amino acid sequence variants are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and previous preparation of antibodies. Cassette mutagenesis by variant or non-mutant form. Sites of most interest for substitutional mutagenesis include hypervariable regions, but FR changes are also expected.

Substantial modification of the biological properties of the antibody is accomplished by selective substitutions that contribute to the maintenance of (a) the structure of the polypeptide backbone, such as a sheet or helical conformation, in the substituted region, (b) the charge or hydrophobicity of the target site of the molecule, or (c) There is a significant difference in the effect of the size of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties:

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

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

(3) acidic: asp, glu;

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

(5) Residues affecting chain orientation: gly, pro; and

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions necessarily involve exchanging a member of one of these classes for another.

Any cysteine residue not involved in maintaining the correct conformation of the antibody can also generally be substituted with serine to improve the oxidative stability of the molecule and to prevent aberrant conjugation. Conversely, cysteine linkages can be added to antibodies to improve their stability (especially if the antibody is an antibody fragment such as an Fv fragment).

To increase the serum half-life of antibodies, salvage receptor binding epitopes can be incorporated into antibodies, especially antibody fragments, for example as described in US 5739277. The term "salvage receptor binding epitope" as used herein refers to an epitope in the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for enhancing the serum half-life of the IgG molecule in vivo (US 2003/0190311, US6821505; US 2003/0190311, US6821505; US 6165745; US 5624821; US 5648260; US 6165745; US 5834597).

A glycosylation variant of an antibody refers to a variant in which the glycosylation pattern of the antibody is altered. Alteration refers to deletion of one or more carbohydrate moieties found in an antibody, addition of one or more carbohydrate moieties to an antibody, alteration of the composition of glycosylation (glycosylation pattern), or degree of glycosylation.

Antibodies can be glycosylated (N-linked or O-linked) at conserved sites in their constant regions (Hse et al (1997) J. Biol. Chem. 272:9062-9070; Jefferis and Lund, (1997) Chem. Immunol. 65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect protein function (Boyd et al (1996) Mol. Immunol. 32: 1311-1318; Wittwe and Howard, (1990) Biochem. 29: 4175-4180), and the relationship between glycoprotein moieties This intramolecular interaction can affect the conformation and provide a three-dimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner (1996) Current Opin. Biotech. 7:409-416). Oligosaccharides can also be used to direct a given glycoprotein to a specific molecule based on a specific recognition structure (Malhotra et al (1995) Nature Med. 1: 237-243; Umana et al (1999) Nature Biotech. 17: 176-180 ). Removal of oligosaccharides can optimize antigen binding and other properties of antibodies (Boyd et al (1996) Mol. Immunol. 32:1311-1318).

Factors that affect glycosylation during recombinant antibody production include growth mode, media formulation, culture density, oxidation, pH, purification protocols, etc. (US 5047335; US 5510261; US 5278299). Glycosylation, or specific types of glycosylation, can be removed enzymatically from glycoproteins, for example using endoglycosidase H (Endo H). In addition, recombinant host cells can be genetically engineered, for example, to be defective in the processing of particular types of polysaccharides. These and similar techniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed by conventional techniques of carbohydrate analysis, including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, sequential enzymatic digestion, and HPAEC- PAD, where HPAEC-PAD is charge-based, uses high-pH anion-exchange chromatography to separate oligosaccharides. Methods to release oligosaccharides for analytical purposes are also known and include, but are not limited to, enzymatic treatment (typically performed using peptide-N-glycosidase F/endo-β-galactosidase), elimination using a caustic environment, To primarily release O-linked structures, and to release N- and O-linked oligosaccharides using anhydrous hydrazine chemistry.

Maytansinoid Drug Portion

Maytansinoid compounds inhibit cell proliferation by inhibiting the formation of polymeric microtubules of tubulin during mitosis (Remillard et al (1975) Science 189:1002-1005; US 5208020). Maytansine and maytansinoids are highly cytotoxic, but their clinical application in cancer therapy has been greatly limited by their severe systemic side effects, mainly attributed to their poor tumor selectivity . Clinical trials of maytansine have been discontinued due to severe side effects on the central nervous system and gastrointestinal system (Issel et al (1978) Can. Treatment. Rev. 5: 199-207).

Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification, derived from fermentation products, (ii) easy to use Derivatization of functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

Maytansinoid compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources according to known methods and produced by genetic engineering techniques (see Yue et al (2002) PNAS 99: 7968-7973), or synthetically prepare maytansinol and maytansinol analogs according to known methods.

Exemplary maytansinoid drug moieties include those with modified aromatic rings such as: C-19-dechloro (US 4256746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20 -Hydroxyl (or C-20-demethylation) +/-C-19-dechlorination (US Patents 4361650 and 4307016) (by demethylation with Streptomyces or Actinomyces or demethylation with LAH chlorine); and C-20-desmethoxy, C-20-acyloxy (-OCOR), +/- dechlorination (US4294757) (prepared by acylation with acid chloride), and modified at other positions of those.

Exemplary maytansinoid drug moieties also include those with _the following modifications: C-9-SH, prepared by reaction of maytansinol with _H2S or _P2S5 (US 4424219); C-14-alkoxy Methyl (desmethoxy/CH2 OR) (US 4331598); C-14-Hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc), prepared from Nocardia (US 4450254); C -15-Hydroxy/acyloxy, prepared by transformation of maytansinol with Streptomyces (US 4364866); C-15-Methoxy, isolated from Trewianudflora (US 4313946 and US 4315929); C-18 -N-demethylated, prepared by demethylating maytansinol with Streptomyces (US 4362663 and US 4322348); and 4,5-deoxy, prepared by titanium trichloride/LAH reduction of maytansinol (US 4371533).

It is known that many sites on maytansine compounds can be used as attachment sites depending on the type of attachment. For example, for forming an ester bond, the C-3 site with a hydroxyl group, the C-14 site modified with a hydroxymethyl group, the C-15 site modified with a hydroxyl group, and the C-20 site with a hydroxyl group are all suitable .

Maytansinoid drug moieties (D) include those having the structure:

Wherein the wavy line represents the covalent attachment of the sulfur atom of D to the antibody-drug conjugate (ADC) linker (L). R independently can be H or C ₁ -C ₆ alkyl. The alkylene chain connecting the amide group to the sulfur atom may be methanel, ethanyl, or propyl, i.e. m is 1, 2, or 3 (US633410, 5208020, Chari et al. al (1992) Cancer Res. 52: 127-131; Liu et al (1996) Proc. Natl. Acad. Sci 93: 8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated for compounds of the invention, ie, any combination of the R and S conformations at the chiral carbon positions of D. In one embodiment, the maytansinoid drug moiety (D) will have the following stereochemistry:

Specific options for D include:

DM1(N ^2' -deacetyl-N ^2' -(3-mercapto-1-oxopropyl)-maytansine) where (CR ₂ ) _m =CH ₂ CH ₂ ;

DM3(N ^2' -deacetyl-N- ^2' (4-mercapto-1-oxopentyl)-maytansine) where (CR ₂ ) _m =CH ₂ CH ₂ CH(CH ₃ );

DM4(N ^2' -deacetyl-N ^2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine) where (CR ₂ ) _m =CH ₂ CH ₂ C(CH ₃ ) ₂ :

The steric hindrance provided by alkyl groups such as methyl groups to the carbons near the sulfur atoms of DM3 and DM4 may affect the intracellular cleavage rate of ADC (US 2004/0235840 A1). Thus the variable alkyl unit (CR ₂ ) _m may affect in vitro and in vivo potency, efficacy, and safety/toxicity.

connector

Linker L links the antibody to the drug moiety via a covalent bond, excluding disulfide groups. Linkers are bifunctional or multifunctional moieties that can be used to link one or more drug moieties (D) and antibody units (Ab) to form antibody-drug conjugates (ADCs) of formula I. Antibody-drug conjugates (ADCs) can be conveniently prepared with linkers having drug-binding and antibody-binding reactive functionality. A cysteine thiol, or amine of an antibody (Ab), such as the N-terminus or an amino acid side chain such as lysine, is capable of forming a bond with a functional group of a linker reagent, drug moiety or drug-linker reagent.

The linker is preferably stable extracellularly. Prior to translocation or delivery into cells, the antibody-drug conjugate (ADC) is preferably stable and intact, ie the antibody is still attached to the drug moiety. The linker is stable outside the target cell and can be cleaved at an efficient rate inside the cell. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. until the conjugate has been delivered or transported to cleavage at its target site; and (iv) maintaining the cytotoxic, cytocidal or cytostatic effect of the maytansinoid drug moiety. The stability of ADCs can be determined by standard analytical techniques such as mass spectrometry, HPLC, and separation/analysis techniques LC/MS.

Covalent attachment of an antibody to a drug moiety requires the linker to have two reactive functional groups, ie to be bivalent in the sense of reactivity. Bivalent linker reagents such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter moieties that can be used to link two or more functional or biologically active moieties are known and methods have been described as well as Conjugates obtained therefrom (Hermanson, GT. (1996) Bioconjugate Techniques; Academic Press: New York, p234-242).

The linker may have a structure selected from:

Wherein the wavy line represents the covalent linkage with Ab and D in any direction; X can have the following structure in any direction:

Wherein R is independently H or C ₁ -C ₆ alkyl; and n is 1 to 12; Y can have the following structure in any direction:

wherein R is independently H or C ₁ -C ₆ alkyl; and n is 1-12.

For example, a linker can have the following structure called SMCC:

In another embodiment, the linker (L) has the following structure:

where the wavy lines represent covalent linkages to Ab and D in any orientation.

For example, a linker can have the following structure known as SIAB:

In another embodiment, the linker (L) has the following structure:

In another embodiment, linkers can be substituted with groups that modify solubility or reactivity. For example, sulfonate substituents can enhance the water solubility of the reagents and facilitate conjugation of linker reagents to antibody or drug moieties, or to facilitate conjugation of Ab-L to D, or D-L to Ab, depending on Synthetic route used to prepare ADC.

In another embodiment, the linker has a reactive functional group comprising a nucleophilic group, ie reactive to an electrophilic group present on the antibody. Useful electrophilic groups on antibodies include, but are not limited to, the carbonyls of aldehydes and ketones. The heteroatom of the nucleophilic group of the linker is capable of reacting with the electrophilic group on the antibody to form a covalent bond with the antibody unit. Useful nucleophilic groups on linkers include, but are not limited to, hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides. Electrophilic groups on antibodies provide convenient sites for linker attachment.

A linker may be a linker of a peptide comprising one or more amino acid units. Peptide linker reagents can be prepared by solid-phase or liquid-phase synthesis on an automated synthesizer such as Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ), or Model 433 (Applied Biosystems, Foster City, CA) (E. Schröder and K. Lübke, The Peptides, volume 1, pp 76-136 (1965) Academic Press), which is peptide chemistry including t-BOC chemistry (Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R .Liss, Inc., 1988, pp.199-218) and Fmoc/HBTU chemistry (Fields, G.and Noble, R. (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int.J.Peptide Protein Res.35:161-214) are well known in the art.

The compound is worded to refer to, but not limited to, ADCs prepared with the following crosslinkers: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfur (sulfo)-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone)benzoate), and include bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO) ₃ , and BM(PEO) ₄ , which are commercially available Available from Pierce Biotechnology, Inc., Customer Service Department, PO Box 117, Rockford, IL. 61105 USA, USA 1-800-874-3723, International +815-968-0747. See pages 467-498, 2003-2004 Applications Handbook and Catalog. Bis-maleimide reagents allow the attachment of free thiol groups of antibody cysteine residues to thiol-containing drug moieties, labels, or linker intermediates in a sequential or simultaneous fashion. In addition to maleimide, other functional groups reactive with thiol groups of antibodies, maytansinoid drug moieties, or linker intermediates include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, Pyridyl disulfides, isocyanates, and isothiocyanates.

BM(PEO) ₃ BM(PEO) ₄

An exemplary antibody-drug conjugate in which DM1 is linked to the thiol group of Trastuzumab via a BMPEO linker has the following structure:

wherein Tr is trastuzumab; n is 0, 1, or 2; and p is 1, 2, 3, or 4.

Useful linker reagents are also available through other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO), or as per Toki et al (2002) J.Org.Chem.67:1866-1872; US6214345 to Firestone et al; WO 02 /088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and synthesized by methods described in WO 04/032828.

The linker can be a dendritic type linker in which more than one drug moiety is covalently attached to the antibody through a branched, multifunctional linker portion (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768; King et al (2002) Tetrahedron Letters 43: 1987-1990). Dendritic linkers can increase the molar ratio of drug to antibody, that is, the ratio of loading, which is related to the potency of ADC. In this way, on antibodies possessing only one reactive cysteine thiol group, many drug moieties can be attached via dendritic linkers.

The following exemplary specific forms of dendritic linker reagents allow up to nine nucleophilic drug moiety reagents to be coupled by reaction with the ethyl chloride mustard functional group:

drug load

In the molecule of formula I, drug loading is represented by p, representing the average number of maytansinoid drugs loaded per antibody. Drug loading can range from 1 to 8 drugs (D) per antibody (Ab), ie where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the antibody. The composition of the ADC of Formula I includes a collection of antibodies conjugated to 1 to 8 drugs. The average number of drugs per antibody in ADC preparations from conjugation reactions can be identified by conventional methods such as mass spectrometry methods, ELISA assays, electrophoresis, and HPLC. The quantitative distribution of the ADC according to p can also be determined. The p-mean in specific ADC preparations can be determined by ELISA (Hamblett et al (2004) Clinical Cancer Res. 10:7063-7070; Sanderson et al (2005) Clinical Cancer Res. 11:843-852). However, the p(drug) value distribution cannot be discerned by the antibody-antigen binding and detection limits of ELISA. Also, ELISA tests for detecting antibody-drug conjugates do not determine where the drug moiety is attached to the antibody, such as heavy or light chain fragments, or specific amino acid residues. In some cases, isolation, purification, and identification of homogeneous ADCs can be achieved by means such as reverse phase HPLC or electrophoresis, where p is a specific value derived from ADC and other drug loads.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, the site of attachment is a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive A thiol group through which the linker can be attached. Higher drug loading, eg, p>5, may lead to aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.

Typically, the moiety of drug coupled to the antibody is less than the theoretical maximum during the conjugation reaction. An antibody may contain, for example, a number of lysine residues that are not reactive with the drug-linker intermediate (D-L) or the linker reagent. Only the most reactive lysine groups are capable of reacting with amine-reactive linker reagents. Likewise, only the most reactive cysteine thiol groups are capable of reacting with thiol-reactive linker reagents. Typically, antibodies do not contain many, if any, free reactive cysteine thiol groups that could be attached to drug moieties. Most cysteine thiol residues in antibody compounds exist as disulfide bonds and must be reduced with reducing agents such as dithiothreitol (DTT) or TCEP under incomplete or complete reducing conditions. In addition, antibodies must be placed under denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine. ADC loading (drug/antibody ratio) can be controlled in several different ways, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to the antibody, (ii) limiting Coupling reaction time or temperature, and (iii) incomplete or limiting reducing conditions for cysteine thiol modification.

More than one nucleophilic or electrophilic group in an antibody reacts with a drug-linker intermediate or linker reagent, followed by a drug moiety reagent, and the resulting product is a mixture of ADC compounds with, for example, 1, 2, 3, etc. attached to the antibody The distribution of drug moieties. Liquid chromatography methods such as polymer reversed-phase (PLRP) and hydrophobic interaction (HIC) enable the separation of compounds in a mixture by drug loading values. ADC preparations with a single drug loading value (p) can be isolated ("Effect of drugloading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Hamblett, K.J., et al, Abstract No. 624, American Association for Cancer Research; 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; "Controlling the Location of Drug Attachment in Antibody-Drug Conjugates", Alley, S.C., et al, AbstractNo.627, American Association for Cancer Research; 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). However, these single-load ADCs may still be heterogeneous mixtures, since drug moieties can be attached to different sites on the antibody via linkers.

Preparation of antibody-drug conjugates

ADCs of Formula I can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of an antibody nucleophilic or electrophilic group with a divalent linker reagent, Formation of an antibody-linker intermediate Ab-L linked by a covalent bond, which is then reacted with an activated drug moiety D; The bonded drug-linker intermediate D-L is then reacted with the nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be applied using a variety of antibodies, drug moieties, and linkers to prepare antibody-drug conjugates of Formula I.

Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) Sugar hydroxyl or amino groups where the antibody is glycosylated. Ammonia, thiols, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) active esters such as NHS Esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyls, and maleimides group. Certain antibodies have reducible interchain disulfide bonds, ie, cysteine bridges. Antibodies can be obtained by using a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride); Getz et al ( 1999) Anal.Biochem.Vol 273:73-80; Soltec Ventures, Beverly, MA) to make it reactive for coupling with linker reagents. In this way, each cysteine disulfide bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by the reaction of lysines with 2-iminothiophene (Traut's reagent), which converts amines to thiols.

Antibody-drug conjugates can also be produced by modifying antibodies to introduce electrophilic moieties capable of reacting with nucleophilic substituents on linker reagents or drugs. For example, sugars of glycosylated antibodies can be oxidized with periodate oxidizing agents to form aldehyde or ketone groups, which can react with amine groups of linker reagents or drug moieties. The resulting imine Schiff base group can form a stable bond, or can be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the carbohydrate moiety of a glycosylated antibody reacts with galactose oxidase or sodium meta-periodate to generate carbonyl groups (aldehydes and ketones) in the protein that are capable of reacting with appropriate groups on the drug. Conjugate reactions (Hermanson, GT. (1996) Bioconjugate Techniques; Academic Press: New York, p234-242). In another embodiment, proteins comprising N-terminal serine or threonine residues are capable of reacting with sodium periodate resulting in substitution of an aldehyde for the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852). Such aldehydes are capable of reacting with drug moieties or linker nucleophiles.

Likewise, nucleophilic groups on drug moieties include, but are not limited to, ammonia, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, These groups are capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acidic Halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehyde, ketone, carboxyl, and maleimide groups.

Maytansine can for example be converted to May-SSCH3, which can be reduced to the free thiol May-SH and reacted with the modified antibody (Chari et al (1992) Cancer Research 52:127-131), together with a disulfide linker Formation of maytansinoid-antibody immunoconjugates. Antibody-maytansinoid conjugates formed with disulfide linkers have been reported (WO 04/016801; US6884874; US 2004/039176 A1; WO 03/068144; US 2004/001838 A1; US6441163; US 5208020; US 5416064; WO 01/024763). The disulfide linker SPP was constructed with the linker reagent N-succinimide 4-(2-pyridylthio)pentanoic acid. The antibody-SPP-DM1 conjugate is represented by the following structure:

For comparison with non-disulfide linker ADCs of the invention, disulfide (S-S) linker antibody-drug conjugates were tested. Trastuzumab-SPP-DM1 was prepared according to Example 3 (Ranson, M. and Sliwkowski M. (2002) Oncology 63 (suppl 1): 17-24). Disulfide antibody-drug conjugates were also tested: trastuzumab-SPDP-DM1, trastuzumab-SPP-DM3, and trastuzumab-SPP-DM4, which had the following structure:

                曲妥单抗-SPDP-DM1

Trastuzumab-SPDP-DM1

Trastuzumab-SPP-DM3

Trastuzumab-SPP-DM4

The ADC of the present invention includes an SMCC linker and a DM1 maytansinoid drug moiety, represented by Ab-SMCC-DM1:

A specific form of Ab-SMCC-DM1 is Trastuzumab-SMCC-DM1, wherein p is 1, 2, 3, or 4 (Ab = Trastuzumab, Tr, WO 2005/037992). Another specific form of ADC is Trastuzumab-SIAB-DM1 (Trastuzumab = Tr), which has the following structure:

Screening of antibody-drug conjugates (ADCs) targeting tumor-associated antigens and cell surface receptors

Transgenic animals and cell lines are particularly useful for screening antibody-drug conjugates (ADCs) with potential for prophylactic or therapeutic treatment of diseases or disorders involving overexpression of tumor-associated antigens and cell surface receptors such as HER2 (US 6632979). Screening for available ADCs may involve administering a range of doses of a candidate ADC to transgenic animals and testing the effect of the ADC on the disease or disorder being assessed at various time points. Alternatively, or in addition, the drug may be administered prior to or concurrently with exposure to the disease-inducing factor, if applicable. Candidate ADCs can be screened serially and separately, or simultaneously, in culture or high-throughput screening arrays. The rate at which ADCs can be screened for use in the prophylactic or therapeutic treatment of a disease or disorder is limited only by the rate of synthesis or screening methodology including detection/assay/data analysis.

A specific protocol for the screening method includes (a) transplanting cells from a stable breast cancer cell line into a non-human animal, (b) administering an ADC candidate drug to the non-human animal and (c) determining that the candidate drug inhibits tumors from the transplanted cells ability to form relationships. The present invention also relates to a method of screening candidate ADCs for the treatment of a disease or disorder characterized by overexpression of a receptor protein comprising (a) combining cells from a stable breast cancer cell line with Candidate drug exposure, and (b) assessing the ability of candidate ADCs to inhibit the growth of stable cell lines.

A specific protocol of the screening method involves (a) contacting cells from a stable breast cancer cell line with an ADC candidate drug, and (b) assessing the ability of the candidate ADC to block ligand activation of HER2. Another embodiment evaluates a candidate ADC for its ability to block heregulin binding. In another embodiment, candidate ADCs are assessed for their ability to block ligand-stimulated tyrosine phosphorylation.

Another embodiment of the screening method involves (a) contacting cells from a stable breast cancer cell line with an ADC candidate drug, and (b) assessing the ability of the candidate ADC to induce cell death. In one embodiment, a candidate ADC is assessed for its ability to induce apoptosis.

Another embodiment of the screening method comprises (a) administering an ADC candidate drug to a transgenic non-human mammal that overexpresses, for example, a native human protein, such as HER2, or a fragment thereof, in its mammary cells, wherein such transgenic mammal has encoded a native The nucleic acid sequence of the human protein or its biologically active fragment of the natural human protein is stably integrated into its genome, the nucleic acid sequence is operably linked to a transcriptional regulatory sequence that directs its expression, and develops a response to antibody therapy such as anti- HER2 antibody non-responsive or poorly responsive tumors, such as breast tumors, or administered to a non-human mammal having a tumor transplanted from said transgenic non-human mammal; and (b) assessing the effect of the candidate ADC on the target disease or disorder . Without limitation, the disease or disorder may be a HER2-overexpressing cancer, such as breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, and bladder cancers. The cancer may be breast cancer expressing HER2 at least about 500,000 copies per cell, or at least about 2,000,000 copies per cell. For example, the ability of an ADC drug candidate to induce cell death and/or apoptosis can be assessed using assay methods well known in the art and described below.

In one embodiment, candidate ADCs are screened by administering a range of doses of the candidate ADC to transgenic animals and evaluating the animal's physiological response to the compound over time. Administration can be oral, or by injection as appropriate, depending on the chemical nature of the compound being assessed. At times, it may be appropriate to administer the compounds with co-factors that enhance the efficacy of the compounds. If the cell line derived from the subject transgenic animal is used to screen for compounds useful in the treatment of different disorders associated with overexpression of certain tumor-associated antigenic proteins or cell surface receptors, such as HER2-overexpression, then at The test compound is added to the cell culture medium at an appropriate time, and the cellular response to the compound is assessed over time using appropriate biochemical and/or histological tests. Sometimes it may be appropriate to apply a compound of interest to the medium together with cofactors that will enhance the potency of the compound.

Accordingly, the present invention provides assays for the identification of ADCs that specifically target and bind to overexpressed HER2 proteins, the presence of which is associated with abnormal cell function and breast cells that are involved in the etiology of breast tumorigenesis. Involved in the pathogenesis of proliferation and/or differentiation.

In order to identify ADCs that block ligand activation of ErbB (e.g., ErbB2) receptors, compounds that block the binding of an ErbB ligand to an expressed ErbB (ErbB2) receptor (e.g., in coupling to another ErbB receptor in which The ability of the ErbB receptor of interest to bind to the cell of said another ErbB receptor to form an ErbB hetero-oligomer). For example, cells isolated from transgenic animals overexpressing HER2 and transfected to express another ErbB receptor with which HER2 forms hetero-oligomers can be incubated with ADCs and then contacted with labeled ErbB ligands . Compounds can then be assessed for their ability to block ligand binding to the ErbB receptor in the ErbB hetero-oligomer.

For example, candidate ADCs that inhibit heregulin (HRG) binding to breast tumor cell lines that overexpress HER2 and are cultured in a 24-well plate on ice as a monolayer can be evaluated for expression by the transgenic non-human mammals herein. (e.g. mice) established. Anti-ErbB2 monoclonal antibody can be added to each well and incubated for 30 minutes. ¹²⁵ I-labeled rHRGβ1 _177-224 (25,000 cpm) can then be added and incubation can be continued for 4 to 16 hours. Dose response curves can be prepared and _IC50 values can be calculated for compounds of interest.

Alternatively, or in addition, ADCs can be assessed for blocking ErbB ligand-stimulated tyrosine phosphorylation of ErbB receptors present in ErbB hetero-oligomers. For example, here a cell line established from a transgenic animal can be incubated with a test ADC and then tested for ErbB ligand-dependent tyrosine with an anti-phosphotyrosine monoclonal antibody, optionally coupled to a detectable label. Phosphorylation activity. The kinase receptor activation assay described in US 5766863 can also be used to determine ErbB receptor activation and blockade of activity by compounds.

In one embodiment, ADCs that inhibit HRG stimulation of pi 80 tyrosine phosphorylation can be screened in MCF7 cells essentially as follows. For example, cell lines established from HER2 transgenic animals can be added to 24-well plates, compounds can be added to each well and incubated at room temperature for 30 minutes; rHRGβ _1177-244 can be added to each well to a final concentration of 0.2 nM Concentration, you can continue to keep warm for about 8 minutes. The medium can be aspirated from each well and 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 [mu]l) can be run on a 4-12% gradient gel (Novex) and then electrophoretically transferred to a polyvinylidene fluoride membrane. Anti-phosphotyrosine (at 1 μg/ml) immunoblots can be developed and the intensity of the major reactive band at Mr-180,000 can be quantified by reflectance densitometry. An alternative method to assess inhibition of receptor phosphorylation is the KIRA (Kinase Receptor Activation) assay (Sadick et al (1998) Jour. of Pharm. and Biomed. Anal. 1-9). In this test, some well-established anti-HER2 monoclonal antibodies, which are known to inhibit HRG-stimulated pl80 tyrosine phosphorylation, can be used as positive controls. Dose response curves for the inhibition of HRG-stimulated pi 80 tyrosine phosphorylation as measured by reflectance densitometry can be prepared and _IC50 's for compounds of interest can be calculated.

Growth inhibitory effects of test ADCs on cell lines derived from HER2 transgenic animals can also be assessed (Schaefer et al (1997) Oncogene 15: 1385-1394). According to this test, cells can be treated with different concentrations of the test compound for 4 days and stained with gentian violet or the redox dye Alamar Blue. Incubation with the compounds showed a growth inhibitory effect on this cell line similar to that shown by the monoclonal antibody 2C4 on MDA-MB-175 cells (Schaefer et al., supra). In another embodiment, exogenous HRG does not significantly reverse this inhibition.

To identify growth inhibitory ADC compounds that specifically target HER2, ADCs that inhibit the growth of HER2-overexpressing cancer cells derived from transgenic animals can be screened (US 5677171). According to this test, HER2 overexpressing cells were cultured in a 1:1 mixture of F12 and DMEM medium supplemented with 10% fetal calf serum, glutamine and penicillin streptomycin. Cells were plated at 20,000 cells (2mls/35mm dish) in 35mm cell culture dishes and test compounds were added at different concentrations. Six days later, the number of cells compared to untreated cells was counted using an electronic COULTER ^™ cell counter. Those ADCs that inhibit cell growth by about 20-100%, or about 50-100%, can be selected as growth inhibitory compounds.

To select ADCs that induce cell death, loss of membrane integrity, indicated by eg PI, trypan blue or 7AAD uptake, can be assessed relative to controls. The PI uptake assay uses cells isolated from mammary tumor tissue of transgenic animals. According to this test, cells were cultured in Dulbecco's Modified Eagle Medium (-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM L-glucose Aminoamide. Thus, the test is performed in the absence of complement and immune effector cells. Cells were seeded in 100 x 20 mm dishes at a density of 3 x ¹⁰⁶ per dish and allowed to attach overnight. The medium was then removed and replaced with fresh medium alone or with medium containing different concentrations of the compound. The cells were incubated for a period of 3 days. After each treatment, cell monolayers were rinsed with PBS and deattached by trypsinization. Cells were then centrifuged at 1200 rpm at 4°C for 5 minutes, and the pellet was resuspended in 3 ml of ice-cold Ca ²⁺ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ), and aliquoted into 35 mm capped strainers 12×75 mm tubes (1 ml per tube, 3 tubes per treatment group) to remove cell clots. PI (10 μg/ml) was then added to the tube. Samples can be analyzed with a FACSCAN( ^TM) flow cytometer and FACSCONVERT ^(TM) CellQuest software (Becton Dickinson). Those compounds that induce statistically significant levels of cell death as measured by PI uptake can be selected as cell death inducing compounds.

To select compounds that induce apoptosis, an annexin binding assay was performed with cells established from mammary tumor tissue of transgenic animals. Cells were cultured and seeded in Petri dishes as discussed in the previous paragraph. The medium was then removed and replaced with fresh medium alone or with medium containing 10 μg/ml antibody-drug conjugate (ADC). After a three-day incubation period, cell monolayers were rinsed with PBS, and attachments were lysed by trypsinization. Cells were then centrifuged, resuspended in Ca2 ⁺ binding buffer, and aliquoted into the aforementioned tubes for cell death assay. Labeled annexin (eg annexin V-FITC) (1 μg/ml) is then added to the tube. Samples can be analyzed with a FACSCAN( ^TM) flow cytometer and FACSCONVERT ^(TM) CellQuest software (Becton Dickinson). Those compounds that induced statistically significant levels of annexin binding relative to controls were selected as apoptosis-inducing compounds.

In Vitro Cell Proliferation Assay

Typically, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is determined by contacting mammalian cells bearing a tumor-associated antigen or receptor protein with antibodies to the ADC in cell culture medium; Cells for a period of about 6 hours to about 5 days; cell viability is determined. Cell-based in vitro assays were used to determine viability, proliferation ( _IC50 ), cytotoxicity ( _EC50 ), and induction of apoptosis (caspase activation) of ADCs.

The in vitro potency of the antibody-drug conjugates was determined by cell proliferation assays (Figures 1-4). CellTiter- ^{Glo_Luminescent} Cell Viability Assay is a commercially available (Promega Corp., Madison, WI) homogeneous assay based on recombinant expression of Coleoptera luciferase (US 5583024 ; US 5674713; US5700670). This cell proliferation assay determines the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; US 6602677). The CellTiter- ^Glo® assay is implemented in 96-well plates, making it amenable to automated high-throughput screening (HTS) (Cree (1995) AntiCancer Drugs 6:398-404). The homogeneous assay method involves the direct addition of a single reagent (CellTiter- ^Glo_Reagent ) to cells cultured in serum-supplemented media. Cell washing, media removal, and multiple pipetting steps are not required. Ten minutes after reagent addition and mixing, the system detected 15 cells/well in a 384-well array.

The homogeneous "add-mix-assay" format results in cell lysis and produces a luminescence signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter- ^Glo® Assay produces a "glow-type" luminescent signal that is generated by a luciferase reaction and typically has a half-life of greater than five hours, depending on the cell type and media used. Viable cells are reflected in relative light units (RLU). The substrate Beetle luciferin is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons. The extended half-life eliminates the need to use a reagent injector and provides flexibility for multi-plate sequential or batch mode processing. The cell proliferation assay can be used in a variety of multiwell arrays, eg, 96 or 384 well arrays. Data can be recorded with a photometer or CCD camera imaging device. Fluorescence output is provided in relative light units (RLU), which are measured over time.

For four different breast tumor cell lines, the anti-proliferation effects of the three antibody-drug conjugates were determined by the above-mentioned cell proliferation and in vitro cell killing tests (Fig. 1-4). Figure 1 shows that 3 days after treatment of SK-BR-3 (HER2 3+) breast tumor cells, the concentrations of trastuzumab-SPP-DM1, trastuzumab-SPDP-DM1 and trastuzumab-SMCC-DM1 were gradually increased time-efficiency measurements. Figure 2 shows that after 3 days of treatment with BT-474 (HER2 3+) breast tumor cells, the concentration of trastuzumab-SPP-DM1, trastuzumab-SPDP-DM1 and trastuzumab-SMCC-DM1 were gradually increased. potency measure. Figure 3 shows potency measurements at increasing concentrations of Trastuzumab-SPP-DM1, Trastuzumab-SPDP-DM, and Trastuzumab-SMCC-DM1 after 3 days of treatment of MCF7 (HER2-low) breast tumor cells . Figure 4 shows that after treatment of MDA-MB-468 (HER2-negative) breast tumor cells for 3 days, when the concentrations of Trastuzumab-SPP-DM1, Trastuzumab-SPDP-DM1 and Trastuzumab-SMCC-DM1 were gradually increased, measures of effectiveness.

_IC50 values were determined for SK-BR-3 and BT-474, which are known to overexpress the HER2 receptor protein. For 6 assays on SK-BR-3 cells, many conjugates, Trastuzumab-SPP-DM1 (2.8 DM1(Drug/Ab) per Trastuzumab) gave an average IC of 14.4 μg/ml ₅₀ , ranging from 9.1 to 22.3 μg/ml, for 4 assays on BT-474 cells, resulting in an average IC ₅₀ of 51.7 μg/ml, ranging from 28.7 to 63.1 μg/ml. 4 assays on SK-BR-3 cells, many conjugates Trastuzumab-SMCC-DM1 (2.7 DM1(Drug/Ab) per Trastuzumab) gave an average _IC50 of 15.2 μg/ml, It ranged from 12.6 to 18.8 μg/ml with an average _IC50 of 94.9 μg/ml for 2 experiments against BT-474 cells, which ranged from 75.2 to 114.6 μg/ml. The conjugate was inactive against HER2 overexpressing cells MCF7 and MDA-MB-468.

Strong in vitro cell killing (IC ₅₀ =<0.25 μg/ml) by anti-CD19-SMCC-DM1 was shown with Raji cells, where naked antibody and control ADC, Trastuzumab-SMCC-DM1 showed no effect in Raji cells. Strong in vitro cell killing (IC ₅₀ =<0.25 μg/ml) of anti-CD79a-SMCC-DM1 and anti-CD79b-SMCC-DM1 was shown with Ramos cells, where naked antibody and control ADC, trastuzumab- SMCC-DM1 showed no effect.

Figure 17 shows the in vitro cell proliferation test (Example 5) implemented by HT1080EphB2 (C8) cells using an anti-EphB2R 2H9 antibody-drug conjugate: 2H9-SPP-DM1 (IC ₅₀ 80ng/ml ), and 2H9-SMCC-DM1 (IC ₅₀ 50ng/ml) treated.

Serum clearance and stability in mice

ADC serum clearance and stability were studied in nude, naive (no tumor received by exogenous transplantation) mice. Figure 5 shows serum clearance in beige nude mice with no Trastuzumab-SMCC-DM1 tumors versus no Trastuzumab-SPP-DM1 tumors, measuring conjugate and total antibody at six time points over a 7-day period serum concentration. The difference in the amount of total antibody and ADC shows cleavage of the linker and separation of the antibody from its DM1 portion. SMCC-linked ADCs remained intact in vivo longer than SPP-linked conjugates, as indicated by higher conjugated antibody serum concentrations at day seven.

Figure 6 shows the stability over time of the following conjugates in tumor-free nude mice: Trastuzumab-SPDP-DM1, Trastuzumab-SPP-DM1, Trastuzumab-SPP-DM3, Trastuzumab Anti-SPP-DM4, and Trastuzumab-SMCC-DM1, serum concentrations were determined at six time points over 7 days. ADCs with SMCC linkers are more stable in vivo than conjugates linked by SPP or SPDP, although trastuzumab-SMCC-DM1 has approximately the same - Same stability as SPP-DM4.

The experiments shown in Figures 5 and 6 were performed in tumor-free nude mice. However, in tumor-bearing nude mice, the SMCC-linked conjugates showed the same enhanced stability compared to the SPP-linked conjugates. As shown in Figure 7, after 7 days of treatment, approximately 72% of the initial trastuzumab-SMCC-DM1 remained as a conjugate, whereas after 7 days of treatment, only approximately 10% of the initial trastuzumab-SPP-DM1 remained For conjugates, the enhanced stability of the non-enzymatically cleavable trastuzumab-SMCC-DM1 conjugate in tumor-bearing nude mice was shown.

Serum clearance and stability in rats

Figures 8 and 9 show the relative stability and clearance profiles of a disulfide linker ADC (trastuzumab-SPP-DM1 ) and a non-disulfide linker ADC (trastuzumab-SMCC-DM1 ) in rats. The parameters studied included: parameter Trastuzumab-SPP-DM1 Trastuzumab-SMCC-DM1 Vd (ml/kg 41 41 Clearance (ml/day/kg) 52 15 T ¹ / ₂ alpha 0.09 0.15 T ¹ / ₂ beta 0.7 0.85 T ¹ / ₂ gamma (day) 2.6 5.5

Compared with disulfide linker ADC Trastuzumab-SPP-DM1 (Figure 8), the non-disulfide linker ADC Trastuzumab-SMCC-DM1 (Figure 9) showed more good stability.

In vivo efficacy

The efficacy of the antibody-drug conjugates of the invention can be determined by implanting cancer cell allografts or xenografts in rodents and treating the tumor with ADC. Depending on the cell line, the antibody binding specificity of the ADC to the receptor present on the cancer cell, the dosing regimen, and other factors, expected results will vary. The in vivo efficacy of anti-HER2 ADCs was determined using a high-expressing HER2 transgenic explant mouse model. Propagation of allografts from Fo5 mmtv transgenic mice that do not respond or respond poorly to HERCEPTIN treatment. Subjects were treated once with ADC and monitored for 3-6 weeks to determine tumor doubling time, log cell kill, and time to tumor shrinkage. Follow dose-response and conduct multiple dose trials.

Tumors readily developed in transgenic mice expressing mutant activating neu, the rat homologue of HER2, but unmutated in overexpressed HER2 in breast cancer, and in transgenic mice overexpressing non-mutated HER2. In mice, tumor formation is much less intense (Webster et al (1994) Semin. Cancer Biol. 5:69-76).

To enhance tumor formation with non-mutated HER2, transgenic mice were produced with a HER2 cDNA plasmid in which the upstream ATG was deleted to prevent translation initiation at this upstream ATG codon, which would otherwise be reduced from the downstream true The frequency with which the HER2 initiation codon initiates translation (see, eg, Child et al (1999) J. Biol. Chem. 274:24335-24341). In addition, the addition of a chimeric intron to the 5' end will also enhance expression levels as previously reported (Neuberger and Williams (1988) Nucleic Acids Res. 16:6713; Buchman and Berg (1988) Mol. Cell Biol. 8: 4395; Brinster et al (1988) Proc. Natl. Acad. Sci. USA 85: 836). The chimeric intron is derived from the Promega vector, pCI-neo mammalian expression vector (bp 890-1022). On one side of the 3' end of the cDNA are human growth hormone exons 4 and 5, and polyadenylation sequences. Additionally, FVB mice were used because this strain is more susceptible to tumor development. A promoter from MMTV-LTR was used to ensure tissue-specific HER2 expression in the mammary gland. Animals were fed AIN 76A diet to enhance susceptibility to tumor formation (Rao et al (1997) Breast Cancer Res. and Treatment 45: 149-158).

Figures 10-13 show that ADCs have strong antitumor activity in allografts of HER2 positive tumors (Fo5) that originally occurred in MMTV-HER2 transgenic mice. In this model, only antibodies (such as trastuzumab) do not have significant antitumor activity (Erickson et al US 6632979). As shown in Figures 10 and 11, treatment with ADC blocked tumor growth compared to control (vehicle) growth levels. Treatment with trastuzumab-SMCC-DM1 and trastuzumab-SIAB-DM1 conjugates slowed tumor growth the most. As shown in Figures 10, 12 and 13, the trastuzumab-SMCC-DM1 conjugate slowed tumor growth more than the conjugate with the SPP linker, i.e. was more potent, both as measured by the time to tumor doubling in nude mice Log Cell Kill was also determined corresponding to the indicated doubling time measurements (Figure 13).

The in vivo efficacy of anti-CD22 ADCs was determined using a mouse tumor xenograft model. On day 1 (unless otherwise specified), quantitative administration of anti-CD22 antibody-drug conjugate or naked (naked) antibody to mice in the test group once, eight SCID mice in each test group, each mouse had 2,000 Ten thousand Bjab-luc (luciferase-expressing Bjab cells) xenograft tumor cells (Example 8).

ADC or Ab μgDM1/m2 Abmg/kg mouse Average Drug Load (p) MTD(day) Tr-SMCC-DM1 200 4.2 3.2 3 7A2-SMCC-DM1 200 3.8 3.6 6 5E8-SMCC-DM1 200 3.8 3.6 10 RFB4-SMCC-DM1 200 3.2 4.25 18 RFB4-SMCC-DM1 (dosed 3X on days 1, 7, 14) 405 10 2.75 55 7A2 - 4 - 3 5E8 - 4 - 3 RFB4 - 4 - 3

The time to tumor size doubling (MTD, mean tumor doubling time) was determined. The three naked anti-CD22 antibodies showed essentially no efficacy relative to the non-specific binding ADC (trastuzumab-SMCC-DM1). The corresponding conjugates all showed obvious effects of retarding tumor growth. The effect of multiple dosing was established with RFB4-SMCC-DM1, where the MTD for mice dosed once was 18 days and the MTD for mice dosed three times on days 1, 7, and 14 was 55 days. Complete tumor regression occurred in all 8 mice in the three dose groups. 7 days after administration of a single dose (400 μg DM1/m2) in SCID mice with 20 million Bjab-luc xenograft tumor cells per mouse, in which antibodies were 12F7, 9A8, 8C9, 8G10, 3F11, 10D2 Other anti-CD22-SMCC-DM1 conjugates of , 6C9, 14D1, and 11H10 showed a reduction in initial tumor volume or a delay in tumor growth relative to the control (trastuzumab-SMCC-DM1 ). In SCID mice with 5 million Ramos RA1 xenograft tumor cells per mouse, anti-CD22 conjugates RFB4-SMCC-DM1, 5E8-SMCC- DM1, and 7A2-SMCC-DM1 were also effective in arresting tumor growth relative to the control (trastuzumab-SMCC-DM1).

The conjugate RFB4-SMCC-DM1 (Example 8) was studied at three different drug loadings on groups of ten SCID mice each bearing Bjab-luc xenografts. The low (1.95) and medium (3.7) drug-loaded conjugates showed significant tumor growth retardation, respectively, with an MTD of about 15 days. The high loading (6.75) conjugate did not show an effect significantly different from the control conjugate GP120-SMCC-DM1, or the naked antibody RFB4.

Anti-CD22 ADC or Ab μgDM1/m2 Ab mg/kg mouse Average Drug Load (p) MTD(day) RFB4 - 10 - 3 RFB4-SMCC-DM1 (low load) 144 5 1.95 15 RFB4-SMCC-DM1 (medium load) 273 5 3.7 15 RFB4-SMCC-DM1 (high load) 497 5 6.75 3 GP120-SMCC-DM1 (high load) 449 5 6.1 3

Anti-CD19-SMCC-DM1 and anti-CD22-SMCC-DM1 conjugates showed no in vivo activity in the Raji cell mouse tumor xenograft model. Other anti-CD19 and anti-CD22 conjugates may have in vivo activity against other cancer cell tumor models.

The in vivo efficacy of anti-CD79a (alpha) and anti-CD79b (beta) ADCs was determined using a mouse tumor xenograft model. Groups of 8 SCID mice each with 20 million Biab-luc xenograft tumor cells per mouse were dosed on Day 1 with the samples in the table below and in Example 8 that follows. ADC, Ab, or control μgDM1/m2 Ab mg/kg mouse Average Drug Load (p) MTD (days) PBS (buffer control) - - - 3.5 Anti-GP120 - 3.2 - 3.5 SN8 anti-CD79b - 3.1 - 4 17A7 anti-CD79b - 3.1 - 4 8H9 anti-CD79a - 4.0 - 3 Anti-GP120-SMCC-DM1 200 3.2 4.2 3.5 SN8 anti-CD79b-SMCC-DM1 200 3.1 4.4 >7 17A7 anti-CD79b-SMCC-DM1 200 3.1 4.4 >7 8H9 anti-CD79a-SMCC-DM1 200 4.0 3.4 >7

After 7 days, the conjugates SN8 anti-CD79b-SMCC-DM1, 17A7 anti-CD79b-SMCC-DM1, and 8H9 anti-CD79a-SMCC-DM1 all showed a reduction in initial tumor volume (160 mm3 on average). In the test group of 8 mice, the conjugate SN8 anti-CD79b-SMCC-DM1 achieved partial regression (PR) in 4 animals and complete regression (CR) in 2 animals. Conjugate 17A7 anti-CD79b-SMCC-DM1 achieved CR in 1 animal. Conjugate 8H9 anti-CD79a-SMCC-DM1 achieved PR in 2 animals and CR in 1 animal. 8 days after administration of a single dose (192 μg DM1/m2) in CB17 ICR SCID mice with 20 million Bjab-luc xenograft tumor cells per mouse, where antibodies were 2F2, 5C3, 7H7, 8D11, 15E4 Other anti-CD79b-SMCC-DM1 conjugates of , , and 16C11 showed reduction in initial tumor volume or arrest of tumor growth relative to control (trastuzumab-SMCC-DM1 ).

The dose-response effect of mice administered anti-CD79b-SMCC-DM1 was determined. A test group of 8 SCID mice each with 20 million Bjab-luc xenograft tumor cells per mouse was dosed on Day 1 with the samples in the table below (Example 8). Anti-CD79b-SMCC-DM1 was dosed at levels of 0.5, 2.0, and 3.64 mg Ab/kg mouse. ADC, Ab or Control μgDM1/m2 Abmg/kg mouse MTD (days) Vehicle Control (PBS) - 4 anti-CD79b-SMCC-DM1 32 0.5 10 anti-CD79b-SMCC-DM1 130 2.0 35 anti-CD79b-SMCC-DM1 236 3.64 >70

The in vivo efficacy of anti-TENB2 ADCs was determined using a mouse tumor xenograft model. TENB2 is a tumor antigen that has been shown to be expressed almost exclusively in human prostate and overexpressed in human prostate tumors (Glynne-Jones et al (2001) Int J Cancer. Oct 15; 94(2): 178-84). PC3-TVA-919cv1:5 is a human prostate cancer cell line expressing high levels of TENB2.

Athymic nude mice were subcutaneously injected with 5 million PC3-TVA-919 high or medium expressing cells in a volume of 0.2 ml per mouse. Cells were suspended in HBSS. When the average tumor size reached 100-200 mm ³ , the mice were randomly divided into 8 groups of 8-10 mice each and given a single IV treatment of the following samples (Example 8).

ADC or control μgDM1/m2 Ab mg/kg mouse Medium expression MTD (days) High expression MTD (days) PBS (vehicle control) - - 30 twenty two Mouse anti-ragweed (ragweed)-SPP-DM1 (negative control) 373 6.68 18 twenty two Mouse anti-ragweed-SMCC-DM1 (negative control) 373 9.34 14 18 Mouse anti-TENB2: 3146-SPP-DM1 373 7.34 58 35 Mouse anti-TENB2: 3146-SMCC-DM1 373 10.6 44 19 Chimeric anti-TENB2-SPP-DM1 373 8.7 57 43 Chimeric anti-TENB2-SMCC-DM1 373 7.63 43 twenty two

The murine anti-TENB2-DM1 conjugate showed antitumor efficacy against PC3-TENB2 tumors relative to negative and vehicle controls. The murine 10H1 anti-NaPi3b-SMCC-DM1 conjugate showed no anti-tumor efficacy against PC3-NaPi3b tumors relative to negative and vehicle controls. Other antibody variants of anti-NaPi3b conjugates may have in vivo activity against PC3-NaPi3b tumor or other cancer cell lines.

Rodent Toxicity

Antibody-drug conjugates and a control "vehicle" without ADC were evaluated in an acute toxicity rat model. Toxicity of ADC was studied by treating female Sprague-Dawley rats with ADC followed by observation and analysis of the effects on various organs. ADC toxicity can be observed, identified, and measured based on gross observations (body weight), clinicopathological parameters (serum chemistry and hematology), and histopathology. Trastuzumab-SMCC-DM1 was found to be associated with less acute toxicity than trastuzumab-SPP-DM1 at equivalent dose levels.

Through a single injection of trastuzumab-SMCC-DM1 (two doses: 1860 and 3260 μg DM1/m ² ), combined with disulfide ADC, trastuzumab-SPP-DM1 (two doses: 1860 and 3260 μg DM1/m ² ), a 5-day acute toxicity study was carried out on adolescent female rats (100-125gms) compared to single injection (day 0) of free DM1 maytansine (thiol) and control vehicle. Body weight was measured daily. Clinical chemistry, serum enzymes, and hematology analyzes were performed on days 3 and 5; conclusions were made along with a complete autopsy for histopathological evaluation. Signs of toxicity included clinical observations of weight loss.

Following dosing of ADC, animal body weight loss, or changes in body weight, relative to animals administered vehicle alone is a gross and general indicator of systemic or local toxicity. Figure 14 shows the change in body weight (grams) over a period of 5 days. Rats receiving the disulfide ADC, Trastuzumab-SPP-DM1, showed significant, dose-dependent toxicity manifested by lethality at higher doses and body weight loss at lower doses. In contrast, rats receiving trastuzumab-SMCC-DM1 gained body weight, and rats given the lower dose showed no reduction in the rate of weight gain relative to rats given the placebo vehicle. Rats given higher levels of trastuzumab-SMCC-DM1 also gained body weight compared to free DM1 cytotoxin.

Hepatotoxicity was measured by increased liver enzymes, increased numbers of mitotic and apoptotic signs, and hepatocyte necrosis. Toxicity to the hematolymphoid system was observed by depletion of leukocytes, major granulocytes (neutrophils), and/or platelets, and involvement of lymphoid organs, ie, atrophic or apoptotic activity. Toxicity was also manifested as gastrointestinal lesions such as increased numbers of mitotic and apoptotic signs and degenerative entercolitis.

Enzyme markers of liver injury studied included:

AST (aspartate aminotransferase)

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

- Liver: plasma ratio of 7000:1

-T1/2: 17 hours

ALT (alanine aminotransferase)

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

- Liver: plasma ratio of 3000:1

-T1/2: 42 hours; diurnal variation

GGT (g-glutamyltransferase)

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

- Weak predictor of hepatic injury; often elevated in biliary disease

None of the three enzymes assayed above were liver specific. It was found that the ADC of the present invention caused a transient, slight increase in the liver enzymes ALT and AST, and a transient decrease in reticulocytes (Figure 15). No effects on peripheral blood granulocytes or platelets were observed (Figure 16).

Figures 15 and 16 show that in this five-day acute toxicity study, rats exposed to 22.3 mg/kg Trastuzumab-SPP-DM1 (group 2) showed the most severe clinical toxicity. These animals showed the most severe signs of weight loss, elevated liver function test values, leukopenia and thrombocytopenia, and morphologic toxicity targeting hematopoietic lymphoid tissue. The degree of toxicity was similar to that obtained in previous studies using the 25 mg/kg dose. In contrast, Group 3 and Group 4 animals administered Trastuzumab-SMCC-DM1 at doses of 10 and 25 mg/kg, respectively, could not be distinguished from vehicle-treated animals on the basis of clinicopathological and body weight data. Morphologically, the livers of these animals showed a slight increase in the number of mitotic figures, however, peripheral lymphoid and hematopoietic tissues were within the normal range.

Group 5 animals at 50 mg/kg Trastuzumab-SMCC-DM1 showed signs of toxicity. However, except for one liver function test (ALT), toxicity was less severe than in animals receiving 50% of the same drug dose as Trastuzumab-SPP-DM1 (Group 2). At approximately the same dose, Trastuzumab-SMCC-DM1 (Group 4, 22.3 mg/kg) exhibited approximately 25% of the AST levels of Trastuzumab-SPP-DM1 (Group 2, 25 mg/kg). By day 5 of the study, animals in Group 5 showed weight gain (following a transient weight loss on days 3 and 4), decreased serum bilirubin and increased platelet counts (Figure 15).

Animals exposed to the free maytansinoid DM1 (group 6) showed the same toxicity pattern as animals treated with the trastuzumab conjugate. This dose of free DM1 was equivalent to the amount of drug given in the previous study at the 10 mg/kg Trastuzumab-SPP-DM1 dose. The severity of toxicity in Group 6 animals was less than that seen in Group 2 animals, but greater than previously seen in animals treated with 10 mg/kg Trastuzumab-SPP-DM1. Recovery appeared to be fairly rapid: in animals treated with free maytansine, spleen sections showed increased numbers of immature hematopoietic components; meanwhile, by day 5, in group 6 animals, LFTs and clinical hematological parameters showed a clear trend toward trend toward normalization.

Macaque Toxicity/Safety

Toxicity and safety of ADC administered to Cynomolgus monkeys can be assessed. Toxicity/safety studies of the antibody-drug conjugate trastuzumab-SMCC-DM1 were performed in macaques. Three groups of monkeys were studied to assess the toxicity of intravenously administered Trastuzumab-SMCC-DM1 relative to the control (vehicle), with escalating doses of Trastuzumab-SMCC-DM1 injected. Group 1 (4 subjects) received only vehicle (PBS, pH 6.5, ADC-depleted formulation) on days 1 and 22, after which necropsy was performed on day 36. Group 2 (4 subjects) received trastuzumab-SMCC-DM1 4900 μg/m2 on days 1 and 22. Group 3 (4 subjects) received Trastuzumab-SMCC-DM1 7200 μg/m2 on day 22.

Hepatotoxicity was inferred from elevated liver enzyme measurements in rodent toxicity studies. Cynomolgus monkeys were dosed with vehicle (group 1) and trastuzumab-SMCC-DM1 (group 2: 4900 μg/m ² ; group 3: 7200 μg/m ² ). In rhesus monkeys, liver enzyme AST, platelet count, white blood cells, absolute neutrophils, erythrocytes and reticulocytes have been measured for trastuzumab-SMCC-DM1, compared with 2 IV administration schemes (embodiment 10 ).

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic antibody-drug conjugates (ADCs) can be administered by any route appropriate to the condition to be treated. ADCs are typically administered parenterally, ie infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, bolus, intratumoral injection or epidurally (Shire et al (2004) J.Pharm. Sciences 93(6):1390-1402). Pharmaceutical formulations of therapeutic antibody-drug conjugates (ADCs) are typically prepared for parenteral administration with a pharmaceutically acceptable parenteral vehicle and are in injectable unit dosage form. The antibody-drug conjugate (ADC) with desired purity is optionally mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer, and can be in the form of a lyophilized preparation or an aqueous solution (Remington's Pharmaceutical Sciences ( 1980) 16th edition, Osol, A.Ed.).

Acceptable parenteral vehicles, diluents, carriers, excipients, and stabilizers are nontoxic to the subject at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other Organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexaquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or Benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low Molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine , arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions such as sodium; metal complexes (eg, zinc-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibody formulations are described in WO 97/04801, which is expressly incorporated herein by reference. An exemplary formulation of ADC such as Trastuzumab-SMCC-DM1 contains about 100 mg/ml trehalose (2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl) Tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol; C ₁₂ H ₂₂ O ₁₁ ; CAS No. 99-20-7) and about 0.1% TWEEN ^TM 20 (Sorberry Alcohol ester 20; 2-[2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl laurate; C ₂₆ H ₅₀ O ₁₀ ; CAS No. 9005-64-5), pH about 6.

A pharmaceutical formulation of a therapeutic antibody-drug conjugate (ADC) may comprise an amount of a non-reactive drug moiety (D), an antibody-linker intermediate (Ab-L), and/or a drug-linker intermediate (DL), It is the result of incomplete purification and separation of excess reagents, impurities, and by-products during ADC preparation; or the result of hydrolysis or degradation affected by time/temperature during bulk ADC storage or formulation ADC composition storage. For example, the ADC formulation Trastuzumab-SMCC-DM1 may contain detectable amounts of free drug DM1. Alternatively, or in addition, it may comprise a detectable amount of the drug-linker intermediate DM1-SMCC. Alternatively, or in addition, it may comprise a detectable amount of the antibody trastuzumab. Exemplary formulations of Trastuzumab-SMCC-DM1 may contain up to 10% molar equivalents of DM1-SMCC. Unexpectedly, compared to free drug DM1 (IC ₅₀ 0.0045 μM), DM1-SMCC (IC ₅₀ 0.05 μM) was approximately 20-fold less potent in cell killing.

For example, active pharmaceutical ingredients can also be packaged in prepared microcapsules by coacervation techniques or interfacial polymerization, for example in hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively, Packaged in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release preparations can be prepared. Examples of suitable sustained release preparations include semipermeable matrices of solid hydrophobic polymers comprising the ADC, wherein the matrix is in the form of a shaped particulate product, such as a film, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methyl acrylate), or polyvinyl alcohol, polylactide (US 3773919), L-glutamic acid, and gamma-ethyl L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPO ^TM (composed of lactic acid-glycolic acid copolymer and leuprolide acetate can be injection microspheres), and poly-D-(-)-3-hydroxybutyrate.

Preparations for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

Such formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are made by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Aqueous suspensions contain the active material (ADC) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia, and dispersing or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of ethylene oxide and fatty acids (e.g., polyoxyethylene stearate), ethylene oxide and long-chain fatty acids Condensation products of alcohols (for example, heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more Sweeteners such as sucrose or saccharin.

The pharmaceutical composition of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or a prepared into freeze-dried powder. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are traditionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are likewise useful in the preparation of injectables.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution for intravenous infusion may contain from about 3 to 500 μg of active ingredient per milliliter of solution to enable an infusion rate of about 30 mL/hr of a suitable volume. Subcutaneous (bolus) administration can be accomplished with a total volume of about 1.5 ml or less and a concentration of about 100 mg ADC per ml. For ADCs that require frequent and chronic administration, the subcutaneous route can be used, such as by prefilled syringe or autoinjector technology.

As a general recommendation, the initial pharmaceutically effective amount per dose of ADC administered will be in the range of about 0.01-100 mg/kg, i.e. about 0.1 to 20 mg/kg of patient body weight per day, with typical initial ranges of compounds used being about 0.3 to 15mg/kg/day. For example, human patients may initially be dosed at about 1.5 mg ADC per kg of patient body weight. The dose may be escalated up to the maximum tolerated dose (MTD). The dosing regimen may be about every 3 weeks, but may be more or less frequent depending on the condition or response being diagnosed. During the course of treatment, the dose may be further adjusted to be at or below the MTD, which may be safely administered in multiple cycles, such as about 4 or more.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended subject; and water and Non-aqueous sterile suspensions may contain suspending and thickening agents.

Although oral administration of protein therapeutics is generally discouraged because of low bioavailability due to limited intestinal (gut) absorption, hydrolysis or denaturation, formulations of ADCs suitable for oral administration can be prepared as discrete units such as each containing a predetermined amount of ADC. Capsules, cachets or tablets.

The formulations may be packaged in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of non-alcohol immediately before use. A liquid carrier such as water is used for injection. Immediate injectable solutions and suspensions are prepared from sterile powders, granules and tablets of the type previously described. Exemplary unit dosage formulations contain a daily dose, or unit daily sub-dose, or an appropriate fraction thereof, of active ingredient.

The invention further provides a veterinary composition comprising at least one active ingredient as defined above together with a veterinary carrier. A veterinary carrier is a material for the purpose of administering the composition, which may be a solid, liquid or gaseous material, and which is inert or acceptable in the veterinary field and which is compatible with the active ingredient. compatible. These veterinary compositions may be administered parenterally, orally or by any other desired route.

Antibody-Drug Conjugate Therapy

The antibody-drug conjugates of the invention will be useful in the treatment of a variety of diseases or disorders, such as cancer and autoimmune disorders. Exemplary conditions or disorders include benign or malignant tumors; leukemia and lymphoid malignancies; other diseases such as neuronal, glial, astrocytic, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocytic (blastocoelic), inflammatory, angiogenic and immunological diseases. Cancers sensitive to ADC therapy include those characterized by overexpression of specific tumor-associated antigens or cell surface receptors such as HER2.

ADC compounds identified in animal models and cell-based assays can be further tested in tumor-bearing primate and human clinical trials. Human clinical trials can be designed similarly to clinical trials testing the efficacy of the anti-HER2 monoclonal antibody HERCEPTIN in patients with HER2-overexpressing metastatic breast cancer who have previously received extensive anticancer therapy, as in Baselga et al. (1996) J. Clin. Oncol. 14:737-744 reported. Clinical trials can be designed to evaluate the efficacy of ADCs in combination with known treatment regimens, such as radiation and/or chemotherapy involving known chemotherapeutic and/or cytotoxic agents (Pegram et al (1999) Oncogene 18:2241-2251).

Typically, the disease or disorder to be treated is cancer. Examples of cancers to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and lung squamous cell carcinoma, peritoneal Carcinoma, hepatocellular carcinoma, gastric or gastric cancer, including gastrointestinal cancer, gastrointestinal stromal tumor (GIST), pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma ), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, hepatic cancer, anal cancer, penile cancer cancer, and head and neck cancer.

The cancer to be treated herein may be a cancer identified by overactivation of ErbB receptors, such as HER2. This hyperactivation may be due to overexpression or increased production of ErbB receptors or ErbB ligands. In one embodiment, a diagnostic or prognostic test will be performed to determine whether the patient's cancer is characterized by overactivation of ErbB receptors. For example, ErbB gene amplification and/or overexpression of ErbB receptors can be assayed in cancer. There are a variety of assays in the art for determining this amplification/overexpression, including the IHC, FISH, and shed antigen tests described above. Alternatively, or in addition, the level of an ErbB ligand, such as TGF-alpha, in or associated with a tumor can be measured according to known methods. Such tests can detect the protein and/or the nucleic acid encoding it in the sample being tested. In one embodiment, ErbB ligand levels in tumors can be determined using immunohistochemistry (IHC); see, eg, Scher et al. (1995) Clin. Cancer Research 1:545-550. Alternatively, or in addition, the level of ErbB ligand-encoding nucleic acid in the sample to be tested can be assessed; for example by FISH, southern blot, or PCR techniques. In one embodiment, ErbB2 overexpression can be analyzed by IHC, for example using HERCEPTEST (Dako). IHC can be performed on paraffin-embedded tissue sections from tumor biopsies based on the following criteria for the intensity of ErbB2 protein staining: score 0, no staining is observed or membranous staining is observed in less than 10% of tumor cells; score 1 +, faint/slightly discernible membrane staining detected in more than 10% of tumor cells with only a fraction of membrane staining; score 2+, weak to moderate membrane staining observed in more than 10% of tumor cells Complete membrane staining; score 3+, moderate to intense complete membrane staining was observed in more than 10% of tumor cells. Those tumors with an ErbB2 overexpression assessment score of 0 or 1+ can be identified as not overexpressing ErbB2, while those with a score of 2+ or 3+ can be identified as overexpressing ErbB2.

Alternatively, or in addition, FISH assays such as INFORM ^™ (Ventana Co., Ariz.) or PATHVISION ^™ (Vysis, 111.) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine tumor The extent (if any) of ErbB2 overexpression in

Alternatively, ErbB receptor or ErbB ligand overexpression or amplification can be assessed using in vivo diagnostic tests, for example by administering a molecule (such as an antibody) that binds to the molecule to be detected and bears a detectable label (such as a radioactive isotope), and externally The patient is scanned to locate the markers.

For the prophylaxis or treatment of a disease, the appropriate dosage of the ADC will depend on the type of disease to be treated, the severity and course of the disease, whether the molecule is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and The response to the antibodies, and the judgment of the attending physician. The molecules are suitable for administration to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, the initial candidate dose administered to the patient is about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of the molecule, whether e.g. in one or more separate administrations, or as a continuous infusion . Typical daily dosages may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Exemplary dosages of ADC to be administered to a patient range from about 0.1 to about 10 mg/kg of patient body weight.

For repeated administrations over several days or longer, depending on the condition, the treatment will be continued until the desired suppression of the desired disease symptoms is achieved. An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of anti-ErbB2 antibody. Other dosing regimens can also be used. The progress of this treatment can be easily monitored by conventional techniques and testing methods.

combination therapy

Antibody-drug conjugates (ADCs) can be combined with a second compound having anticancer properties as a pharmaceutical combination formulation, or as a dosing regimen for combination therapy. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the ADCs in the combination so that they do not adversely affect each other.

The second compound can be a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an aromatase inhibitor, a protein kinase inhibitor, a lipid kinase inhibitor, an antiandrogen, an antisense oligonucleotide Acids, ribozymes, gene therapy vaccines, antiangiogenic and/or cardioprotective agents. Such molecules are suitably provided in combination medicaments in amounts effective for the intended purpose. Pharmaceutical compositions comprising ADCs may also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin formation inhibitor, a topoisomerase inhibitor, or a DNA conjugate.

Alternatively, or in addition, the second compound may be an antibody that binds ErbB2 and blocks ligand activation of the ErbB receptor. The second antibody may be the monoclonal antibody 2C4 or the humanized 2C4 "Omnitarg" (WO 01/00245). The second antibody can be moiety conjugated to a cytotoxic or chemotherapeutic agent, such as maytansinoid, auristatin, calicheamicin, or 1,8 di-naphthalimide. For example, it may be desirable to further provide in a formulation or dosing regimen an antibody that binds EGFR, ErbB2, ErbB3, ErbB4, or vascular endothelial factor (VEGF).

Other treatment regimens may be combined with the administration of anticancer agents identified according to the present invention. Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. Co-administration includes simultaneous administration, using separate formulations or a single pharmaceutical formulation, as well as sequential administration in any order, where there is preferably a period in which both (or all) active agents exert their biological activity simultaneously.

In one embodiment, treatment with an ADC of the invention involves administration of an anticancer agent identified herein in combination with one or more chemotherapeutic or growth inhibitory agents, including coadministration of a cocktail-like mixture of different chemotherapeutic agents, optionally with Anti-ErbB2 antibodies such as trastuzumab are treated together. Chemotherapeutic agents include Erlotinib hydrochloride (CP-358774, TARCEVA ^™ ; Genentech/OSI), taxanes (such as paclitaxel and docetaxel), and/or anthracyclines. Preparation and dosing regimens of such chemotherapeutic agents may be used according to manufacturer's instructions, or as determined empirically by the skilled artisan. Preparation and dosing regimens for such chemotherapeutic agents are also described in Chemotherapy Service Ed., MC Perry, Williams & Wilkins, Baltimore, Md. (1992).

Anticancer agents may be combined with antihormonal compounds; for example antiestrogens such as tamoxifen; antiprogestins such as onapristone (EP 616812); or antiandrogens such as flutamide (flutamide), wherein these molecules Known doses are used. Where the cancer to be treated is a hormone-independent cancer, the patient may have previously received anti-hormonal therapy and, after the cancer has become hormone-independent, the patient may be administered an anti-ErbB2 antibody (and optionally an anti-ErbB2 antibody as described herein). other reagents). It may be beneficial to co-administer a cardioprotective agent (to prevent or reduce treatment-related myocardial dysfunction) or one or more cytokines to the patient. In addition to the treatments described above, patients may be treated with surgery to remove cancer cells, and/or with radiation therapy.

Suitable dosages of any of the above co-administered agents are those currently used, and may be reduced due to the combined action (synergy) of newly identified agents and other chemotherapeutic agents or treatments.

The combination therapy may provide "synergy" and prove "synergistic", ie the effect obtained when the active ingredients are used together is greater than the sum of the effects obtained with the compounds separately. Synergy may be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) administered by some other regimen effect. When delivered in alternation therapy, a synergistic effect may be obtained when the compounds are administered or delivered sequentially, for example by separate injections in different syringes. In general, during alternation therapy, effective doses of each active ingredient are administered sequentially, ie sequentially, while in combination therapy, effective doses of two or more active ingredients are administered together.

Metabolites of antibody-drug conjugates

In vivo metabolites of the ADC compounds described herein also fall within the scope of the invention, to the extent such products are novel and non-obvious relative to the prior art. Such products may, for example, result from oxidation, reduction, hydrolysis, amidation, esterification, enzymatic cleavage, etc. of the administered compound. Accordingly, the invention includes novel and non-obvious compounds produced by the method comprising contacting a compound of the invention with a mammal for a time sufficient to produce a metabolite thereof.

ADCs can be administered parenterally to animals such ^as rats, mice, ^guinea pigs, monkeys or administered to In humans, metabolites are identified by providing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating their conversion products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated by the use of antibodies capable of binding epitopes retained in metabolites). Metabolite structures are determined in a conventional manner, eg by MS, LC/MS or NMR analysis. In general, analysis of metabolites is done in the same manner as traditional drug metabolism studies well known to those skilled in the art. The conversion products, as long as they are not found in vivo, can be used in diagnostic tests for the therapeutic administration of ADC compounds.

Metabolites include products of in vivo cleavage of the ADC, where cleavage occurs at any linkages linking the drug moiety to the antibody. Thus metabolic cleavage can yield naked antibodies, or antibody fragments. Antibody metabolites can be attached to a portion of the linker, or to the entire linker. Metabolic cleavage may also result in the production of a drug moiety or a portion of the drug moiety. Metabolites of the drug moiety can be attached to a portion of the linker, or to the entire linker.

products

In another embodiment, an article of manufacture, or "kit," comprising an ADC and materials useful in the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or film packs. The container can be constructed from a variety of materials such as glass or plastic. The container contains an antibody-drug conjugate (ADC) composition effective in treating the condition of choice and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial, the tube vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used to treat the condition of choice, such as cancer. In one embodiment, the label or package insert indicates that the composition comprising an ErbB2-binding antibody is useful for treating a cancer expressing an ErbB receptor selected from the group consisting of epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4. Additionally, the label or package insert may indicate that the patient to be treated has a cancer characterized by overactivation of an ErbB receptor selected from EGFR, ErbB2, ErbB3, or ErbB4. For example, the cancer may be one that overexpresses these receptors and/or overexpresses one of the ErbB ligands such as TGF-α. The label or package insert may also indicate that the composition is useful for treating cancer, wherein the cancer is not characterized by overexpression of the ErbB2 receptor. In other embodiments, the package insert may indicate that the ADC composition may also be used to treat hormone-independent cancer, prostate cancer, colon cancer, or colorectal cancer.

The article of manufacture may comprise (a) a first container having a compound therein, wherein the compound comprises an ADC of the invention, wherein the antibody to the ADC is a primary antibody that binds ErbB2 and inhibits growth of ErbB2-overexpressing cancer cells; and (b ) a second container containing therein a compound comprising a second antibody that binds ErbB2 and blocks ligand activation of the ErbB receptor, or a conjugate of the second antibody and a maytansinoid. The article of manufacture of this embodiment may further comprise a package insert indicating that the first and second compounds are useful for treating cancer. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer solution and dextrose solution. It may further include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Example

Example 1 - Production, Characterization and Humanization of Anti-ErbB2 Monoclonal Antibody 4D5

The murine monoclonal antibody 4D5, which specifically binds the extracellular domain of ErbB2, was produced as described by Fendly et al (1990) Cancer Research 50: 1550-1558. Briefly, NIH 3T3/HER2-3 produced as described by Hudziak et al (1987) Proc. ₄₀₀ cells (expressing approximately 1 x ¹⁰⁵ ErbB2 molecules/cell) were used to immunize BALB/c mice. Mice were injected ip with ¹⁰⁷ cells in 0.5 ml PBS at weeks 0, 2, 5 and 7. Wheat germ agglutinin-sepharose (WGA) purified ErbB2 membrane extracts were injected ip into mice with immunoprecipitated ^32p -labeled ErbB2 at weeks 9 and 13. antiserum. Thereafter 0.1 ml of the ErbB2 preparation was injected iv to fuse the splenocytes with the mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for ErbB2-binding by ELISA and radioimmunoprecipitation.

The murine monoclonal antibody 4D5 was humanized using a "gene conversion mutagenesis" strategy as described in US 5821337, the entire contents of which are expressly incorporated herein by reference. The humanized monoclonal antibody 4D5 used in the experiments below is called huMAb4D5-8. The antibody is of the IgG1 isotype.

Purification of embodiment 2-trastuzumab

A vial containing 440mg HERCEPTIN_(huMAb4D5-8, rhuMAb HER2, US5821337) antibody) was dissolved in 50mL MES buffer (25mM MES, 50mM NaCl, pH 5.6), and the sample was loaded in the same buffer equilibrated On a cation exchange column (Sepharose S, 15cm×1.7cm). The column was then rinsed with the same buffer (5 column volumes). Trastuzumab was eluted by increasing the NaCl concentration of the buffer to 200 mM. Fractions containing the antibody were collected, diluted to 10 mg/ml, and dialyzed into a buffer containing 50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.

Example 3-Antibody-Drug Conjugate: Preparation of Trastuzumab-SPP-DM1:

Purified trastuzumab was derivatized with N-succinimide-4-(2-pyridylthio)pentanoate to introduce dithiopyridyl groups. Treat trastuzumab with SPP (5.3 molar equivalents in 2.3 mL ethanol), trastuzumab (376.0 mg, 8 mg/ml) dissolved in 44.7 mL of 50 mM potassium phosphate buffer containing NaCl (50 mM) and EDTA (1 mM) (pH 6.5). After incubation at ambient temperature under argon for 90 minutes, the reaction mixture was gel filtered through a Sephadex G25 column equilibrated with 35 mM sodium citrate, 154 mM NaCl, 2 mM EDTA. Antibody-containing fractions were collected and tested. The degree of modification of the antibody was determined as described above. The recovery of the modified antibody (trastuzumab-SPP-Py) was 337 mg (89.7%), in which 4.5 releasable 2-thiopyridyl groups were attached to each antibody (P').

Trastuzumab-SPP-Py (337.0 mg, 9.5 μmols of releasable 2-thiopyridine group) was diluted to a final concentration of 2.5 mg/mL with the above 35 mM sodium citrate buffer, pH 6.5. DM1(N ^2' -deacetyl-N ^2' -(3-mercapto-1- Oxopropionyl)-maytansine) (1.7 equivalents, 16.1 μmols), the structure of which is shown in Figure 1. The reaction was carried out at ambient temperature under argon for 20 hours.

The reaction solution was loaded on a Sephacryl S300 gel filtration column (5.0cm×90.0cm, 1.77L) equilibrated with 35mM sodium citrate, 154mM NaCl, pH 6.5. The flow rate was 5.0 mL/min and 65 fractions (20.0 mL each) were collected. The main peak is centered on fraction No. 47 (Fig. 3). The main peak includes monomeric Trastuzumab-SPP-DM1. Fractions 44-51 were collected and tested. The number of DM1 drug molecules attached per antibody molecule (p') was determined by measuring the absorbance at 252 nm and 280 nm and was found to be 3.7 drug molecules per antibody molecule.

Example 4-Antibody-Drug Conjugate: Preparation of Trastuzumab-SMCC-DM1:

Purified trastuzumab was derivatized with (succinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, SMCC, Pierce Biotechnology, Inc) to introduce the SMCC linker. Trastuzumab was purified from HERCEPTIN® as described in Example 2 and chlorinated with 7.5 to 10 molar equivalents of SMCC (20 mM in DMSO or DMA (dimethylacetamide), 6.7 mg/mL) in 50 mM potassium phosphate/50 mM Sodium/2mM EDTA, pH 6.5 with 20mg/mL buffer exchange treatment. After stirring at ambient temperature under argon for 2 to 4 hours, the reaction mixture was filtered through a Sephadex G25 column equilibrated with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5. Alternatively, the reaction mixture was gel filtered at pH 6 with 30 mM citrate and 150 mM sodium chloride. Antibody-containing fractions were collected and tested. The recovery of Trastuzumab-SMCC was 88%.

The drug-linker intermediate trastuzumab-SMCC obtained above was diluted to a final concentration of 10 mg/mL with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5, and mixed with a solution of 10 mM DM1 in dimethylacetamide ( 1.7 equiv has 5 SMCC/trastuzumab, 7.37 mg/ml) responses. The reaction was stirred under argon at ambient temperature for 4 to about 16 hours. The coupling reaction mixture was filtered through a Sephadex G25 gel filtration column (1.5×4.9 cm) with 1×PBS at pH 6.5. Alternatively, the reaction mixture was gel filtered at pH 5 with 10 mM succinate and 150 mM sodium chloride. The DM1/trastuzumab ratio (p) was 3.1 as measured by absorbance at 252 nm and 280 nm. The drug to antibody ratio (p) can also be determined by mass spectrometry. Coupling can also be monitored by SDS polyacrylamide gel electrophoresis. Aggregation can be assessed by laser light scattering analysis.

Antibody-SMCC-DM1 Conjugate:

Following this protocol, other antibody-drug conjugates were prepared using the SMCC linker and DM1 drug moiety, including: Antibody-Drug Conjugates (ADCs) Antibody (Ab) antigen Average drug load (DM1/Ab) 10H-SMCC-DM1 10H1 (mouse) anti-NaPi3b NaPi3b 5.0 2H9-SMCC-DM1 2H9 Mab anti-EphB2R 4.0 Ragweed (ragweed)-SMCC-DM1 ragweed 2.7 Anti-TENB2.SMCC-DM1 Anti-TENB2-3416#2 Mouse MAb TENB2 2.5 Anti-TENB2 20D1 Fc chimera-SMCC-DM1 Chimeric human mouse anti-TENB2 TENB2 3.32 anti-CD19-SMCC-DM1 Anti-CD19 (mIgG1 isotype) CD19 5.05 2H7-SMCC-DM1 Anti-CD20 (mIgG2a isotype) CD20 2.44, 3.84 (two batches) CD20 LC-SMCC-DM1 Anti-CD20 (Rituxan_) CD20 HB5-SMCC-DM1 Anti-CD21 (mIgG2a isotype) CD21 4.05 RFB4-SMCC-DM1 anti-CD22 CD22 1.95-6.75 (multiple batches) Cy34.1.2-SMCC-DM1 anti-CD22 CD22 2.70 12F7.1.5-SMCC-DM1 anti-CD22 CD22 5.90 9A8.1.1-SMCC-DM1 anti-CD22 CD22 5.00 8C9.1.2-SMCC-DM1 anti-CD22 CD22 2.90 14B3.3.1-SMCC-DM1 anti-CD22 CD22 5.20 8G10.4.2-SMCC-DM1 anti-CD22 CD22 3.60 2B4.1.4-SMCC-DM1 anti-CD22 CD22 5.20 7A2.4.1-SMCC-DM1 anti-CD22 CD22 3.60 4H3.2.2-SMCC-DM1 anti-CD22 CD22 3.60 5E8.1.8-SMCC-DM1 anti-CD22 CD22 3.60 3F11.2.1-SMCC-DM1 anti-CD22 CD22 4.50 6C9.1.3-SMCC-DM1 anti-CD22 CD22 3.70 10D2.4.3-SMCC-DM1 anti-CD22 CD22 3.65 10D6.8.1-SMCC-DM1 anti-CD72 CD72 2.90 ZL7-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.60 5C3-SMCC-DM1 Anti-CD79a(alpha) CD79a 1.85 6G1-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.50 7H7-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.25 8D11-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.60 8H9-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.40 11E5-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.60 15E4-SMCC-DM1 Anti-CD79a(alpha) CD79a 3.25 16C11-SMCC-DM1 Anti-CD79a(alpha) CD79a 2.50 2F2-SMCC-DM1 Anti-CD79b(beta) CD79b 3.20 17A7-SMCC-DM1 Anti-CD79b(beta) CD79b 4.40 SN8-SMCC-DM1 Anti-CD79b(beta) CD79b 4.40, 0.80 (two batches) 1F9-SMCC-DM1 anti-FcRH1 FcRH1 4.20 7A2-SMCC-DM1 anti-FcRH2 FcRH2 4.20, 3.95 (two batches) 7G7-SMCC-DM1 anti-FcRH2 FcRH2 3.80 c11D6-SMCC-DM1 anti-FcRH2 FcRH2 2.20 51505.111-SMCC-DM1 anti-CXCR5 CXCR5 6.10 12C7 Fc chimera-SMCC-DM1 Anti-Brevican brevican 4.60 H2: PSCA hlog-SMCC-DM1 anti-PSCA hlog PSCA 3 H6: xPSCAhlog-SMCC-DM1 anti-PSCA hlog PSCA 2.6 xFcRH5: 39097D11.1.1-SMCC-DM1 anti-IRTA2 IRTA2 2.6 xFCRH5.7D11-SMCC-DM1 anti-IRTA2 IRTA2 4.4 11D10 LC-SMCC-DM1 mouse anti-CA 0772PMAb CA 0772PMUC16 xNCA LC-SMCC-DM1 mouse anti-CEACAM6 MAb CEACAM6NCA

Antibody-BMPEO-DM1 Conjugate:

For cysteine conjugation, antibodies can be reduced with reducing agents such as dithiothreitol (DTT) or TCEP in the presence of EDTA. After incubation at 37°C for about 1 hour, the pH was adjusted to about 7 with 100 mM potassium phosphate. The reduced antibody was modified with the bis-maleimide reagent BM(PEO)4 (Pierce Chemical), leaving unreacted maleimide groups on the antibody surface. This can be achieved by dissolving BM(PEO)4 in a 50% ethanol/water mixture to a concentration of 10 mM and adding a ten-fold molar excess to approximately 1.6 mg/ml (10 μM) in phosphate-buffered saline. The antibody-containing solution was allowed to react for 1 hour to form Ab-BMPEO. Excess BM(PEO) was removed by gel filtration (HiTrap column, Pharmacia) in a buffer of 30 mM citrate, pH 6, and 150 mM NaCl. An approximately 10-fold molar excess of DM1 was dissolved in dimethylacetamide (DMA) and added to the Ab-BMPEO intermediate. Dimethylformamide (DMF) can also be used to dissolve drug moiety reagents. The reaction mixture was allowed to react overnight before gel filtration or dialysis into PBS to remove unreacted DM1. Gel filtration on a S200 column in PBS to remove high molecular weight aggregates and provide purified Ab-BMPEO-DM1.

Following this protocol, other antibody-drug conjugates were prepared using the BMPEO linker and DM1 drug moiety, including: Antibody-Drug Conjugates (ADCs) Antibody (Ab) antigen Average drug load (DM1/Ab) Trastuzumab-BM(PEO)-DM1 Trastuzumab (Herceptin_) HER2 2.94 CD120-BMPEO4-DM1 mouse anti-GP120 MAb GP120 2 RFB4-BMPEO4-DM1 anti-CD22 CD22 3.7 and 4.25 (two batches)

Example 5 - In Vitro Cell Proliferation Test

ADC efficacy was determined by cell proliferation assay (Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res. 62:5485-5488) using the following protocol:

1. Place a 100 μl aliquot of cell culture containing approximately ¹⁰⁴ cells (SKBR-3, BT474, MCF7, or MDA-MB-468) in medium into each well of a 96-well opaque walled plate middle.

2. Control wells containing media without cells were prepared.

3. ADC was added to the test wells and incubated for 3-5 days.

4. Equilibrate the plate to room temperature for approximately 30 minutes.

5. Add the same volume of CellTiter-Glo reagent as the cell culture medium present in each well.

6. The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis.

7. Incubate the plate at room temperature for 10 minutes to stabilize the fluorescent signal.

8. Luminescence is recorded and reflected in the graph as RLU = Relative Luminescence Units.

Example 6-Mouse serum clearance and stability

Six groups of beige mutant, nude mice (without tumors) were studied, with four animals in each group. On day 0, each mouse received a single dose of ADC at 2 mg/kg in 200 μl of aqueous vehicle, except for the vehicle group which received vehicle alone. Blood was collected by cardiac puncture under anesthesia at each time point (5 minutes, 1 hour, 6 hours, 24 hours, 72 hours, and 168 hours) after administration. Serum was separated and assayed for antibodies and ADC.

Group 1: Vehicle (PBS, pH 6.5, no ADC)

Group 2: Trastuzumab-SMCC-DM1

Group 3: Trastuzumab-SPP-DM1

Group 4: Trastuzumab-SPDP-DM1

Group 5: Trastuzumab-SPP-DM3

Group 5: Trastuzumab-SPP-DM4

Example 7 - Serum Stability in Rats

Six dose groups of 6 Sprague-Dawley rats (100-125 gms each) per dose group were studied. On day 0, animals were administered a single IV dose of vehicle, 10 mg/kg Trastuzumab-SPP-DM1, or 10 mg/kg Trastuzumab-SMCC- DM1. At each time point of 0 (pre-dose), 10, and 30 minutes; 1, 2, 4, 8, 24, and 36 hours; and 2, 3, 4, 7, 14, 21, and 28 days post-dose Approximately 300 μl of whole blood was collected.

Example 8 - Tumor volume in vivo efficacy

Highly expressing HER2 transgenic explant mice:

Animals suitable for transgenic testing can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many strains are suitable, but FVB female mice are preferred due to higher susceptibility to tumor formation. FVB males were used for mating and vasectomy CD.1 mice were used to stimulate pseudopregnancy. Vasectomized mice can be obtained from any commercial supplier. Founders were bred with FVB mice or 129/BL6xFVB p53 heterozygous mice. Mice heterozygous for the p53 allele are used to potentially increase tumor formation. However, this has proven unnecessary. Therefore, some F1 generation tumors are mixed strains. Create mouse tumors with only FVB. Six established mice were obtained with some growing tumors and no pups.

Animals with tumors (allografts expanded from Fo5 mmtv transgenic mice) were treated with a single injection of trastuzumab-DM1 maytansinoid conjugate (10 mg/kg dose), post-injection Tumor volumes were assessed over 20 days.

Bjab-luc xenograft SCID mice:

Experimental groups of eight to ten SCID mice with 20 million Bjab-luc xenograft tumor cells per mouse were administered antibody-drug conjugates, or naked antibody, on day 1. Groups of eight mice each were tested with anti-CD22 ADC, naked anti-CD22 antibody, and control. The control ADC (trastuzumab-SMCC-DM1; loading: DM1/trastuzumab = 3.2) was not a specific binder and administered at 200 μg DM1/m2, 4.2 trastuzumab/kg mice resulted in tumor doubling on average The time is about 3 days. Anti-CD22 conjugates 7A2-SMCC-DM1 (loading: DM1/Ab=3.6), 5E8-SMCC-DM1 (loading: DM1/Ab=3.6), and RFB4-SMCC-DM1 (loading: DM1/Ab=4.3) . Naked antibodies 7A2, 5E8, and RFB4 were administered at 4 mg/kg mice.

Example 9 - Toxicity to Rats

The acute toxicity profile of Trastuzumab-SPP-DM1 (disulfide linker) compared to free DM1 and Trastuzumab-SMCC-DM1 (non-disulfide linker) was assessed. Animals were injected on day 1, complete chemical and hematological profiles at baseline positions were obtained on days 3 and 5, and a complete necropsy was performed on day 5. For each group, three animals were randomly used for routine histological analysis of the following tissues: sternum, liver, kidney, thymus, spleen, and large and small intestine. The test groups are as follows:

Group 1: vehicle (10mM sodium succinate, 100mg/mL sucrose, 0.1% Tween 20, pH5.0)

Group 2: Trastuzumab-SPP-DM1, 22.3 mg/kg

Group 3: Trastuzumab-SMCC-DM1, 10 mg/kg

Group 4: Trastuzumab-SMCC-DM1, 25 mg/kg

Group 5: Trastuzumab-SMCC-DM1, 50mg/kg

Group 6: Free DM1, 160 μg/kg

Example 10 - Toxicity/Safety to Cynomolgus Monkeys

Three groups of four (2 male, 2 female) juvenile Macaca fascicularis (Cynomolgus monkeys) per group were studied.

Group 1: (4 animals) received vehicle only (PBS, pH 6.5, ADC-depleted formulation) on days 1 and 22, after which necropsy was performed on day 36.

Group 2: (4 animals) received Trastuzumab-SMCC-DM1 4900 μg/m2 on day 1 and day 22.

Group 3: (4 animals) received Trastuzumab-SMCC-DM1 7200 μg/m2 on day 22.

Dosing is expressed in surface area of the animal so as to correspond to other species, ie doses in μg/m2 regardless of species, and thus can be compared between species. Trastuzumab-SMCC-DM1 formulations used in Group 2 and Group 3 studies contained PBS, 5.4 mM sodium phosphate, 4.2 mM potassium phosphate, 140 mM sodium chloride, pH 6.5.

Before the first dose (acclimation period) and on days 3, 7, 11, and 14 after the first dose (groups 1 and 2) and on days 3, 7, 11, and 14 after the second dose Blood was collected on days 14, and 21 (Groups 1, 2 and 3) for hematology analysis. Red blood cell (RBC) and platelet (PLT) counts were determined by light scattering. White blood cell (WBC) counts were determined by the peroxidase/basophil method. Reticulocyte counts were determined by light scattering using cationic dyes. Cell counts were determined on an Advia 120 instrument. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in U/L by UV/NADH; IFCC methodology, Total Ab ELISA-ECD/GxhuFc-HRP.Conj.Ab ELISA on Olympus AU400 instrument -xDM1/ECD-Bio/SA-HRP test assay.

All patents, patent applications, and literature references cited throughout this specification are expressly incorporated by reference.

Claims (67)

1. the antibody-drug coupling compound comprises the antibody that is covalently attached to one or more maytansinoid drug moieties by joint, and described chemical compound has formula I:

Ab-(L-D) _p I

Or its pharmaceutically acceptable salt or solvate, wherein:

Ab is in conjunction with the ErbB receptor, and perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor, described tumor associated antigen or cell surface receptor are selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (prostate is striden the film epithelium antigen six times, Genbank accession number NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier, mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), the member 2, the phosphate cotransporter albumen 3b that II type sodium relies on, Genbank accession number NM 006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen 5b Hlog, the sema domain, seven thrombospondins repeat (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

(9) ETBR (endothelin receptor B, Genbank accession number AY275463);

(10) MSG783 (RNF124, imaginary albumen FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers because of 1, the carcinoma of prostate associated protein 1, prostaticly stride film epithelium antigen 2 for six times, stride film prostatein, Genbank accession number AF455138 six times);

(12) TrpM4 (member 4, Genbank accession number NM_017636 for BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor, subfamily M);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor, Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus receptor) or Hs.73792Genbank accession number M26004);

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (the phosphatase anchorin 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20R α (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, NP_443177.1);

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

(28) CD79a (CD79A, CD79 α, the immunoglobulin alpha that is correlated with, with Ig beta (CD79B) covalent interaction and form the B cell-specific proteins that complex, transduction relate to the signal of B cell differentiation, Genbank accession number NP_001774.1 with Ig M molecule from the teeth outwards);

(29) CXCR5 (Burkitt lymphoma receptor 1, by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and may in AIDS, lymphoma, myeloma and leukemic generation, play a role Genbank accession number NP_001707.1 at HIV-2);

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte, Genbank accession number NP_002111.1 with it);

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neural the generation, its defective may cause IDI's Pathophysiology situation, Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the I type memebrane protein family that leucine repeats (LRR), regulate B cell activation and apoptosis, the forfeiture of function is relevant with the enhancing of Patients with SLE disease activity, Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, immunoglobulin Fc domain infer receptor, comprise C2 type Ig sample and ITAM domain, may in the bone-marrow-derived lymphocyte differentiation, work Genbank accession number NP_443170.1);

(35) (transposition related immune globulin superfamily receptors 2, the immunity receptor of inferring may work in B cell development and lymphoma generation IRTA2; The gene imbalance that is caused by transposition betides in some B cell malignant diseases Genbank accession number NP_112571.1); And

(36) TENB2 (the transmembrane protein polysaccharide of inferring, relevant with the accent protein family and the follistatin of the EGF/ somatomedin of somatomedin, Genbank accession number AF179274;

Condition is that described antibody is not TA.1;

L is non-disulphide joint;

D is the maytansinoid drug moiety; And

P is 1 to 8.

2. the antibody-drug coupling compound of claim 1, wherein Ab is in conjunction with being selected from (1)-(16) and one or more tumor associated antigens of (18)-(36) or the antibody of cell surface receptor.

3. the antibody-drug coupling compound of claim 1 or claim 2, wherein L is the joint that is selected from following structure:

Wherein wave is represented covalently bound with Ab and D;

X is:

-(CH ₂) _n-，-(CH ₂CH ₂O) _n-

Y is:

R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12.

4. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein D is selected from structure:

Wherein wave is represented covalently bound with L,

R is H or C independently ₁-C ₆Alkyl, and

M is 1,2 or 3.

5. the antibody-drug coupling compound of claim 4, wherein m is 2, and R is H.

6. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein the maytansinoid drug moiety is DM1, has structure:

7. the antibody-drug coupling compound of claim 3, it has structure:

8. the antibody-drug coupling compound of claim 7, it has structure:

9. the antibody-drug coupling compound of claim 3, it has structure:

10. the antibody-drug coupling compound of claim 1, it has structure:

11. the antibody-drug conjugates of claim 10, wherein Ab is a trastuzumab, and p is 1,2,3 or 4.

12. the antibody-drug coupling compound of claim 1, it has structure:

Wherein Tr is a trastuzumab, and p is 1,2,3 or 4.

13. the antibody-drug coupling compound of claim 1, it has structure:

Wherein n is 0,1 or 2; And p is 1,2,3 or 4.

14. the antibody-drug coupling compound of claim 13, wherein Ab is a trastuzumab.

15. the antibody-drug coupling compound of claim 1, wherein p is 1,2,3 or 4.

16. the antibody-drug coupling compound of claim 1, wherein said antibodies is by the receptor of ErbB gene code.

17. the antibody-drug coupling compound of claim 16, wherein said receptor is selected from EGFR, HER2, HER3 and HER4.

18. the antibody-drug coupling compound of claim 17, wherein said antibody are specifically in conjunction with the HER2 receptor.

19. the antibody-drug coupling compound of claim 1, it is specifically in conjunction with the ectodomain of HER2 receptor and suppressed to express the growth of the tumor cell of HER2 receptor.

20. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein said antibody is selected from monoclonal antibody, antibody fragment, chimeric antibody and humanized antibody.

21. the antibody-drug coupling compound of claim 1, wherein said antibody is for being selected from the humanized antibody of huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

22. the antibody-drug coupling compound of claim 21, wherein said antibody are huMAb4D5-8 (trastuzumab).

23. the antibody-drug conjugates of claim 20, wherein said antibody are the Fab fragment.

24. the antibody-drug coupling compound of claim 1, wherein said antibody is connected in joint by the cysteine mercaptan of antibody.

25. the antibody-drug coupling compound of claim 24, wherein p is 1,2,3 or 4.

26. comprise the antibody-drug coupling compound of claim 1 or claim 2 or the pharmaceutical composition of its pharmaceutically acceptable salt and pharmaceutically acceptable diluent, carrier or excipient.

27. the pharmaceutical composition of claim 26, it further comprises the chemotherapeutics for the treatment of effective dose, and described chemotherapeutics is selected from: Erlotinib, Bortezomib, fulvestrant, Sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, formyl tetrahydrofolic acid, rapamycin, Lapatinib, Lonafarnib, Sorafenib and Gefitinib.

28. the pharmaceutical composition of claim 26, it further comprises the anti-angiogenic rebirth agent for the treatment of effective dose.

29. the pharmaceutical composition of claim 26, it further comprises the bevacizumab for the treatment of effective dose.

30. suppress the method for cell proliferation, comprise with the mammalian cell in the antibody-drug coupling compound processing cell culture medium of claim 1 or claim 2, suppress the propagation of cell thus.

31. the method for claim 30, wherein said mammalian cell have the HER2 receptor protein at the antibody-drug coupling compound.

32. the method for claim 31, wherein said mammalian cell is a breast tumor cell.

33. the method for claim 30, wherein said antibody-drug coupling compound cytotoxicity is stronger than the maytansinoid chemical compound of the maytansinoid part that comprises the antibody-drug coupling compound.

34. the method for claim 30, wherein said antibody-drug conjugates is apoptosis-induced.

35. the treatment method for cancer is included as the patient and uses the antibody-drug coupling compound of claim 1 or claim 2 and the preparation of pharmaceutically acceptable diluent, carrier or excipient.

36. the method for claim 35, wherein said cancer is selected from mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, colorectum, thyroid, pancreas, prostate and bladder cancer.

37. the method for claim 36, wherein said cancer is for crossing the breast carcinoma of expressing ErbB2 with 2+ or higher level.

38. the method for claim 35, wherein the amount of the antibody-drug coupling compound of using for the patient at every dose about 0.1 to about 10mg/kg weight in patients scope.

39. the method for claim 35 is wherein with the interval administration of antibodies-drug conjugates in about three weeks.

40. the method for claim 35 is wherein by infusion administration of antibodies-drug conjugates.

41. the method for claim 35, wherein said antibody-drug conjugates is prepared with pharmaceutically acceptable parenteral carrier.

42. the method for claim 41, wherein said antibody-drug conjugates is formulated as injectable unit dosage form.

43. the method for claim 42, wherein intravenous administration of antibodies-drug conjugates.

44. the method for claim 35 is wherein with the combination of antibody-drug coupling compound, for the patient uses growth inhibited antibody.

45. the method for claim 35 is wherein with antibody-drug coupling compound combination, for the patient uses in conjunction with ErbB2 and blocks second kind of antibody of the ligand activation of ErbB receptor.

46. the method for claim 45, wherein second kind of antibody comprises monoclonal antibody 2C4 or humanization 2C4.

47. the method for claim 46, wherein said second kind of antibody and cytotoxic reagent coupling.

48. the method for claim 35, wherein make up with the antibody-drug coupling compound, for the patient uses chemotherapeutics, wherein said chemotherapeutics is selected from: Erlotinib, Bortezomib, fulvestrant, Sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, formyl tetrahydrofolic acid, rapamycin, Lapatinib, Lonafamib, Sorafenib and Gefitinib.

49. the method for claim 35, wherein with the combination of antibody-drug coupling compound, for the patient uses the anti-angiogenic rebirth agent, wherein said anti-angiogenic rebirth agent is selected from bevacizumab.

50. suppressed to express the method for the growth of tumour cell of growth factor receptors, be included as antibody-drug conjugates and chemotherapeutics that the patient uses claim 1, described antibody-drug conjugates is specifically in conjunction with the growth factor receptors that is selected from HER2 or EGF, and wherein said antibody-drug conjugates and described chemotherapeutics are used with the amount of effective inhibition patient tumors cell growth respectively.

51. the method for treatment human patients, described human patients is to crossing the disease sensitivity that is expressed as feature or suffering from described disease after diagnosing with the ErbB2 receptor, described method comprises antibody-drug conjugates and the chemotherapeutics or the growth inhibitor of co-administered claim 1.

52. the method for claim 51, wherein the antibody of antibody-drug conjugates is anti-ErbB antibody.

53. the method for claim 52, wherein said anti-ErbB antibody has the biological property of 4D5 monoclonal antibody.

54. the method for claim 52, the epi-position that wherein said anti-ErbB antibodies is substantially the same with the 4D5 monoclonal antibody.

55. the method for claim 52, wherein said anti-ErbB antibody is selected from humanized antibody: huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

56. be used to detect the method for testing of cancerous cell, comprise:

(a) cell is contacted with the antibody-drug coupling compound of claim 1 or claim 2; With

(b) measure the degree of antibody-drug coupling compound in conjunction with cell.

57. the method for testing of claim 56, wherein said cell is a breast tumor cell.

58. the method for testing of claim 56, wherein said combination degree detects by the level of measuring the ErbB-code nucleic acid with fluorescence in situ hybridization (FISH).

59. the method for testing of claim 56, wherein said combination degree is measured by SABC (IHC).

60. goods comprise:

The antibody-drug coupling compound of claim 1 or claim 2;

Container; With

Package insert or label, it indicates described chemical compound and can be used for treating with the ErbB receptor and cross the cancer that is expressed as feature.

Can be used for treating and cross the cancer that is expressed as feature 61. the goods of claim 60, wherein said package insert or label indicate described chemical compound with the ErbB2 receptor.

62. the goods of claim 61, wherein said cancer are breast carcinoma.

63. the goods of claim 62, wherein said cancer is expressed as feature with the mistake of ErbB2 receptor 2+ or above level.

64. prepare the method for antibody-drug coupling compound, comprise by joint antibody is covalently attached to one or more maytansinoid drug moieties that described chemical compound has formula I:

Ab-(L-D) _p I

Or its pharmaceutically acceptable salt or solvate, wherein:

Ab is in conjunction with the ErbB receptor, and perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor, described tumor associated antigen or cell surface receptor are selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (prostate is striden the film epithelium antigen six times, Genbank accession number NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier, mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), the member 2, the phosphate cotransporter albumen 3b that II type sodium relies on, Genbank accession number NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen 5b Hlog, the sema domain, seven thrombospondins repeat (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, PIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

(9) ETBR (endothelin receptor B, Genbank accession number AY275463);

(10) MSG783 (RNF124, imaginary albumen (hypothetical protein) FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers because of 1, the carcinoma of prostate associated protein 1, prostaticly stride film epithelium antigen 2 for six times, stride film prostatein, Genbank accession number AF455138 six times);

(12) TrpM4 (member 4, Genbank accession number NM_017636 for BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor, subfamily M);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor, Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus receptor) or Hs.73792Genbank accession number M26004);

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (the phosphatase anchorin 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20R α (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, NP_443177.1);

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

(28) CD79a (CD79A, CD79 α, the immunoglobulin alpha that is correlated with is with Ig beta (CD79B) covalent interaction and form complex with Ig M molecule from the teeth outwards, transduction relates to the B cell-specific proteins of the signal of B cell differentiation, Genbank accession number NP_001774.1);

(29) CXCR5 (Burkitt lymphoma receptor 1, by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and may in AIDS, lymphoma, myeloma and leukemia, play a role Genbank accession number NP_001707.1 at HIV-2);

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte, Genbank accession number NP_002111.1 with it);

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neuranagenesis, its defective may cause IDI's Pathophysiology situation, Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the I type memebrane protein family that leucine repeats (LRR), regulate B cell activation and apoptosis, the forfeiture of function is relevant with the enhancing of Patients with SLE disease activity, Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, the immunoglobulin Fc domain of inferring comprise C2 type Ig sample and ITAM domain, may work Genbank accession number NP_443170.1 in the bone-marrow-derived lymphocyte differentiation);

(35) (transposition related immune globulin superfamily receptors 2, the immunity receptor of inferring may work in B cell development and lymphoma generation IRTA2; The gene imbalance that is caused by transposition betides in some B cell malignant diseases Genbank accession number NP_112571.1); And

(36) (the transmembrane protein polysaccharide of inferring transfers protein family relevant with follistatin with the EGF/ of somatomedin, Genbank accession number AF179274 to TENB2;

Condition is that described antibody is not TA.1;

L is the joint that is selected from following structure:

Wherein wave is represented covalently bound with Ab and D;

X is:

-(CH ₂) _n-，-(CH ₂CH ₂O) _n-

Y is:

Wherein R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12;

D is the maytansinoid drug moiety that is selected from following structure:

Wherein wave is represented covalently bound with L; R is H or C independently ₁-C ₆Alkyl; M is 1,2 or 3; And p is 1 to 8;

Wherein said method comprises:

Make Ab and joint reagent reacting form antibody-joint intermediate A b-L, make Ab-L and drug moiety D reaction form antibody-drug conjugates then; Perhaps

Make drug moiety D and joint reagent reacting form medicine-joint intermediate D-L, make D-L and Ab reaction form antibody-drug conjugates then.

65. the method for claim 64, wherein Ab is selected from (1)-(16) and one or more tumor associated antigens of (18)-(36) or the antibody of cell surface receptor for combination.

66. the method for claim 64 or claim 65, wherein said joint reagent is SMCC.

67. the method for claim 64 or claim 65, wherein said joint reagent is for being selected from DTME, BMB, BMDB, BMH, BMOE, BM (PEO) ₃And BM (PEO) ₄Two-maleimide reagent.

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