Monomethylvaline compounds capable of conjugation to ligands

1. TITLE OF THE INVENTION

MONOMETHYLVALINE COMPOUNDS CAPABLE OF CONJUGATION TO LIGANDS

2. APPLICANT (S) (a) NAME : SEATTLE GENETICS, INC.

(b) NATIONALITY : U.S.A.

(c) ADDRESS : 21823-30th Drive S.E.

Bothell, WA 98021 U.S.A.

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

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MONOMETHYLVALINE COMPOUNDS CAPABLE OF CONJUGATION TO LIGANDS

ABSTRACT

Auristatin peptides, including MeVal-Val-Dil-Dap-Norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were prepared and attached to Ligands through various linkers, including maleimidocaproyl-val-cit-PAB. The resulting ligand drug conjugates were active in vitro and in vivo.

380

The present application is a divisional application of a patent application number 2111/DELNP/2006 filed on 18/04/2006

CONTINUITY

X

This application claims the benefit of U.S. Provisional Patent Application No. 60/518,534, filed November 6, 2003; U.S. Provisional Patent Application No. 60/557,116, filed March 26, 2004; U.S. Provisional Patent Application No. 60/598,899, filed August 4, 2004; and U.S. Provisional Patent Application No. 60/622,455, filed October 27, 2004; the disclosures of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is directed to a Drug Compound and more particularly to Drug-Linker-Ligand Conjugates, Drug-Linker Compounds, and Drug-Ligand Conjugates, to compositions including the same, and to methods for using the same to treat cancer, an autoimmune disease or an infectious disease. The present invention is also directed to antibody-drug conjugates, to compositions including the same, and to methods for using the same to treat cancer, an autoimmune disease or an infectious, disease. The invention also relates to methods of using antibody-drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Improving the delivery of drugs and other agents to target cells, tissues and tumors to achieve maximal efficacy and minimal toxicity has been the focus, of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.

Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., chemotherapeutic (anticancer), cytotoxic, enzyme inhibitor agents and antiviral or antimicrobial drugs) that can be administered. By comparison, although oral administration of drugs is considered to be a convenient and economical mode of administration, it shares the same concerns of non-specific toxicity to unaffected cells once the drug has been absorbed into the systemic circulation. Further complications involve problems with oral bioavailability and residence of drug in the gut leading to additional exposure of gut to the drug and hence risk of gut toxicities. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. The benefits of such treatment include avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells. Intracellular targeting may be achieved by methods, compounds and formulations which allow accumulation or retention of biologically active agents, i.e. active metabolites, inside cells.

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

The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4975278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986):603-05; Thorpe, 1985, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84:

Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784; Mandler et al. (2000) Jour, of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic &amp; Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). The toxins may affect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition (Meyer, D.L. and Senter, P.D. “Recent Advances in Antibody Drug Conjugates for Cancer Therapy” in Annual Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237). Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibodyradioisotope conjugate composed of a murine IgGl kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and ⁱⁿIn or ⁹⁰Y radioisotope bound by a thiourea linker-chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):245363; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cell non-Hodgkin’s Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686: U.S. Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1, is advancing into Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors. The same maytansinoid drug moiety, DM1, was linked through a non-disulfide linker, SMCC, to a mouse murine monoclonal antibody, TA.l (Chari etal. (1992) Cancer Research 52:127-131). This conjugate was reported to be 200-fold less potent than the corresponding disulfide linker conjugate. The SMCC linker was considered therein to be “noncleavable.”

Several short peptidic compounds have been isolated from the marine mollusc Dolabella auricularia and found to have biological activity (Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al. (1995) Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour. Org Chem. 60:4474). Analogs of these compounds have also been prepared, and some were found to have biological activity (for a review, see Pettit et al. (1998) Anti-Cancer Drug Design 13:243277). For example, auristatin E (U.S. Patent No. 5635483) is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anticancer drug vincristine (G. R. Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds, and a C-terminal amide.

The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to: (i) chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cAClO which is specific to CD30 on hematological malignancies (Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation to Ligands”; Francisco et al. (2003) Blood 102(4): 1458-1465; U.S. Publication 2004/0018194; (iii) anti-CD20 antibodies such as RITUXAN® (WO 04/032828) for the treatment of CD20-expressing cancers and immune disorders; (iv) anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectal cancer (Mao, et al. (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583).

Auristatin E conjugated to monoclonal antibodies are disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004.

Despite in vitro data for compounds of the dolastatin class and its analogs, significant general toxicities at doses required for achieving a therapeutic effect compromise their efficacy in clinical studies. Accordingly, there is a clear need in the art for dolastatin/auristatin derivatives having significantly lower toxicity, yet useful therapeutic efficiency. These and other limitations and problems of the past are addressed by the present invention.

The ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbBl, HER1), HER2 (ErbB2 or p 185”™), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol. 9(3): 1165-1172. Maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize ErbB2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-α (U.S. Patent No. 5677171). The anti-ErbB2 antibodies ’ discussed in Hudziak et al. are further characterized in 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): 117127; 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 etal. Proc. Natl. Acad. Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al. (1997) Oncogene 15:1385-1394.

Other anti-ErbB2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); W094/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); 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; U.S. Patent No. 5783186; and Klapper etal. (1997) Oncogene 14:2099-2109.

Homology screening has resulted in the identification of two other ErbB receptor family members; ErbB3 (U.S. Patent No. 5,183,884; U.S. Patent No. 5,480,968; KraU.S. etal. (1989) Proc. Natl. Acad. Sci. USA 86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750; and Plowman et al. (1993) Nature 366:473-475). Both of these receptors display increased expression on at least some breast cancer cell lines.

HERCEPTIN® (Trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody that selectively binds with high affinity in a cellbased assay (Kd = 5 nM) to the extracellular domain of the human epidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S. Patent No. 5821337; U.S. Patent No. 6054297; U.S. Patent No. 6407213; U.S. Patent No. 6639055;

Coussens L, et al. (1985) Science 230:1132-9; SlamonDJ, etal. (1989) Science 244:707-12). Trastuzumab is an IgGl kappa antibody that contains human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HERZ. Trastuzumab binds to the HER2 antigen and thus, inhibits the growth of cancerous cells. Because Trastuzumab is a humanized antibody, it minimizes any HAMA response in patients. The humanized antibody against HER2 is produced by a mammalian cell (Chinese Hamster Ovary, CHO) suspension culture. The HER2 (or c-erbB2) proto-oncogene encodes a transmembrane receptor protein of 185kDa, which 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 an immunohistochemistry based assessment of fixed tumor blocks (Press MF, et al. (1993) Cancer Res 53:4960-70. Trastuzumab has been shown, in both in vitro assays and in animals, to inhibit the proliferation of human tumor cells that overexpress HER2 (Hudziak RM, et al. (1989) Mol Cell Biol 9:1165-72; Lewis GD, et al. (1993) Cancer Immunol Immunother; 37:255-63; Baselga J, et al. (1998) Cancer Res. 58:2825-2831). Trastuzumab is a mediator of antibodydependent cellular cytotoxicity, ADCC (Hotaling TE, et al. (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD, et al. (1997) [abstract]. Proc Am Assoc Cancer Res; 38:602). In vitro, Trastuzumab mediated ADCC has been shown to be preferentially exerted on HER2 overexpressing cancer cells compared with cancer cells that do not overexpress HER2. HERCEPTIN® as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have received one or more chemotherapy regimens for their metastatic disease. HERCEPTIN® in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for their metastatic disease. HERCEPTIN® is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al, (1996) J. Clin. Oncol. 14:737-744).

The murine monoclonal anti-HER2 antibody inhibits the growth of breast cancer cell lines that overexpress HER2 at the 2+ and 3+ (1-2 x 10⁶ HER2 receptors per cell) level, but has no activity on cells that express lower levels of HER2 (Lewis et al., (1993) Cancer Immunol. Immunother. 37:255-263). Based on this observation, antibody 4D5 was humanized (huMAb4D5-8, rhuMAb HER2, U.S. Patent No. 5821337; Carter et al., (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289) and tested in breast cancer patients whose tumors overexpress HER2 but who had progressed after conventional chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17: 2639-2648).

Although HERCEPTIN is a breakthrough in treating patients with ErbB2-overexpressing breast cancers that have received extensive prior anticancer therapy, some patients in this population fail to respond or respond only poorly to HERCEPTIN treatment.

Therefore, there is a significant clinical need for developing further HER2-directed cancer therapies for those patients with HER2-overexpressing tumors or other diseases associated with HER2 expression that do not respond, or respond poorly, to HERCEPTIN treatment.

The recitation of any reference in this application is not an admission that the reference is prior art to this application.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides Drug-Linker-Ligand compounds having the Formula la:

L-(-A_a-W_w-Y_y-D) _p la or a pharmaceutically acceptable salt or solvate thereof wherein,

L- is a Ligand unit;

-A_a-W_w-Y_y- is a Linker unit (LU), wherein the Linker unit includes:

-A- is a Stretcher unit, a is 0 or 1, each -W- is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

-Y- is a Spacer unit, and y is 0,1 or 2;

p ranges from 1 to about 20; and

<img file="IN520DEN2012A_D0002.tif" />

wherein, independently at each location:

R² is selected from H and Ci-C₈ alkyl;

R³ is selected from H, CpCg alkyl, C3-C8 carbocycle, aryl, CpCg alkyl-aryl, CpCg alkyl-(C₃-C₈ carbocycle), C3-C8 heterocycle and CpCg alkyl(C₃-Cg heterocycle);

R⁴ is selected from H, Ci-C₈ alkyl, C₃-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C3-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-Cg heterocycle);

. ^r5 is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from H, Ci-Cg alkyl and C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Ci-Cs alkyl;

R⁷ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and Ci-Cg alkyl(C3-C8 heterocycle);

each R⁸ is independently selected from H, OH, Ci-Cs alkyl, C3-C8 carbocycle and O-(Ci-C8 alkyl);

R⁹ is selected from H and Ci-Cs alkyl;

R¹⁰ is selected from aryl or C3-C8 heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-Cg alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴is H or Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO₃-C_l-C8 alkyl;

each occurrence of R¹⁶ is independently H, Ci-Cg alkyl, or -(CH₂)_n-COOH; where; n is an integer ranging from 0 to 6; and

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C₃-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C3-C8 carbocycle).

In another aspect, Drug Compounds having the Formula lb are provided:

<img file="IN520DEN2012A_D0003.tif" />

Ib or pharmaceutically acceptable salts or solvates thereof, wherein:

R² is selected from hydrogen and -Ci-Cs alkyl;

R³ is selected from hydrogen, -Cj-Cg alkyl, -C3-C8 carbocycle, aryl, -Ci-Cg alkyl-aryl, -Ci-Cg alkyl-(C3-Cs carbocycle), -C3-C8 heterocycle and -Ci-Cs alkyl-(C3-C8 heterocycle);

R⁴ is selected from hydrogen, -Ci-Cs alkyl, -C3-C8 carbocycle, aryl, -Ci-Cs alkyl-aryl, -Ci-Cs alkyl-(C3-C8 carbocycle), -C3-C8 heterocycle and Cj-Cs alkyl-(C3-C8 heterocycle) wherein R⁵ is selected from -H and -methyl; or R⁴and R⁵ jointly, have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from -H, -Ci-Cs alkyl and -C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;

R⁶ is selected from H and -Ci-Cs alkyl;

R⁷ is selected from H, -Ci-Cg alkyl, -C3-C8 carbocycle, aryl, -Ci-Cg alkyl-aryl, -C]-C₈ alkyl-(C3-Cs carbocycle), -C3-C8 heterocycle and -Ci-Cg alkyl(C3-C8 heterocycle);

each R⁸ is independently selected from H, -OH, -Ci-Cs alkyl, -C3Cs carbocycle and -O-(Ci-Cs alkyl);

R⁹ is selected from H and -Ci-Cs alkyl;

R¹⁰ is selected from aryl group or -C3-C8 heterocycle;

Z is -0-, -S-, -NH-, or -NR¹²-, wherein R¹² is Ci-Cg alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, -C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is -C₂-C₈ alkyl;

R¹⁴isHor-Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, -COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, -Ci-C₈ alkyl, or (CH₂)_n-COOH; and n is an integer ranging from 0 to 6.

The compounds of Formula (lb) are useful for treating cancer, an autoimmune disease or an infectious disease in a patient or useful as an intermediate for the synthesis of a Drug-Linker, Drug-Linker-Ligand Conjugate, and Drug-Ligand Conjugate having a cleavable Drug unit.

In another aspect, compositions are provided including an effective amount of a Drug-Linker-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.

In still another aspect, the invention provides pharmaceutical compositions comprising an effective amount of a Drug-Linker Compound and a pharmaceutically acceptable carrier or vehicle.

In still another aspect, the invention provides compositions comprising an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.

In yet another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugLinker Compound.

In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugLinker-Ligand Conjugate.

In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugLigand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for treating an infectious, disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

' In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the DrugLigand Conjugate.

In still another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugLinker Compound.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-LinkerLigand Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate. ..-.

In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.

In another aspect, a Drug Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker Compound having a cleavable Drug unit from the Drug-Ligand Conjugate.

In another aspect, a Drug-Linker Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.

In another aspect, compounds having having Formula la’ are provided:

Ab-(-A_a-W_w-Y_y—D)_p la’ or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab includes an antibody including one which binds to to

CD30, CD40, CD70, and Lewis Y antigen,

A is a Stretcher unit, a is 0 or 1, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

Y is a Spacer unit, and y is 0, 1 or 2, p ranges from 1 to about 20, and

D is a Drug unit selected from Formulas De and D_F:

<img file="IN520DEN2012A_D0004.tif" />

<img file="IN520DEN2012A_D0005.tif" />

R⁸ O

De

<img file="IN520DEN2012A_D0006.tif" />

Dp wherein, independently at each location:

R² is selected from H and CpCg alkyl;

R^? is selected from H, CpCg alkyl, C3-Cg carbocycle, aryl, CpCg alkyl-aryl, CpCg alkyl-(C3-C₈ carbocycle), C3-Cg heterocycle and CpCg alkyl(C₃-C₈ heterocycle);

R⁴ is selected from H, CpCg alkyl, C3-Cg carbocycle, aryl, CpCg alkyl-aryl, CpCg alkyl-(C3-Cg carbocycle), C3-Cg heterocycle and CpCg alkyl(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)n- wherein R^a and R^b are independently selected from H, CpCg alkyl and C3-C₈ carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and CpCg alkyl;

R⁷ is selected from H, CpCg alkyl, C3-Cg carbocycle, aryl, CpCg alkyl-aryl, CpCg alkyl-(C3-Cg carbocycle), C3-Cg heterocycle and CpCg alkyl(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, CpCg alkyl, C3-Cg carbocycle and O-(CpCg alkyl);

R⁹ is selected from H and CpCg alkyl;

R¹⁰ is selected from aryl or C3-Cg heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is CpCg alkyl;

R¹¹ is selected from H, CpC2o alkyl, aryl, C3-Cg heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴isHorCi-C₈alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R^I6)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, Ci-C₈ alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C₃-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C₃-C₈ carbocycle); and n is an integer ranging from 0 to 6.

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

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);

(10) MSG783 (RNF124, hypothetical protein FU20315, Genbank accession no. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);

(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764);

(17) HER2 (Genbank accession no. Ml 1730);

(18) NCA (Genbank accession no. Ml8728);

(19) MDP (Genbank accession no. BC017023);

(20) IL20Ra (Genbank accession no. AF184971);

(21) Brevican (Genbank accession no. AF229053);

(22) Ephb2R (Genbank accession no. NM_004442);

(23) ASLG659 (Genbank accession no. AX092328);

(24) PSCA (Genbank accession no. AJ297436);

(25) GEDA (Genbank accession no. AY260763);

(26) BAFF-R (Genbank accession no. NP_443177.1);

(27) CD22 (Genbank accession no. NP-001762.1);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in Bcell differentiation, Genbank accession No. NP_001774.1);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);

(30) HLA-DOB (Beta subunit of MHC class Π molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession No. NP_002111.1);

(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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);

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

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_OO5573.1);

(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); or (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1).

In still another aspect, the invention provides pharmaceutical compositions comprising an effective amount of a Drug-Linker-Antibody Conjugate and a pharmaceutically acceptable carrier or vehicle.

In still another aspect, the invention provides compositions comprising an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit (moiety) from the Drug-Antibody Conjugate and a pharmaceutically acceptable carrier or vehicle.

In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the DrugAntibody Conjugate.

In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugLinker-Antibody Conjugate.

In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a DrugAntibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient· in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-LinkerAntibody Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, a Drug Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker Compound having a cleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, a Drug-Linker Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker-Antibody Conjugate.

In one aspect, the present invention provides Drug-LinkerAntibody Conjugates (also referred to as antibody-drug conjugates) having Formula Ic:

Ab-(-A_a-W_w- Y_y-D)_{p r} or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antibody which binds to one or more of the antigens (1)(35):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);

(7) Serna 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);

(12) TrpM4 (BR22450, FLJ20041,1RPM4, IRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM.003212);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);

(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764);

(17) HER2 (Genbank accession no. Ml 1730);

(18) NCA (Genbank accession no. M18728);

(19) MDP (Genbank accession no. BC017023);

. (20) IL20Ra (Genbank accession no. AF184971);

(21) Brevican (Genbank accession no. AF229053);

(22) Ephb2R (Genbank accession no. NM_004442);

(23) ASLG659 (Genbank accession no. AX092328);

(24) PSCA (Genbank accession no. AJ297436);

(25) GEDA (Genbank accession no. AY260763);

(26) BAFF-R (Genbank accession no. NP_443177.1);

(27) CD22 (Genbank accession no. NP-001762.1);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in Bcell differentiation, Genbank accession No. NP_001774.1);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);

(30) HLA-DOB (Beta subunit of MHC class II molecule (la ’ antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession No. NP_002111.1);

(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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);

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

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); or (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1);

A is a Stretcher unit, a is 0 or 1, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

Y is a Spacer unit, and y is 0,1 or 2, p ranges from 1 to about 20, and

D is a Drug moiety selected from Formulas D_E and Df:

<img file="IN520DEN2012A_D0007.tif" />

<img file="IN520DEN2012A_D0008.tif" />

wherein the wavy line of D_E and D_F indicates the covalent attachment site to A, W, or Y, and independently at each location:

- R² is selected from H and Ci.C8 alkyl;

R³ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle and Ci-Cg alkyl(C3-C8 heterocycle);

R⁴ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle and Ci-Cg alkyl(C3-C8 heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from H, Ci-Cg alkyl and C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Ci-Cg alkyl;

R⁷ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle and Ci-Cg alkyl(C3-C8 heterocycle);

each R⁸ is independently selected from H, OH, Ci-Cg alkyl, C3-C8 carbocycle and O-(Ci-Cg alkyl);

R⁹ is selected from H and Ci-Cs alkyl;

Rio is selected from aryl or C3-C8 heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-Cg alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ isHorC*-C⁸alkyl;

each occurrence of R¹⁵ is independently H, COOH, ~(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)n-SO₃-Ci-C8 alkyl;

each occurrence of R¹⁶ is independently H, Ci-Cg alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C³-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C3-Cg carbocycle); and n is an integer ranging from 0 to 6.

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

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

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

In another aspect, the invention includes a method for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB2 receptor and does not respond, or responds poorly, to treatment with an anti-ErbB2 antibody, comprising administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound of the invention.

In another aspect, a substantial amount of the drug moiety is not cleaved from the antibody until the antibody-drug conjugate compound enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibodydrug conjugate does enter the cell.

In another aspect, the bioavailability of the antibody-drug conjugate compound or an intracellular metabolite of the compound in a mammal is improved when compared to a drug compound comprising the drug moiety of the antibody-drug conjugate compound, or when compared to an analog of the compound not having the drug moiety.

In another aspect, the drug moiety is intracellularly cleaved in a mammal from the antibody of the compound, or an intracellular metabolite of the compound.

In another aspect, the invention includes a pharmaceutical composition comprising an effective amount of the antibody-drug conjugate compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. The composition may further comprise a therapeutically effective amount of chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, and a DNA binder.

In another aspect, the invention includes a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating tumor cells or cancer cells with an amount of the antibody-drug conjugate compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.

In another aspect, the invention includes a method of inhibiting cellular proliferation comprising exposing mammalian cells in a cell culture medium to an antibody drug conjugate compound of the invention, wherein the antibody drug conjugate compound enters the cells and the drug is cleaved from the remainder of the antibody drug conjugate compound; whereby proliferation of the cells is inhibited.

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

In another aspect, the invention includes an assay for detecting cancer cells comprising:

(a) exposing cells to an antibody-drug conjugate compound of the invention; and (b) determining the extent of binding of the antibody-drug conjugate compound to the cells.

The invention will best be understood by reference to the following detailed description of the exemplary embodiments, taken in conjunction with the accompanying drawings, figures, and schemes. The discussion below is descriptive, illustrative and exemplary and is not to be taken as limiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an in vivo, single dose, efficacy assay of cAClOmcMMAF in subcutaneous Karpas-299 ALCL xenografts.

Figure 2 shows an in vivo, single dose, efficacy assay of cAClOmcMMAF in subcutaneous L540cy. For this study there were 4 mice in the untreated group and 10 in each of the treatment groups.

Figures 3a and 3b show in vivo efficacy of cBR96-mcMMAF in subcutaneous L2987. The filed triangles in Figure 3 a and arrows in Figure 3b indicate the days of therapy.

Figures 4a and 4b show in vitro activity o/cAC10-antibody-drug conjugates against CD30⁺ cell lines.

Figures 5a and 5b show in vitro activity of cBR96-antibody-drug conjugates against Le^y+ cell lines.

Figures 6a and 6b show in vitro activity of clF6-antibody-drug conjugates against CD70⁺ renal cell carcinoma cell lines.

Figure 7 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with antibody drug conjugates (ADC): -·- Trastuzumab-MC-vcPAB-MMAF, 3.8 MMAF/Ab, -o- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.8 MMAF/Ab, measured in Relative Fluorescence Units (RLU) versus gg/ml concentration of ADC. H = Trastuzumab where H is linked via a cysteine [cys].

Figure 8 shows an in vitro, cell proliferation assay with BT-474 cells treated with ADC: -·- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, —o~ Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MCMMAF, 4.8 MMAF/Ab.

Figure 9 shows an in vitro, cell proliferation assay with MCF-7 cells treated with ADC: -·- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, —o— Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab, and -ΔTrastuzumab-MC-MMAF, 4.1 MMAF/Ab.

Figure 10 shows an in vitro, cell proliferation assay with MDAMB-468 cells treated with ADC: -·- Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab, -o- Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab, and -ΔTrastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.

Figure 11 shows a plasma concentration clearance study after administration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats: The administered dose was 2 mg of ADC per kg of rat. Concentrations of total antibody and ADC were measured over time. (H = Trastuzumab).

Figure 12 shows a plasma concentration clearance study after administration of H-MC-vc-MMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg administered at day 1 and day 21. Concentrations of total antibody and ADC were measured over time. (H = Trastuzumab).

Figure 13 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 gg/m²) and Trastuzumab-MC-vc-PAB-MMAF (555 gg/m²). (H = Trastuzumab).

Figure 14 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660 gg/m²) of Trastuzumab-MC-MMAE and 1250 gg/m²Trastuzumab-MC-vc-PAB-MMAE.

Figure 15 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with Vehicle and 650 gg/m² trastuzumab-MC-MMAF.

Figure 16 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on

Day 0 with Vehicle and 350 gg/m² of four trastuzumab-MC-MMAF conjugates where the MMAF/trastuzumab (H) ratio is 2,4, 5.9 and 6.

Figure 17 shows the Group mean change, with error bars, in animal (rat) body weights (Meant SD) after administration of Vehicle, trastuzumab-MCval-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MCMMAF and trastuzumab-MC-val-cit-PAB-MMAF.

Figure 18 shows the Group mean change in animal (rat) body weights (Meant SD) after administration of 9.94 mg/kg H-MC-vc-MMAF, 24.90 mg/kg H-MC-vc-MMAF, 10.69 mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg H-MC(Me)-vc-PAB-MMAF, 10.17 mg/kg H-MC-MMAF, 25.50 mg/kg H-MCMMAF, and 21.85 mg/kg H-MC-vc-PAB-MMAF. H = trastuzumab. The MC linker is attached via a cysteine of trastuzumab for each conjugate.

Figure 19 shows the Group mean change, with error bars, in Sprague Dawley rat body weights (Meant SD) after administration of trastuzumab (H)-MC-MMAF at doses of 2105, 3158, and 4210 gg/m². The MC linker is attached via a cysteine of trastuzumab for each conjugate.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

DEFINITIONS AND ABBREVIATIONS

Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

When trade names are used herein, applicants intend to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.

The term “antibody” herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. Described in terms of its structure, an antibody typically has a Y-shaped protein consisting of four amino acid chains, two heavy and two light. Each antibody has primarily two regions: a variable region and a constant region. The variable region, located on the ends of the arms of the Y, binds to and interacts with the target antigen. This variable region includes a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region, located on the tail of the Y, is recognized by and interacts with the immune system (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.

The term “antibody” as used herein, also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin 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 can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin. In another aspect, the antibodies are polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR’s, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies,

i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, U.S. Patent No. 4816567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent mp/ 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855).

Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Another method to prepare MAbs¹· uses genetic engineering including recombinant DNA techniques. Monoclonal antibodies made from these techniques include, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines DNA encoding regions from more than one type of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human. A humanized antibody comes predominantly from a human, even though it contains nonhuman portions. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. The nonhuman, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial to allow the antibody to recognize and bind to a specific antigen.

As noted, murine antibodies can be used. While useful for diagnostics and short-term therapies, murine antibodies cannot be administered to people long-term without increasing the risk of a deleterious immunogenic response. This response, called Human Anti-Mouse Antibody (HAMA), occurs when a human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death.

Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the nonhuman portions of administered antibodies. Furthermore, chimeric and humanized antibodies have the additional benefit of activating secondary human immune responses, such as antibody dependent cellular cytotoxicity.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 andCH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

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

The expressions “ErbB2” and “HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al., Proc. Natl. Acad. Sci. USA, 82:6497-6501 (1985) and Yamamoto etal., (1986) Nature, 319:230-234 (Genebank accession number X03363). The term “erbB2” refers to the gene encoding human ErbB2 and “neu” refers to the gene encoding rat pl85neu. Preferred ErbB2 is native sequence human ErbB2.

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

By “ErbB ligand” is meant a polypeptide which binds to and/or activates an ErbB receptor. The ErbB ligand may 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 243:10741076; Kimura et al., Nature, 348:257-260 (1990); and Cook et al., Mol. Cell. Biol., 11:2547-2557 (1991)); betacellulin (Shing et al., Science, 259:1604-1607 (1993); and Sasada et al., Biochem. Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science, 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem., 270:7495-7500 (1995); and Komurasaki et al., Oncogene, 15:2841-2848 (1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway etal., Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari etal., Oncogene, 18:2681-89 (1999)) or cripto (CR-1) (Kannan et al., J. Biol. Chem., 272(6):3330-3335 (1997)). ErbB ligands which bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HBEGF and epiregulin. ErbB ligands which bind ErbB3 include heregulins. ErbB ligands capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulins. The ErbB ligand may also be a synthetic ErbB ligand. The synthetic ligand may be specific for a particular ErbB receptor, or may recognize particular ErbB receptor complexes. An example of a synthetic ligand is the synthetic heregulin/EGF chimera biregulin (see, for example, Jones etal., (1999) FEBSLetters, 447:227-231, which is incorporated by reference).

“Heregulin” (HRG) refers to a polypeptide encoded by the heregulin gene product as disclosed in U.S. Patent No. 5641869 or Marchionni et al., Nature, 362:312-318 (1993). Examples of heregulins include heregulin-a, heregulin-βΐ, heregulin-P2 and heregulin-p3 (Holmes et al., Science, 256:12051210 (1992); and U.S. Patent Nno. 5641869); neu differentiation factor (NDF) (Peles etal., Cell 69: 205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (Falls et al. (1993) Cell 72:801-815); glial growth factors (GGFs) (Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al., J. Biol. Chem., 270:14523-14532 (1995)); γheregulin (Schaefer et al., Oncogene, 15:1385-1394 (1997)). The term includes biologically active* fragments and/or amino acid sequence variants of a native sequence HRG polypeptide, such as an EGF-like domain fragment thereof (e.g., HRGpl 177-244).

“ErbB hetero-oligomer” is a noncovalently associated oligomer comprising at least two different ErbB receptors. An “ErbB dimer” is a noncovalently associated oligomer that comprises two different ErbB receptors. Such complexes may form when a cell expressing two or more ErbB receptors is exposed to an ErbB ligand. ErbB oligomers, such as ErbB dimers, can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described in Sliwkowski etal.,J. Biol. Chem., 269(20):14661-14665 (1994), for example. Examples of such ErbB hetero-oligomers include EGFR-ErbB2 (also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes. Moreover, the ErbB hetero-oligomer may comprise two or more ErbB2 receptors combined with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR (ErbB 1). Other proteins, such as a cytokine receptor subunit (e.g., gpl30) may be included in the hetero-oligomer.

A “native sequence” polypeptide is one which has the same amino acid sequence as a polypeptide, e.g., tumor-associated antigen receptor, derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of naturally-occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.

The term “amino acid sequence variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% homology with at least one receptor binding domain of a native ligand, or with at least one ligand binding domain of a native receptor, such as a tumorassociated antigen, and preferably, they will be at least about 80%, more preferably, at least about 90% homologous with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

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

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRHL FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, (1991) Annu. Rev. Immunol, 9:457-92. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5500362 or 5821337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Prco. Natl. Acad. Sci. USA, 95:652-656 (1998).

The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and Fey RHI subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ΓΠΜ) in its cytoplasmic domain. (See review M. in Daerott, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas etal., J. Lab. Clin. Med., 126:33041 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.

The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the βsheet structure. The hypervariable regions in each chain are held together in close - proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Rabat et al. supra) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 5052 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigenbinding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab’)₂ antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

&lt;··’·· “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as described in Table 3 of U.S. Patent No. 5821337 expressly incorporated herein by reference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies as described herein below.

An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of Nterminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. 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 is one capable of binding that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen.

An antibody which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.

A “disorder” is any condition that would benefit from treatment of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant tumors; leukemia and lymphoid malignancies, in particular breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.

The term “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the-case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The term “substantial amount” refers to a majority, i.e. &gt;50% of a population, of a collection or a sample.

The term “intracellular metabolite” refers to a compound resulting from a metabolic process or reaction inside a cell on an antibody drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker of the ADC, or hydrolysis of a functional group such as a hydrazone, ester, or amide. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after entry, diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on an Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate, an an antibody drug conjugate (ADC) or the like whereby the covalent attachment, e.g., the linker, between the drug moiety (D) and the antibody (Ab) is broken, resulting in the free drug dissociated from the antibody inside the cell. The cleaved moieties of the DrugLigand Conjugate, a Drug-Linker-Ligand Conjugate or ADC are thus intracellular metabolites.

The term bioavailability refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form. · .....

The term “cytotoxic activity” refers to a cell-killing, cytostatic or anti-proliferation effect of an antibody drug conjugate compound or an intracellular metabolite of an antibody drug conjugate compound. Cytotoxic activity may be expressed as the IC50 value which is the concentration (molar or mass) per unit volume at which half the cells survive.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

An “ErbB2-expressing cancer” is one which produces sufficient levels of ErbB2 at the surface of cells thereof, such that an anti-ErbB2 antibody can bind thereto and have a therapeutic effect with respect to the cancer.

A cancer “characterized by excessive activation” of an ErbB2 receptor is one in which the extent of ErbB2 receptor activation in cancer cells significantly exceeds the level of activation of that receptor in non-cancerous cells of the same tissue type. Such excessive activation may result from overexpression of the ErbB2 receptor and/or greater than normal levels of an ErbB2 ligand available for activating the ErbB2 receptor in the cancer cells. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of an ErbB2 receptor is occurring which results in such excessive activation of the ErbB2 receptor. Alternatively, or additionally, the cancer may be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB2 ligand is occurring in the cancer which attributes to excessive activation of the receptor. In a subset of such cancers, excessive activation of the receptor may result from an autocrine stimulatory pathway.

A cancer which “overexpresses” an ErbB2 receptor is one which has significantly higher levels of an ErbB2 receptor at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. ErbB2 receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the ErbB2 protein present on the surface of a cell (e.g., via an immunohistochemistry assay; IHC). Alternatively, or additionally, one may measure levels of ErbB2-encoding nucleic acid in the cell, e.g., via fluorescent in situ hybridization (FISH; see WO 98/45479), southern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). Overexpression of the ErbB2 ligand, may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g., in a tumor biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR or in vivo assays described above. One may also study ErbB2 receptor overexpression by measuring shed antigen (e.g., ErbB2 extracellular domain) in a biological fluid such as serum (see, e.g., U.S. Patent No. 4933294; WO 91/05264; U.S. Patent No. 5401638; and Sias et al., (1990) J. Immunol. Methods, 132: 73-80). Aside from the above assays, various other in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.

The tumors overexpressing HER2 are rated by immunohistochemical scores corresponding to the number of copies of HER2 molecules expressed per cell, and can been determined biochemically: 0 = ΟΙ 0,000 copies/cell, 1+ = at least about 200,000 copies/cell, 2+ = at least about 500,000 copies/cell, 3+ = about 1-2 x 10⁶ copies/cell. Overexpression of HER2 at the 3+ level, which leads to ligand-independent activation of the tyrosine kinase (Hudziak etal., (1987) Proc. Natl. Acad. Sci. USA, 84:7159-7163), occurs in approximately 30% of breast cancers, and in these patients, relapse-free survival and overall survival are diminished (Slamon et al., (1989) Science, 244:707-712; Slamone/a/., (1987) Science, 235:177-182).

Conversely, a cancer which is “not characterized by overexpression of the ErbB2 receptor” is one which, in a diagnostic assay, does not express higher than normal levels of ErbB2 receptor compared to a noncancerous cell of the same tissue type.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., ^2I1At, ¹³¹1,¹²⁵I, ^Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, and radioactive isotopes of Lu), 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. In one aspect, the term is not intended to include radioactive isotopes.

A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA™); aeetogenins (especially bullatacin and bullatacinone); delta-9tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol;

colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;

androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as ALIMTA®, LY231514pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, anti-metabolites such as

5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX^rm, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 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); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloid; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKCalpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGER activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No. 4943533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies that bind type Π mutant EGFR (U.S. Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. Patent No. 5891996; and human antibodies that bind EGFR, such as ABX-EGF (see WO 98/50433, Abgenix). The anti-EGFR antibody may be conjugated with a cyotoxic agent, thus generating an immunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSA™; Astra Zeneca), Erlotinib HC1 (CP-358774, TARCEVA™; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).

A “tyrosine kinase inhibitor” is a molecule which inhibits to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor. Examples of such inhibitors include the EGFR-targeted drugs noted in the preceding paragraph as well as quinazolines such as PD 153035,4-(3chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines, curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoding nucleic acid); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI166 (Novartis); GW2016 (Glaxo SrnithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: U.S. Patent No. 5804396; WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels. The antiangiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic factor is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).

The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -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-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically or hydrolytically activated or converted into the 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, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are hot limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptidecontaining prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, βlactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as including the anti-CD30, CD40, CD70 or Lewis Y antibodies and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic ¹ oligonucleotide adaptors or linkers can be used in accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be

I clear from the context.· “

An “autoimmune disease” herein is a disease or disorder arising from and directed against an individual’s own tissues or a co-segregate or manifestation thereof or resulting condition therefrom. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), and IBD with co-segregate of pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and/or episcleritis), respiratory distress syndrome, including adult respiratory distress syndrome (ARDS), meningitis, IgEmediated diseases such as anaphylaxis and allergic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN, membranous proliferative GN (MPGN), including Type I and Type Π, and rapidly progressive GN, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis (MS) such as spino-optical MS, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener’s granulomatosis, agranulocytosis, vasculitis (including Large Vessel vasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu’s) Arteritis), Medium Vessel vasculitis (including Kawasaki’s Disease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis , such as Churg-Strauss vasculitis or syndrome (CSS)), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AffiLA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman’s syndrome, Goodpasture’s Syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, StevensJohnson syndrome, solid organ transplant rejection (including pretreatment for high panel reactive antibody titers, IgA deposit in tissues, and rejection arising from renal transplantation, liver transplantation, intestinal transplantation, cardiac transplantation, etc.), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune complex nephritis, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), thrombocytopenia (as developed by myocardial infarction patients, for example), including autoimmune thrombocytopenia, autoimmune disease of the testis and ovaiy including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto’s Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison’s disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM), including pediatric IDDM, and Sheehan’s syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome, Berger’s Disease (IgA nephropathy), primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractory sprue with co-segregate dermatitis herpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery * &gt; · disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, monoclonal gammopathy of uncertain/unknown significance (MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism, inflammatory myopathy, and focal segmental glomerulosclerosis (FSGS).

Alkyl is Cl-Ci 8 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, CH(CH3)₂), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l-propyl (iBu, i-butyl, -CH2CH(CH3)₂), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (CH(CH2CH3)₂), 2-methyl-2-butyl (-C(CH3)₂CH2CH3), 3-methyl-2-butyl (CH(CH3)CH(CH3)₂), 3-methyl-l-butyl (-CH2CH2CH(CH3)₂), 2-methyl-l-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)₂), 3methyl-3-pentyl (-C(CH3)(CH2CH3)₂), 2-methyl-3-pentyl (CH(CH2CH3)CH(CH3)₂), 2,3-dimethyl-2-butyl (-C(CH3)₂CH(CH3)₂), 3,3dimethyl-2-butyl (-CH(CH3)C(CH3)3.

“Alkenyl” is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carboncarbon, sp² double bond. Examples include, but are not limited to: ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH₂), cyclopentenyl (-C5H7), and 5-hexenyl (-CH₂ CH₂CH₂CH₂CH=CH₂).

.. “Alkynyl” is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon carbon, sp triple bond. Examples include, but are not limited to: acetylenic (-OCH) and propargyl (-CH₂C=CH).

Alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-) 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

“Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH=CH-).

“Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene (-C=C-), propargyl (-CH₂OC-), and 4pentynyl (-CH₂CH₂CH₂C=CH-).

Aryl means 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. Some aryl groups are represented in the exemplary structures as “Ar”. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.

Arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan1-yl and the like. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.

Heteroarylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S. The heteroaryl moiety of the heteroarylalkyl group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

Substituted alkyl”, “substituted aryl, and substituted arylalkyl mean alkyl, aryl, and arylalkyl respectively, in which one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, -O', -OR, -SR, -S’, -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO₂, =N₂, -N₃, NC(=O)R, -C(=O)R, c(=o)nr₂, -so₃, -so₃h, -s(=o)₂r, -os(=o)₂or, -s(=o)₂nr, -s(=o)r, OP(=O)(OR)₂, -P(=O)(OR)₂, -PO 3, -PO₃H₂, -C(=O)R, -C(=0)X, -C(=S)R, -co₂r, -CO₂, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR₂, -C(=S)NR₂, -C(=NR)NR₂, where each X is independently a halogen: F, Cl, Br, or I; and each R is independently -H, C₂-Ci8 alkyl, C6-C₂o aryl, C3-C14 heterocycle, protecting group or prodrug moiety. Alkylene, alkenylene, and alkynylene groups as described above may also be similarly substituted.

“Heteroaryl” and Heterocycle refer to a ring system in which one or more ring atoms is a heteroatom, e.g., nitrogen, oxygen, and sulfur. The heterocycle radical comprises 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S. A heterocycle may be a monocycle having 3 ta7^!' ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

- Heterocycles are described in Paquette, Leo A.; Principles of · Modem Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A series of Monographs (John Wiley &amp; Sons, New York, 1950 to present), in particular Volumes 13,14, 16,19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Examples of heterocycles include by way of example and not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bistetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-l,2,5-thiadiazinyI, 2H,6H-l,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, ΙΗ-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, βcarbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2,4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2,3, 4, or 5 of-a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2,4, or 5 of an oxazole, imidazole or thiazole, position 3,4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3,4, 5,6,7, or 8 of a quinoline or position 1, 3,4,5, 6, 7, or 8 of an isoquinoline. Still more typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,

4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, ΙΗ-indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or β-carboline. Still more typically, nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5], [5.5] , [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6.6] system. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, l-cyclopent-2-enyl, l-cyclopent-3enyl, cyclohexyl, 1-cyclohex-1-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cycloheptyl, and cyclooctyl.

Linker, “Linker Unit”, or “link” means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, a linker is specified as LU. Linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: -(CR2)_nO(CR2)_n-, repeating units of alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.

The term chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term achiral refers to molecules which are superimposable on their mirror image partner.

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

Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.

Enantiomers refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally follow 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 &amp; Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

Examples of a “patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human.

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a heterocyclic aromatic group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-Cs alkyl, -O-(Ci-C₈ alkyl), -aryl, -C(O)R’, -OC(O)R’, -C(O)OR’, -C(O)NH₂, -C(O)NHR’, -C(O)N(R’)₂ -NHC(O)R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃, -NH₂, NH(R’), -N(R’)₂ and -CN; wherein each R’ is independently selected from H, Ci-Cg alkyl and aryl.

The term “Ci-Cg alkyl,” as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “Ci-Cg alkyl” groups include, but are not limited to, -methyl, ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -ndecyl; while branched Ci-Cg alkyls include, but are not limited to, -isopropyl, sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated Ci-Cg alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl- 1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, 1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A Ci-Cg alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-C₈ alkyl, -O-(Ci-C₈ alkyl), -aryl, -C(O)R’, -OC(O)R’, C(O)OR’, -C(O)NH₂ , -C(O)NHR’, -C(O)N(R’)₂ -NHC(O)R’, -SO₃R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃, -NH₂, -NH(R’), -N(R’)₂ and -CN; where each R’ is independently selected from H, -Ci-C₈ alkyl and aryl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C₃-C₈ carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl, and -cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-C₈ alkyl, -O-(Ci-C₈ alkyl), -aryl, -C(O)R’, -OC(O)R’, -C(O)OR’, -C(O)NH₂ , -C(O)NHR’, -C(O)N(R’)₂ -NHC(O)R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃, -NH₂, NH(R’), -N(R’)₂ and -CN; where each R’ is independently selected from H, -CiC₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined above wherein one of the carbocycle groups’ hydrogen atoms is replaced with a bond.

A “Ci-Cio alkylene” is a straight chain, saturated hydrocarbon group of the formula -(CH₂)i_io-. Examples of a Ci-Cio alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.

An “arylene” is an aryl group which has two covalent bonds and can be in the ortho, meta, or para configurations as shown in the following structures:

<img file="IN520DEN2012A_D0009.tif" />

in which the phenyl group can be unsubstituted or substituted with up to four groups including, but not limited to, -Ci-Cg alkyl, -O-(Ci-Cg alkyl), -aryl, C(O)R’, -OC(O)R’, -C(O)OR’, -C(O)NH₂ , -C(O)NHR’, -C(O)N(R’)₂ NHC(O)R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃, -NH₂, -NH(R’), -N(R’)₂ and -CN; wherein each R’ is independently selected from H, -Ci-Cg alkyl and aryl.

A “C3-C8 heterocycle” refers to an aromatic or non-aromatic C₃-Cg carbocycle in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a C₃-Cg heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. A C₃-Cg heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, -Ci-Cg alkyl, O-(Ci-C₈ alkyl), -aryl, -C(0)R’, -0C(0)R’, -C(0)0R’, -C(0)NH₂ , -C(O)NHR’, C(0)N(R’)₂ -NHC(0)R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃ , -NH₂, -NH(R’), -N(R’)₂ and -CN; wherein each R’ is independently selected from H, -Ci-Cg alkyl and aryl.

“C₃-Cg heterocyclo” refers to a C₃-Cg heterocycle group defined above wherein one of the heterocycle group’s hydrogen atoms is replaced with a bond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up to six groups including, but not limited to, -Ci-Cg alkyl, -O-(Ci-Cg alkyl), -aryl, C(O)R’, -OC(O)R’, -C(O)OR’, -C(0)NH₂ , -C(0)NHR’, -C(O)N(R’)₂ NHC(0)R’, -S(O)₂R’, -S(O)R’, -OH, -halogen, -N₃, -NH₂, -NH(R’), -N(R’)₂ and -CN; wherein each R’ is independently selected from H, -Ci-Cg alkyl and aryl.

An “Exemplary Compound” is a Drug Compound or a DrugLinker Compound.

An “Exemplary Conjugate” is a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate or a Drug-Linker-Ligand Conjugate. 68

In some embodiments, the Exemplary Compounds and Exemplary Conjugates are in isolated or purified form. As used herein, “isolated” means separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95 %, and in another aspect at least 98%, of Exemplary Compound or Exemplary Conjugate by weight of the isolate.

Examples of a “hydroxyl protecting group” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.

The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an Exemplary Compound or Exemplary Conjugate. The Exemplary Compounds and Exemplary Conjugates contain at least one amino group, and accordingly acid addition salts can be formed with this amino group. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., Ι,Γ-methylene-bis -(2-hydroxy-3naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.

The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a compound of the invention, e.g., an Exemplary Compound or Exemplary Conjugate. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The following abbreviations are used herein and have the indicated definitions: AE is auristatin E, Boc is N-(r-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA is MN-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is MN-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine, dov is ΛζΝ-dimethylvaline, DTNB is 5,5’dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline, ESMS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is /V-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is O-(7-azabenzotriazol-1 -yl)-/V,/V,^V’,/V’-tetramethyluronium hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH3CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or 4-methoxytrityl),nor is (IS, 2R)-(+)norephedrine, PAB is p-aminobenzyl, PBS is phosphate-buffered saline (pH 7.4),

PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromo ZrA-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusion chromatography, Su is succinimide, TBTU is G-benzotriazol-l-yl-N,/V,N,/V-tetramethyluronium tetrafluoroborate, TFA is trifiuoroacetic acid, TLC is thin layer chromatography, UV is ultraviolet, and val is valine.

The following linker abbreviations are used herein and have the indicated definitions: Val Cit is a valine-citrulline, dipeptide site in protease cleavable linker; PAB is p-aminobenzylcarbamoyl; (Me)vc is N-methyl-valine citrulline, where the linker peptide bond has been modified to prevent its cleavage by cathepsin B; MC(PEG)6-OH is maleimidocaproyl- polyethylene glycol; SPP is N-Succinimidyl 4-(2-pyridylthio) pentanoate; and SMCC is N-Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-1 carboxylate.

The terms “treat” or “treatment,” unless otherwise indicated by context, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean 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 prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

In the context of cancer, the term “treating” includes any or all of: preventing growth of tumor cells, cancer cells, or of a tumor; preventing replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.

The following cytotoxic drug abbreviations are used herein and have the indicated definitions: MMAE is mono-methyi auristatin E (MW 718); MMAF is N-methyl valine- valine-dolaisoleuine-dolaproine-phenylalanine (MW 731.5); MMAF-DMAEA is MMAF with DMAEA (dimethylaminoethylamine) in an amide linkage to the C-terminal phenylalanine (MW 801.5); MMAF-TEG is MMAF with tetraethylene glycol esterified to the phenylalanine; MMAF-NtBu is N-t-butyl, attached as an amide to C-terminus of MMAF; AEVB is auristatin E valeryl benzylhydrazone, acid labile linker through the C-terminus of AE (MW 732); and AFP is Monoamide of p-phenylene diamine with C-terminal Phenylalanine of Auristatin F (MW 732).

THE COMPOUNDS OF THE INVENTION

4.2.1 THE COMPOUNDS OF FORMULA (la)

In one aspect, the invention provides Drug-Linker-Ligand Conjugates having Formula la:

t^-Aa-W^Yy^)) _p la or a pharmaceutically acceptable salt or solvate thereof wherein,

L- is a Ligand unit;

-Aa-W_w-Yy- is a Linker unit (LU), wherein the Linker unit includes:

-A- is a Stretcher unit, a is 0 or 1, each -W- is independently an Amino Acid unit, w is an integer ranging from 0 to 12, -Y- is a Spacer unit, and y is 0, 1 or 2;

p ranges from 1 to about 20; and

<img file="IN520DEN2012A_D0010.tif" />

wherein, independently at each location:

R² is selected from H and CpCg alkyl;

R³ is selected from H, Ci-Cg alkyl, Cj-Cg carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-Cg carbocycle), C3-Cg heterocycle and Ci-Cg alkyl(C₃-C₈ heterocycle);

R⁴ is selected from H, Ci-C₈ alkyl, C3-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C3-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)n- wherein R^a and R^b are independently selected from H, Ci-C8 alkyl and C₃-C₈ carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Ci-C₈ alkyl;

R⁷ is selected from H, Ci-C₈ alkyl, C3-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C3-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, Ci-C₈ alkyl, C3-C₈carbocycle and O-(Ci-C₈ alkyl);

R⁹ is selected from H and Ci-C₈ alkyl;

R¹⁰ is selected from aryl or C3-Q heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-C₈ alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)2;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴is H or Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, Ci-C₈ alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C3-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C3-C₈ carbocycle); and n is an integer ranging from 0 to 6.

In another embodiment, the present invention provides Drug Compounds having the Formula lb:

<img file="IN520DEN2012A_D0011.tif" />

Ib or pharmaceutically acceptable salts or solvates thereof, wherein:

R² is selected from hydrogen and -Ci-C₈ alkyl;

R³ is selected from hydrogen, -Ci-C₈ alkyl, -C₃-C₈ carbocycle, aryl, -Ci-C₈ alkyl-aryl, -Ci-C₈ alkyl-(C₃-C₈ carbocycle), -C₃-C₈ heterocycle and -Ci-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from hydrogen, -Ci-C8 alkyl, -C3-C8 carbocycle, aryl, -Ci-C8 alkyl-aryl, -Ci-C8 alkyl-(C3-C8 carbocycle), -C3-C8 heterocycle and Ci-C8 alkyl-(C3-C8 heterocycle) wherein R⁵ is selected from -H and -methyl; or R⁴and R⁵ jointly, have the formula -(CR^aR^b)n- wherein R^a and R^b are independently selected from -H, -Ci-C₈ alkyl and -C₃-C₈ carbocycle and n is selected from 2, 3,

4,5 and 6, and form a ring with the carbon atom to which they are attached;

R⁶ is selected from H and -Ci-C₈ alkyl;

R⁷ is selected from H, -Ci-C₈ alkyl, -C₃-C₈ carbocycle, aryl, -Ci-C₈alkyl-aryl, -Ci-C₈ alkyl-(C₃-C₈ carbocycle), -C₃-C₈ heterocycle and -Ci-C₈ alkyl(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, -OH, -Ci-C₈ alkyl, -C₃C₈ carbocycle and -O-(Ci-C₈ alkyl);

R⁹ is selected from H and -Ci-C₈ alkyl;

R¹⁰ is selected from aryl group or -C₃-C₈ heterocycle;

Z is -0-, -S-, -NH-, or -NR¹²-, wherein R¹² is Ci-C₈ alkyl;

R¹¹ is selected from H, Ci-C2o alkyl, aryl, -C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)2;

m is an integer ranging from 1-1000;

R¹³ is -C₂-C₈ alkyl;

R¹⁴is H or -Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, -COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, -Ci-C₈ alkyl, or (CH₂)_n-COOH; and n is an integer ranging from 0 to 6.

In yet another embodiment, the invention provides Drug-LinkerLigand Conjugates having the Formula la’:

Ab-(-A_a- W_w-Y_y-D)_p

Formula la’ or pharmaceutically acceptable salts or solvates thereof.

wherein:

Ab is an antibody,

A is a Stretcher unit, a is 0 or 1, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

Y is a Spacer unit, and y is 0,1 or 2, p ranges from 1 to about 20, and

D is a Drug moiety selected from Formulas De and Df:

<img file="IN520DEN2012A_D0012.tif" />

ο

CH₃ R⁹ Ο

1 1

<img file="IN520DEN2012A_D0013.tif" />

Ν

R wherein, independently at each location:

R² is selected from H and Ci-Cg alkyl;

R³ is selected from H, Ci-Cs alkyl, C3-C8 carbocycle, aryl, Ci-Cs alkyl-aryl, Ci-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle and Ci-C₈ alkyl(C3-C8 heterocycle);

R⁴ is selected from H, Ci-Cs alkyl, C3-C8 carbocycle, aryl, Ci-Cs alkyl-aryl, Ci-Cs alkyl-(C3-Cs carbocycle), C3-C8 heterocycle and Ci-Cs alkyl(C3-C8 heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from H, Ci-Cs alkyl and C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Ci-Cs alkyl;

R⁷ is selected from H, Ci-C₈ alkyl, C3-C8 carbocycle, aryl, CpCs alkyl-aryl, Ci-Cs alkyl-(C3-Cs carbocycle), C3-C8 heterocycle and Ci-Cs alkyl(C3-C8 heterocycle);

each R⁸ is independently selected from H, OH, Ci-Cs alkyl, C3-C8 carbocycle and O-(Ci-Cs alkyl);

R⁹ is selected from H and Ci-Cs alkyl;

R¹⁰ is selected from aryl or C3-C8 heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-Cs alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, Ci-C₈ alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C₃-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C₃-C₈ carbocycle); and n is an integer ranging from 0 to 6.

Ab is any antibody covalently attached to one or more drug units. Ab includes an antibody which binds to CD30, CD40, CD70, Lewis Y antigen. In another embodiment, Ab does not include an antibody which binds to an ErbB receptor or to one or more of receptors (1)-(35):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);

(7) Serna 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);

(9) ETBR (Endothelin type B receptor, Genbank accession no.

AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);

(12) TrpM4 (BR22450, FU20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);

(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764);

(17) HER2 (Genbank accession no. Ml 1730);

(18) NCA (Genbank accession no. M18728);

(19) MDP (Genbank accession no. BCO17023);

(20) IL20Ra (Genbank accession no. AF184971);

(21) Brevican (Genbank accession no. AF229053);

(22) Ephb2R (Genbank accession no. NM_004442);

(23) ASLG659 (Genbank accession no. AX092328);

(24) PSCA (Genbank accession no. AJ297436);

(25) GEDA (Genbank accession no. AY260763);

(26) BAFF-R (Genbank accession no. NP_443177.1);

(27) CD22 (Genbank accession no. NP-001762.1);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in Bcell differentiation, Genbank accession No. NP_001774.1);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);

(30) HLA-DOB (Beta subunit of MHC class Π molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession No. NP_002111.1);

(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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);

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

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and IT AM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); and/or (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and tymphomagenesis; deregulation of the gene by translocation occurs iri Some B cell malignancies, Genbank accession No. NP_112571.1).

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

In another embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ is -H. In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is -H, and R⁷ is sec-butyl. In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is -H.

In still another embodiment, each occurrence of R⁸ is -OCH3.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH3, and R⁹ is -H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -0-, R¹¹ is -H, methyl or t-butyl.

In one embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵is -(CH2)n-N(R¹⁶)2, and R¹⁶ is -Ci-C₈ alkyl or -(CH₂)_n-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵ is -(CH2)_n-SO3H.

In one aspect, Ab is cAClO, cBR96, cS2C6, clF6, c2F2, hAClO, hBR96, hS2C6, hlF6, and h2F2.

Exemplary embodiments of Formula la have the following

<img file="IN520DEN2012A_D0014.tif" />

<img file="IN520DEN2012A_D0015.tif" />

mA-S. b

O

<img file="IN520DEN2012A_D0016.tif" />

L-MC-MMAF wherein L is an antibody, Vai is valine, and Cit is citrulline.

The drug loading is represented by p, the average number of drug molecules per antibody in a molecule (e.g., of Formula la, la’ and Ic). Drug loading may range from 1 to 20 drugs (D) per Ligand (eg. Ab or inAb). Compositions of Formula la and Formula la’ include collections of antibodies conjugated with a range of drugs, from 1 to 20. The average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Ligand-Drug-Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Ligand-Drug-conjugates where p is a certain value from LigandDrug-Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

4.2.2 THE DRUG COMPOUNDS OF FORMULA (lb)

In another aspect, the present invention provides Drug Compounds having the Formula (lb):

<img file="IN520DEN2012A_D0017.tif" />

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R² is selected from -hydrogen and -Ci-Cs alkyl;

R³ is selected from -hydrogen, -Ci-Cs alkyl, -C3-C8 carbocycle, aryl, -Ci-Cs alkyl-aryl, -Ci-Cs alkyl-(C3-Cs carbocycle), -C3-C8 heterocycle and Ci-Ce alkyl-(C3-Cs heterocycle);

R⁴ is selected from -hydrogen, -Cj-Cg alkyl, -C3-C8 carbocycle, aryl, -Ci-Cs alkyl-aryl, -Ci-Cs alkyl-(C3-Cs carbocycle), -C3-C8 heterocycle and Ci-Cs alkyl-(C3-C8 heterocycle) wherein R⁵ is selected from -H and -methyl; or R⁴and R⁵ jointly, have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from -H, -Ci-Cs alkyl and -C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;

R⁶ is selected from -H and -Ci-Cs alkyl;

R⁷ is selected from -H, -Ci-C₈ alkyl, -C3-C8 carbocycle, aryl, -CiC₈ alkyl-aryl, -Ci-Cs alkyl-(C3-Cs carbocycle), -C3-C8 heterocycle and -Ci-Cs alkyl-(C3-Cs heterocycle);

each R⁸ is independently selected from -H, -OH, -Ci-Cs alkyl, -C3Cs carbocycle and -O-(Ci-Cs alkyl);

R⁹ is selected from -H and -Ci-Cs alkyl;

R¹⁰ is selected from aryl group or -C3-C8 heterocycle;

Z is -0-, -S-, -NH-, or -NR¹²-, wherein R¹² is Ci-Cs alkyl;

R¹¹ is selected from -H, C1-C20 alkyl, aryl, -C3-C8 heterocycle, (R¹³O)_m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)2;

m is an integer ranging from 1-1000;

R¹³ is -C₂-C₈ alkyl;

R¹⁴is -H or-Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently -H, -COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently -H, -Ci-C₈ alkyl, or (CH₂)_n-COOH; and n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ is -H. In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is -H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is -H.

In still another embodiment, each occurrence of R⁸ is -OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH₃, and R⁹ is -H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -0-, R¹¹ is -H, methyl or t-butyl.

In one embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵is -(CH2)n-N(R¹⁶)2, and R¹⁶ is -Ci-C₈ alkyl or -(CH₂)_n-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵ is -(CH2)_n-SO₃H.

Illustrative Compounds of Formula (lb), each of which may be used as drug moieties (D) in ADC, include compounds having the following structures:

<img file="IN520DEN2012A_D0018.tif" />

ο

<img file="IN520DEN2012A_D0019.tif" />

W

<img file="IN520DEN2012A_D0020.tif" />

<img file="IN520DEN2012A_D0021.tif" />

<img file="IN520DEN2012A_D0022.tif" />

<img file="IN520DEN2012A_D0023.tif" />

<img file="IN520DEN2012A_D0024.tif" />

<img file="IN520DEN2012A_D0025.tif" />

<img file="IN520DEN2012A_D0026.tif" />

and pharmaceutically acceptable salts or solvates thereof.

THE COMPOUNDS OF FORMULA (Ic)

In another aspect, the invention provides antibody-drug conjugate compounds (ADC) having Formula Ic:

Ab-(-A_a- W_w-Y_y-D)_{p k} comprising an antibody covalently attached to one or more drug units (moieites). The antibody-drug conjugate compounds include pharmaceutically acceptable salts or solvates thereof.

Formula Ic compounds are defined wherein:

Ab is an antibody which binds to one or more tumor-associated antigen receptors (1)-(35):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);

(7) Serna 5b (FU10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004);

(15) CD79b (CD79B, Οϋ79β, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764);

(17) HER2 (Genbank accession no. Ml 1730);

(18) NCA (Genbank accession no. Ml8728);

(19) MDP (Genbank accession no. BCO17023);

(20) IL20Ra (Genbank accession no. AF184971);

(21) Brevican (Genbank accession no. AF229053);

(22) Ephb2R (Genbank accession no. NM_004442);

(23) ASLG659 (Genbank accession no. AX092328);

(24) PSCA (Genbank accession no. AJ297436);

(25) GEDA (Genbank accession no. AY260763;

(26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3,

BR3, NP_443177.1); · (27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in Bcell differentiation, Genbank accession No. NP_001774.1);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);

(30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession No. NP_002111.1);

(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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);

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

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); and (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1).

A is a Stretcher unit, a is 0 or 1, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

Y is a Spacer unit, and y is 0, 1 or 2, p ranges from 1 to about 8, and

D is a Drug moiety selected from Formulas De and D_F:

<img file="IN520DEN2012A_D0027.tif" />

wherein the wavy line of De and D_F indicates the covalent attachment site to A, W, or Y, and independently at each location:

R² is selected from H and Ci-C₈ alkyl;

R³ is selected from H, Ci-C₈ alkyl, C₃-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

R⁴ is selected from H, Ci-C₈ alkyl, C₃-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from H, Ci-Cg alkyl and C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Cj-C₈ alkyl;

R⁷ is selected from H, Ci-C₈ alkyl, C3-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C₃-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, Ci-C₈ alkyl, C3-C₈carbocycle and O-(Ci-C₈ alkyl);

R⁹ is selected from H and Ci-C₈ alkyl;

R¹⁰ is selected from aryl or C3-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-C₈ alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)2;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R^I4isHorC_rC₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or-(CH₂)_n-SO₃-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, Ci-C₈ alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C₃-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C3-C₈ carbocycle); and n is an integer ranging from 0 to 6.

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

In another embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ is -H. In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is -H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is H. .....

In still another embodiment, each occurrence of R⁸ is -OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH3, and R⁹ is -H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -O-, R¹¹ is -H, methyl or t-butyl.

In one embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵is -(CH2)_n-N(R¹⁶)2, and R¹⁶ is -CrC₈ alkyl or -(CH₂)_n-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵ is -(CH2)_n-SO₃H.

Exemplary embodiments of Formula Ic ADC have the following structures:

<img file="IN520DEN2012A_D0028.tif" />

<img file="IN520DEN2012A_D0029.tif" />

<img file="IN520DEN2012A_D0030.tif" />

<img file="IN520DEN2012A_D0031.tif" />

Ab-MC-MMAE

<img file="IN520DEN2012A_D0032.tif" />

wherein Ab is an antibody which binds to one or more tumor-associated antigen receptors (l)-(35); Vai is valine; and Cit is citrulline.

The drug loading is represented by p, the average number of drugs per antibody in a molecule of Formula I. Drug loading may range from 1 to 20 drugs (D) per antibody (Ab or mAb). Compositions of ADC of Formula I include collections of antibodies conjugated with a range of drugs, from 1 to 20. The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV/visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of ADC in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

For some antibody drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.

Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds of the invention exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). Additionally, the antibody must be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by dual ELISA antibody assay, specific for antibody and specific for the drug. Individual ADC molecules may be identified in the mixture by mass spectroscopy, and separated by HPLC, e.g., hydrophobic interaction chromatography (“Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibodydrug conjugate”, Hamblett, K.J., et al, Abstract No. 624, American Association for Cancer Research; 2004Annual 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, Abstract No. 627, American Association for Cancer Research; 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). Thus, a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

4.3 THE LINKER UNIT

A “Linker unit” (LU) is a bifunctional compound which can be used to link a Drug unit and an Ligand unit to form Drug-Linker-Ligand Conjugates, or which are useful in the formation of immunoconjugates directed against tumor associated antigens. Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells.

In one embodiment, the Linker unit of the Drug-Linker Compound and DrugLinker-Ligand Conjugate has the formula:

-A_a—W_w—Y_ywherein:

-A- is a Stretcher unit;

a is 0 or 1;

each -W- is independently an Amino Acid unit; w is independently an integer ranging from 0 to 12;

-Y- is a Spacer unit; and y is 0, 1 or 2.

In the Drug-Linker-Ligand Conjugate, the Linker is capable of linking the Drug moiety and the Ligand unit.

THE STRETCHER UNIT

The Stretcher unit (-A-), when present, is capable of linking a Ligand unit to an amino acid unit (-W-). In this regard a Ligand (L) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a ligand, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. In one aspect, the Ligand functional groups are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand. Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a Ligand using 2-iminothiolane (Traut’s reagent) or another sulfhydryl generating reagent.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the Ligand unit. The sulfur atom can be derived from a sulfhydryl group of a Ligand. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Illa and Illb, wherein L-, -W-, -Y-, -D, w and y are as defined above, and R¹⁷ is selected from -C1-C10 alkylene-, -C3-C8 carbocyclo-, -O-(Ci-C₈ alkyl)-, -arylene-, -C1-C10 alkylene-arylene-, -arylene-CiC10 alkylene-, -C1-C10 alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-Ci-Cio alkylene-, -C3-C8 heterocyclo-, -Ci-Cioalkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-Ci-Cio alkylene-, -(CH₂CH₂O)_r-, and -(CH2CH₂O)_r-CH2-; and r is an integer ranging from 1-10. It is to be understood from all the exemplary embodiments of Formula la, such as III-VI, that even where not denoted expressly, from 1 to 20 drug moieties are linked to a Ligand (p = 1-20).

<img file="IN520DEN2012A_D0033.tif" />

Illa

<img file="IN520DEN2012A_D0034.tif" />

Illb

An illustrative Stretcher unit is that of Formula Illa wherein R¹⁷ is

-(CH₂)₅-:

<img file="IN520DEN2012A_D0035.tif" />

Another illustrative Stretcher unit is that of Formula Illa wherein

R¹⁷ is -(CH₂CH₂O)_r-CH₂-; and r is 2:

<img file="IN520DEN2012A_D0036.tif" />

Still another illustrative Stretcher unit is that of Formula Illb wherein R¹⁷ is -(CH2)s-:

O

<img file="IN520DEN2012A_D0037.tif" />

<img file="IN520DEN2012A_D0038.tif" />

o ·

In another embodiment, the Stretcher unit is linked to the Ligand unit via a disulfide bond between a sulfur atom of the Ligand unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is depicted within the square brackets of Formula IV, wherein R¹⁷, L-, -W-, -Y-, -D, w and y are as defined above.

L-i-S-R¹⁷-C(O)- -W_w-Y_y—D

IV

In yet another embodiment, the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of a Ligand. Example of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Va and Vb, wherein -R¹⁷-, L-, -W-, -Y-, -D, w and y are as defined above;

C(O)NH-R¹⁷-C(O)—W_w—Y_y— D

Va

Yy^-D

Vb

In yet another aspect, the reactive group of the Stretcher contains a reactive site that is reactive to a modified carbohydrate’s (-CHO) group that can be present on a Ligand. For example, a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Via, VIb, and Vic, wherein -R¹⁷-, L-, -W-, -Y-, -D, w and y are as defined above.

<img file="IN520DEN2012A_D0039.tif" />

Via

<img file="IN520DEN2012A_D0040.tif" />

VIb ο

<img file="IN520DEN2012A_D0041.tif" />

Vic

THE AMINO ACID UNIT

The Amino Acid unit (-W-), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug moiety if the Spacer unit is absent, and links the Ligand unit to the Drug unit if the Stretcher unit and Spacer unit are absent.

W_w- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each -W- unit independently has the formula denoted below in the square brackets, and w is an integer ranging from 0 to 12:

<img file="IN520DEN2012A_D0042.tif" />

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, phydroxybenzyl, -CH₂OH, -CH(OH)CH₃, -CH₂CH₂SCH₃, -CH₂CONH₂, CH₂COOH, -CH₂CH₂CONH₂, -CH₂CH₂COOH, -(CH₂)₃NHC(=NH)NH₂, (CH₂)₃NH₂, -(CH₂)₃NHCOCH₃, -(CH₂)₃NHCHO, -(CH₂)4NHC(=NH)NH₂, (CH₂)4NH₂, -(CH₂)4NHCOCH₃, -(CH₂)4NHCHO, -(CH₂)₃NHCONH₂, (CH₂)4NHCONH₂, -CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

<img file="IN520DEN2012A_D0043.tif" />

The Amino Acid unit can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).

Illustrative W_w units are represented by formulas (VII)-(IX):

<img file="IN520DEN2012A_D0044.tif" />

wherein R²⁰ and R²¹ are as follows:

R²⁰

R²¹

<td> benzyl methyl isopropyl isopropyl</td><td> (CH₂)4NH₂; (CH₂)4NH₂; (CH₂)₄NH₂; (CH₂)₃NHCONH₂;</td>

100 benzyl isobutyl sec-butyl

<img file="IN520DEN2012A_D0045.tif" />

benzyl benzyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂;

methyl; and (CH₂)₃NHC(=NH)NH₂;

<img file="IN520DEN2012A_D0046.tif" />

wherein R²⁰, R²¹ and R²² are as follows:

<td> R²⁰</td><td> R*</td><td> R^</td>

<td> benzyl</td><td> benzyl</td><td> (CH₂)4NH₂;</td>

<td> isopropyl</td><td> benzyl</td><td> (CH₂)4NH₂; and</td>

<td> H</td><td> benzyl</td><td> (CH₂)4NH₂;</td>

<img file="IN520DEN2012A_D0047.tif" />

wherein R²⁰, R²¹, R²² and R²³ are as follows:

<td> ^20</td><td> g21</td><td> g22</td><td> £23</td>

<td> H</td><td> benzyl</td><td> isobutyl</td><td> H; and</td>

<td> methyl</td><td> isobutyl</td><td> methyl</td><td> isobutyl.</td>

Exemplary Amino Acid units include, but are not limited to, units of formula (VII) where: R²⁰ is benzyl and R²¹ is -(CH₂)4NH₂; R²⁰ isopropyl and

101

R²¹ is -(CH2)4NH2; R²⁰ isopropyl and R²¹ is -(CH2)₃NHCONH₂. Another exemplary Amino Acid unit is a unit of formula (VIII) wherein R²⁰ is benzyl, R²¹is benzyl, and R²² is -(CH₂)4NH₂.

Useful -W_w- units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumorassociated protease. In one embodiment, a -W_w - unit is that whose cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.

In one embodiment, -W_w- is a dipeptide, tripeptide, tetrapeptide or pentapeptide.

When R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, the carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is independently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit is valine-citrulline. In another aspect, the Amino Acid unit is phenylalanine-lysine (i.e. fk). In yet another aspect of the Amino Acid unit, the Amino Acid unit is Nmethylvaline-citrulline. In yet another aspect, the Amino Acid unit is 5aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alariine lysine, glycine serine valine glutamine and isonepecotic acid.

In certain embodiments, the Amino Acid unit can comprise natural amino acids. In other embodiments, the Amino Acid unit can comprise nonnatural amino acids.

THE SPACER UNIT

The Spacer unit (-Y-), when present, links an Amino Acid unit to the Drug moiety when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug moiety when the Amino Acid unit is absent. The Spacer unit also links the Drug moiety to the Ligand unit when both the Amino Acid unit and Stretcher unit are absent.

102

Spacer units are of two general types: self-immolative and non self-immolative. A non self-immolative Spacer unit is one in which part or all of the Spacer unit remains bound to the Drug moiety after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immolative Spacer unit include, but are not limited to a (glycine-glycine) Spacer unit and a glycine Spacer unit (both depicted in Scheme 1) (infra). When an Exemplary Compound containing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycineDrug moiety is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction takes place within the target cell, cleaving the glycine-Drug moiety bond and liberating the Drug.

In another embodiment, -Y_y- is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) whose phenylene portion is substituted with Q_mwherein Q is -Ci-Cg alkyl, -O-(C|-C₈ alkyl), -halogen,- nitro or -cyano; and m is an integer ranging from 0-4.

Scheme 1

<img file="IN520DEN2012A_D0048.tif" />

enzymatic cleavage

Gly-D hydrolysis

Drug

Ab-f-Aa-Ww-Gly-Glyj-D enzymatic cleavage ,

Gly-Gly-D hydrolysis

Drug

In one embodiment, a non self-immolative Spacer unit (-Y-) is Gly-Gly-.

In another embodiment, a non self-immolative the Spacer unit (-Y-) is -Gly-.

103

In one embodiment, a Drug-Linker Compound or a Drug-Linker Ligand Conjugate is provided in which the Spacer unit is absent (y=0), or a pharmaceutically acceptable salt or solvate thereof.

Alternatively, an Exemplary Compound containing a selfimmolative Spacer unit can release -D without the need for a separate hydrolysis step. In this embodiment, -Y- is a PAB group that is linked to -W_w - via the amino nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group. Without being bound by any particular theory or mechanism, Scheme 2 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via a carbamate or carbonate group espoused by Toki et al. (2002) J Org. Chem. 67:1866-1872.

<img file="IN520DEN2012A_D0049.tif" />

Qm ^nh?-£%?__cZ1_d

II

O

1,6-elimination

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

104

Without being bound by any particular theory or mechanism,

Scheme 3 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via an ether or amine linkage.

Scheme 3

<img file="IN520DEN2012A_D0050.tif" />

enzymatic cleavage

1,6-elimination

<img file="IN520DEN2012A_D0051.tif" />

NH + Drug wherein Q is -Ci-C₈ alkyl, -O-(Ci-C₈ alkyl), -halogen,- nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.

Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology,

105

1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972,94, 5815) and 2aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacer useful in Exemplary Compounds.

In one embodiment, the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs.

Scheme 4

<img file="IN520DEN2012A_D0052.tif" />

Qm CH₂(O(C(O)))_n-D \ ^CH₂(O(C(O)))_n-D enzymatic cleavage

<img file="IN520DEN2012A_D0053.tif" />

drugs wherein Q is -Ci-Cg alkyl, -O-(Ci-Cg alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1 to about 20.

In one embodiment, the -D moieties are the same. In yet another embodiment, the -D moieties are different.

In one aspect, Spacer units (-Y_y-) are represented by Formulas (X)(XII):

<img file="IN520DEN2012A_D0054.tif" />

wherein Q is -Ci-Cg alkyl, -O-(Ci-C₈ alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4;

106

HN-CH₂-CO-l xi and —NHCH₂C(O)-NHCH₂C(O)s XII.

Embodiments of the Formula la’ and Ic antibody-drug conjugate compounds include:

<img file="IN520DEN2012A_D0055.tif" />

and,

<img file="IN520DEN2012A_D0056.tif" />

<img file="IN520DEN2012A_D0057.tif" />

wherein w and y are each 0,

107

<img file="IN520DEN2012A_D0058.tif" />

THE DRUG UNIT (MOIETY)

The drug moiety (D) of the antibody drug conjugates (ADC) are of the dolastatin/auristatin type (U.S. Patent Nos. 5635483; 5780588) which have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear

108 and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965)

D is a Drug unit (moiety) having a nitrogen atom that can form a bond with the Spacer unit when y=l or 2, with the C-terminal carboxyl group of an Amino Acid unit when y=0, with the carboxyl group of a Stretcher unit when w and y =0, and with the carboxyl group of a Drug unit when a, w, and y =0. It is to be understood that the terms “drug unit” and “drug moiety” are synonymous and used interchangeably herein.

In one embodiment, -D is either formula De or Df:

<img file="IN520DEN2012A_D0059.tif" />

<img file="IN520DEN2012A_D0060.tif" />

D_f wherein, independently at each location:

R² is selected from H and Ci-C₈ alkyl;

R³ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Cj-C₈alkyl-aryl, Ci-C₈ alkyl-(C3-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

R⁴ is selected from H, Ci-C₈ alkyl, C3-C₈ carbocycle, aryl, Ci-C₈alkyl-aryl, Ci-C₈ alkyl-(C3-C₈ carbocycle), C3-C₈ heterocycle and Ci-C₈ alkyl(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

109 or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(CR^aR^b)_n- wherein R^a and R^b are independently selected from H, Ci-Cg alkyl and C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6;

R⁶ is selected from H and Ci-Cg alkyl;

R⁷ is selected from H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, Ci-Cg alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and Ci-Cg alkyl(C3-C8 heterocycle);

each R⁸ is independently selected from H, OH, Ci-Cg alkyl, C3-C8 carbocycle and O-(Ci-Cs alkyl);

R⁹ is selected from H and Ci-Cg alkyl;

R¹⁰ is selected from aryl or C3-C8 heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is Ci-Cg alkyl;

R¹¹ is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, (R¹³O)m-R¹⁴, or -(R¹³O)m-CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴is H or Ci-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)nN(R¹⁶)2, -(CH₂)_n-SO₃H, or -(CH₂)_n-SO3-Ci-C₈ alkyl;

each occurrence of R¹⁶ is independently H, Ci-Cg alkyl, or -(CH₂)_n-COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)2-C(R⁸)2-(C3-C₈heterocycle), and -C(R⁸)2-C(R⁸)2-(C3-C8 carbocycle); and n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ is -H. In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is H.

In still another embodiment, each occurrence of R⁸ is -OCH₃.

110

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is H,R⁷ is sec-butyl, each occurrence of R⁸ is -OCH3, and R⁹ is H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -O-, R¹¹ is H, methyl or tbutyl.

In one embodiment, when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵is -(CH2)n-N(R¹⁶)2, and R¹⁶ is -Ci-C₈ alkyl or -(CH₂)_n-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R^I5)2, wherein R¹⁵ is -(CH2)_n-SO₃H.

Illustrative Drug units (-D) include the drug units having the following structures:

<img file="IN520DEN2012A_D0061.tif" />

MMAE

<img file="IN520DEN2012A_D0062.tif" />

MMAF

111

<img file="IN520DEN2012A_D0063.tif" />

<img file="IN520DEN2012A_D0064.tif" />

<img file="IN520DEN2012A_D0065.tif" />

<img file="IN520DEN2012A_D0066.tif" />

<img file="IN520DEN2012A_D0067.tif" />

<img file="IN520DEN2012A_D0068.tif" />

<img file="IN520DEN2012A_D0069.tif" />

<img file="IN520DEN2012A_D0070.tif" />

112

<img file="IN520DEN2012A_D0071.tif" />

<img file="IN520DEN2012A_D0072.tif" />

<img file="IN520DEN2012A_D0073.tif" />

and pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited to triethylene glycol esters (TEG), as shown above, can be attached to the Drug Unit

113 at R¹¹. Without being bound by theory, the hydrophilic groups assist in the internalization and non-agglomeration of the Drug Unit.

THE LIGAND UNIT

The Ligand unit (L-) includes within its scope any unit of a Ligand (L) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. A Ligand is a molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. In one aspect, the Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit reacts. Such Ligands include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substance.

A Ligand unit can form a bond to a Stretcher unit, an Amino Acid unit, a Spacer Unit, or a Drug Unit. A Ligand unit can form a bond to a Linker unit via a heteroatom of the Ligand. Heteroatoms that may be present on a Ligand unit include sulfur (in one embodiment, from a sulfhydryl group of a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of a Ligand) and nitrogen (in one embodiment, from a primary or secondary amino group of a Ligand). These heteroatoms can be present on the Ligand in the Ligand’s natural state, for example a naturally-occurring antibody, or can be introduced into the Ligand via chemical modification.

In one embodiment, a Ligand has a sulfhydryl group and the Ligand bonds to the Linker unit via the sulfhydryl group’s sulfur atom.

In yet another aspect, the Ligand has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The Ligand unit bonds to the Linker unit via the sulfhydryl group’s sulfur atom. The·^f '· reagents that can be used to modify lysines include, but are not limited to, N

114 succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut’s Reagent).

In another embodiment, the Ligand can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. The Ligand unit bonds to the Linker Unit, such as the Stretcher Unit, via the sulfhydryl group’s sulfur atom.

In yet another embodiment, the Ligand can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The corresponding aldehyde can form a bond with a Reactive Site on a Stretcher. Reactive sites on a Stretcher that can react with a carbonyl group on a Ligand include, but are not limited to, hydrazine and hydroxylamine. Other protocols for the modification of proteins for the attachment or association of Drug Units are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley &amp; Sons (2002), incorporated herein by reference.

Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to, transferrin, epidermal growth factors (“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF’), such as TGF-α and TGFβ, vaccinia growth factor (“VGF’), insulin and insulin-like growth factors I and Π, lectins and apoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund’s (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions,

115 peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAh) to an antigenof-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256,495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80, 73087312; Kozbor etal., 1983, Immunology Today 4, 72-79; and Olsson etal., 1982, Meth. Enzymol. 92, 3-16).

The antibody can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art. Traditional production of fulllength bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the

116 correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, Ch2, and Ch3 regions. It is preferred to have the first heavy-chain constant region (ChI) containing the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In an embodiment of this approach, the bispecific antibodies have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation (International Publication No. WO 94/04690) which is incorporated herein by reference in its entirety.

For further details for generating bispecific antibodies see, for example, Suresh et ak, Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio/Technology

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10:163-167; Carter et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Using such techniques, bispecific antibodies can be prepared for use in the treatment or prevention of disease as defined herein.

Bifunctional antibodies are also described, in European Patent Publication No. EPA 0 105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed for example, in International Publication WO 83/03679, and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.

The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens or other antibodies bound to tumor cells or matrix. In this regard, “functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, for e.g., Rabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Rabat E et al., 1980, J. of Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but not limited to, F(ab’)i fragments, which contain the variable region, the light chain constant region and the CHI-domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be

118 generated by reducing the disulfide bridges of the F(ab’)2 fragments. Other useful antibodies are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent No. 4946778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:54454), or any other molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Patent No. 4816567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171496; European Patent Publication No. 173494; International Publication No. WO 86/01533; U.S. Patent No. 4816567; European Patent Publication No.12,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura etal., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988,

J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et a/., 1986, BioTechniques 4:214; U.S. Patent No. 5225539; Jones et al., 1986/ Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et

119 al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.

Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies. See, e.g., U.S. Patent Nos. 5625126; 5633425; 5569825; 5661016; 5545806; each of which is incorporated herein by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Quan, Μ. P. and Carter, P. 2002. The rise of monoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease, Jardieu, P. M. and Fick Jr./R. B, eds., Marcel Dekker, New York, NY, Chapter 20, pp, 427-469).

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

Antibodies include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.

The antibodies include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, CA) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank

121 database or a database like it, the literature publications, or by routine cloning and sequencing.

In a specific embodiment, known antibodies for the treatment or prevention of cancer can be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, humanized anti-HER2 monoclonal antibody, HERCEPTIN® (trastuzumab; Genentech) for the treatment of patients with metastatic breast cancer; Rituxan® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin’s lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG_2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; Vitaxin (Medlmmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, MA) which is a humanized IgGi antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) which is a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin’s lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma; Oncolym (Techniclone, Inc., CA) which is a radiolabeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin’s lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or nonHodgkin’s lymphoma; Avastin (Genentech, Inc., CA) which is an anti-VEGF

122 humanized antibody for the treatment of lung and colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for the treatment of non-Hodgkin’s lymphoma; and CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA153 (carcinomas), CAI9-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE -4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1KLH (breast cancer), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin’s lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas). Some specific, useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, PA, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, Μ. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, Μ. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo

123 antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb, and mAbs against the CD30 antigen, such as AC 10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151,5896-5906, 1993: Wahl et al., 2002 Cancer Res. 62(13):3736-42). Many other internalizing antibodies that bind to tumor associated antigens can be used and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000,15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

In certain embodiments, the antibody is not Trastuzumab (full length, humanized anti-HER2 (MW 145167)), HerceptinF(ab’)₂ (derived from anti-HER2 enzymatically (MW 100000)), 4D5 (full-length, murine antiHER2, from hybridoma), rhu4D5 (transiently expressed, full-length humanized antibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8 (fulllength, murine antiHER2, with mutated FcRn binding domain), or Hg (“Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore non-glycosylated)).

In another specific embodiment, known antibodies for the treatment or prevention of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. In another embodiment, useful antibodies are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, AntiCardiolipin Antibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SM Antibody; Anti-Mitochondrial Antibody; Thyroid Antibody; Microsomal

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Antibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo; Anti-UiRNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital Cells Antibody; Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti-GBM Antibody.

In certain embodiments, useful antibodies can bind to both a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/0X40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD1 la, CD1 lb, CD1 lc, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Nonlimiting examples of suitable lectins are C-type, S-type, and I-type lectin.

In one embodiment, the Ligand binds to an activated lymphocyte that is associated with an autoimmune disease.

In another specific embodiment, useful Ligands immunospecific for a viral or a microbial antigen are monoclonal antibodies. The antibodies may be chimeric, humanized or human monoclonal antibodies. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi,

125 pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.

Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from BD Biosciences (San Francisco, CA), Chemicon International, Inc. (Temecula, CA), or Vector Laboratories, Inc. (Burlingame, CA) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, literature publications, or by routine cloning and sequencing.

In a specific embodiment, useful Ligands are those that are useful for the treatment or prevention of viral or microbial infection in accordance with the methods disclosed herein. Examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, Synagis (Medlmmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenies) which is a CD4 fusion antibody useful for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; Protovir (Protein Design Labs, Inc., CA) which is a humanized IgGi antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.

Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,

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Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

Other antibodies useful in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.

In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor

127 associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.

Antibodies which comprise Ab in Formula Ic antibody drug conjugates (ADC) and which may be useful in the treatment of cancer include, but are not limited to, antibodies against tumor-associated antigens (TAA). Such tumor-associated antigens are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art. Examples of TAA include (1)-(35), but are not limited to TAA (1)-(35) listed below. For convenience, information relating to these antigens, all of which are known in the art, is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s). Tumor-associated antigens targetted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the corresponding sequences listed (SEQ ID NOS: 1-35) or the sequences identified in the cited references. In some embodiments, TAA having amino acid sequence variants exhibit substantially the same biological properties or characteristics as a TAA having the sequence found in the corresponding sequences listed (SEQ ID NOS: 1-35). For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed. The sequences and disclosure specifically recited herein are expressly incorporated by reference.

TUMOR-ASSOCIATED ANTIGENS (1)-(35):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203, tenDijke,P., etal. Science 264 (5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); W02004063362 (Claim 2); W02003042661 (Claim 12); US2003134790-Al (Page 38-39); W02002102235 (Claim 13; Page 296); W02003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530); W02003029421 (Claim 6); W02003024392 (Claim 2; Fig 112); WO200298358 (Claim 1; Page 183); W0200254940 (Page 100-101);

128

WO200259377(Page 349-350); W0200230268 (Claim 27; Page 376); W0200148204 (Example; Fig 4)

NP_001194 bone morphogenetic protein receptor, type IB /pid=NP_001194.1 Cross-references: MIM:603248; NP_001194.1; NM_001203_l

502 aa

MLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIE

ED Ό3σΐ,ΡννΤ3θαΕσΕΕΟ3ϋΡ&lt;20ΕϋΤΡΙΡΗζ)ΡΡ3ΙΕεσΤΕΕΝΕ0ΝΚΌΙ,ΗΡΤΕΡΡΙ,ΚΝΡΌΡ VD σΡΙΗΗΕΑϋΙ,ΙδντνΟΒΙ.ΒΙ.νΓΙΐυΌΥΕΡΥΚΡΟΕΤΡΡΡΥδΙΟΙ,ΕΟηΕΤΥΙΡΡσΕβΙΛυ) LI EQSQSSGSGSGLPLLVQRTIAKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFFTTEE AS WFRETEIYQTVLMRHENILGFIAADIKGTGSWTQLYLITDYHENGSLYDYLKSTTLDA KS

ΜΚΚΙ/ΑΥΒενεσίιΟΗΙ,ΗΤΕΙΡβΤΟσΚΡΑΙΑΗΡΟΙιΚΒΚΝΙΙύνΚΚΝΟΤσΟΙΑΟΕΟΙιΑνΚΡΙ

SD TNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFQSYIMADMYSFGLILWEVARRCVSGG IV EEYQLPYHDLVPSDPSYEDMREIVCIKKLRPSFPNRWSSDECLRQMGKLMTECWAHNP AS

RLTALRVKKTLAKMSESQDIKL (SEQ ID NO: 1) (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486); Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch,H.W., etal. (1992) J. Biol. Chem. 267 (16):11267-11273); W02004048938 (Example 2); W02004032842 (Example IV); W02003042661 (Claim 12); W02003016475 (Claim 1); WO200278524 (Example 2); W0200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); W02003003906 (Claim 10; Page 293); WO200264798 (Claim 33; Page 93-95); W0200014228 (Claim 5; Page 133-136); US2003224454 (Fig 3); W02003025138 (Claim 12; Page 150);

NP_003477 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 /pid=NP_003477.3 - Homo sapiens

129

Cross-references: MIM:600182; NP_003477.3; NM_015923;

NM_003486_l

507 aa

MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAI

IV

GTIIGSGIFVTPTGVLKEAGSPGLAL^ATWAACGVFSIVGALCYAELGTTISKSGGDYA

YM

ΕΕνΥσδΕΡΑΕΙιΚΕνίΙΕΕΕΙΙΗΡΕΕΟΥίνΑΙινΕΑΤΥΙιΕΚΡΕΕΡΤαΡνΡΕΕΑΑΚΕνΑΟΙισ

VL

ΕΙίΤΑνΝΟΥενΚΑΑΤΗνΟΟΑΕΑΑΑΚΕΕΑΕΑΙιΙΙΕΕΟΕνΟΙΟΚσννΕΝΕΌΡΝΕΕΕΕσΤΚΙι

DV

GNI νΕΑΙ,ΥΕΟΕΕΑΥΟσΜΝΥΕΝΕνΤΕΕΜΙΝΡΥΚΝΕΡΙιΑΙ 1ISLPIVTLVYVLTNLAYFT

TL δΤΕΟΜΕδδΕΑνΑνΏΕΟΝΥΗΕσνΜδΜΙΙΡνΡνσΕδΟΕσδνΝαδΕΡΤδδΗΕΕΕνΟδΚΕσΗ LP

SILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWL

RH

ΗΚΡΕΕΕΗΡΙΚνΝΕΑΕΡνΕΕΙΕΑεΕΡΕΙΑνδΡννΚΤΡνΕΟαίΟΕΤΙΙΕδΟΕΡνΥΕΕσνΜΜ

KN

KPKWLLQGIFSTTVLCQKLMQWPQET (SEQ ID NO: 2) (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449

Cancer Res. 61 (15), 5857-5860 (2001), Hubert,R.S., etal. (1999) Proc. Natl. Acad. Sci. USA. 96 (25): 14523-14528); W02004065577 (Claim 6);

W02004027049 (Fig IL); EP1394274 (Example 11); W02004016225 (Claim 2); W02003042661 (Claim 12); US2003157089 (Example 5); US2OO318583O (Example 5); US2003064397 (Fig 2); WO200289747 (Example 5; Page 618619); W02003022995 (Example 9; Fig 13A, Example 53; Page 173, Example 2; Fig 2A);

NP_036581 six transmembrane epithelial antigen of the prostate Cross-references: MIM:604415; NP_036581.1; NM_012449_l 339 aa ,

ΜΕδΗΚΌΙΤΝΟΕΕΕΚΚΜΚΡΕΚΝΕΕΕΌΟΥΕΗΚΌΤσΕΤδΜΕΚΕΡνΕΕΗΕΗΟΤΑΗΑΌΕΕΌΟΡ

SE

130

LQHTQELFPQWHIjPIKIAAIIASLTFLYTIjLREVIHPLATSHQQYFYKIPILVINKVL

PM

VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIY

SLi

ΞΥΡΜΚΕεΥΕΥΚΕΙΛΜΑΥςονΟΟΝΚΕΠΑΜΙΕΗΟνΝΚΜΕΙΥνΕΙΌϊνσΕΑΙΕΑΙ,ΕΑνΤΞΙ PS

VSDSLTWREFHYIQSKLGIVSLLIjGTIHALjIFAWNKWIDIKQFVWYTPPTFMIAVFLP

IV νΕΙΕΚΞΙΕΕΙιΡΟΙιΕΚΚΙΙιΚΙΚΗσΜΕΏντΚΙΝΚΤΕΙΟβΟΙ, (SEQ ID NO 3) (4) O772P (CA125, MUC16, Genbank accession no. AF361486

J. Biol. Chem. 276 (29):27371-27375 (2001)); W02004045553 (Claim 14); WO200292836 (Claim 6; Fig 12); WO200283866 (Claim 15; Page 116-121); US2003124140 (Example 16); US2003091580 (Claim 6); W0200206317 (Claim 6; Page 400-408);

Cross-references: GI:34501467; AAK74120.3; AF361486_1

6995 aa

PVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTA

VT

NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTS

SL

TSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPD

IS

TEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFS

HS

EMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTA

LL

TLGPVKTTDMLRTSSEPETSSPPNIjSSTSAEIIjATSEVTKDREKIHPSSNTPWNVGT

VI

YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLRE

IS

TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITR IS tpltttgsaemtitpktghsgassqgtftldtssraswpgthsaathrsphsgmttpm

SR σΡΕΟνεΜΡΞΕΡΞνΕΚΤΞΡΡΞεΕνεΕΕΑντεΡεΡΕΥεΤΡΞΕΒεΗΒΒΡΕΕνΤΒΕΡΤΡνΜΜ KT

TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEF

131

YS

SYPGLPEPSKVTSPWTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQE IH

SATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKT ES

TEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPW LS

ΗΡΞνΕΕΑΞΞΑΒΕβΙ,ΞΕΡνΜΤΕΕΞΡνΕΞΤΕΡΟδΙΗΞΕΕΙ,ΡνΤΕΙ,ΙιΤΞΟΙίνΚΤΤΕΕΙιΟΤΞ SE

PETSSPPNLSSTSAEILATTEVTTDTEKLEMTNWTSGYTHESPSSVLADSVTTKATS SM

GITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSV PT gattevsrteaisssrtsipgpaqstmssdtsmetitristpltrkestdmaitpktg PS

GATSQGTFTLDSSSTASWPGTHSATTQRFPRSWTTPMSRGPEDVSWPSPLSVEKNSP PS slvssssvtspsplystpsgsshsspvpvtslftsimmkatdmldaslepettsapnm NI tsdeslaaskatteteaihvfentaashvettsateelyssspgfseptkvispwts ss irdnmvsttmpgssgitrieiesmssltpglretrtsqditsstetstvlykmpsgat PE

VSRTEVMPSSRTSIPGPAQSTMSLDISDEWTRLSTSPIMTESAEITITTQTGYSLAT SQ νΤΏΡΕσΤ3ΜΤΡΏ30ΤΗ3ΤΜ3ς0Ι.3Η5ΕΜΤΝ1,Μ3ΡαΡΕ3Β3νΐΤ5ΡΡΡνΕΤΤΕ3333ΕΤ3 LP

LTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVE IP

ATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVEDT TV

FTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLFGVPTSATTEVSMT El

MSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLT LA

TTTAPL·ARTHSTVPPRFL·HSEMTTLMSRSPENPSWKSSPFVEKTSSSSSL·LSL·PVTTS PS ν33ΤΒΡ03ΙΡ333Ε3νΤ3νΕΤΡσΜνΚΤΤΠΤ3ΤΕΡΟΤ3Ε3ΡΝΒ3αΤ3νΕΙΏΑΑ3ΕνΤΤΌ TE

KIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANF ET

TGFEAEPFSHLTSGLRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSP DW

132

PPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAET IS

TGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPF

SS

STFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTS

SS

PILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGP QT εΤΒΡΑεΡΚσΕΗΤσσΤΚΡΜΕΤΤΤΤΑΕΚΤΤΤΤΑΕΚΤΤΞΡΑΤΕΤΤΞνΥΤΡΤΕσΤΕΤΡΕΝΑΒ MQ

MASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLV SR

SGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSH GA

ΟνΞΞΑΙΡΤΝΙΞΡεΕΕΏΑΕΤΡΕντίεσΤϋΤεΤΤΓΡΤΕΤΚεΡΗΕΤΕΤΡΤΤίΛίΕΤΗΡΑΕΤεΞ TI

PRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTI PT

LTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALT NS

PGEPATTVSLVTHSAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVST EV

PGWTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPG W

ΤΒΕντεΞΕΑντεΤΤΙΡΙΙιΤΕεί,σΕΡΕΤΤΡΒΜΑΤεΗΟΤΕΑΟεΑνΡΤνΕΡΕνΡσΜντεΕν AS

SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGWTSLVTSSSGV NS

TSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGWTPLVTSSRAVTSTT

IP

ILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTI

SS ΏΕΡΕΤΤΤεΕνΤΗΞΕΑΚΜίεΑΙΡΤΕΟνΞΡΤνΟΟΕνΤΒΕντεεσΞΕΤεΑΡεΝΕΤνΑεεΟΡ ET

IDSWVAHPGTEASSWPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTM

PS

TVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVT HP

AVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSG TD

TSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPDASKMLTSLVISSGTDST TT

FPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSA

133 vs

TTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTI PN

FSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGWTSQVTSSATDTSTAIPTLTPS PG

EPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSS PD

ATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTA LL sthprtetsktfpastvfpqvsettasltirpgaetstalptqttsslftllvtgtsr

VD

ΕΞΡΤΑεΡσνεΑΚΤΑΡΕΞΤΗΡσΤΕΤεΤΜΙΡΤεΤΕΞΕσΕΕΕΤΤσΕΕΑΤεΞΞΑΕΤΞΤΞΤΕΤ LT

VSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPT PP

KTSHGEGVS PTTILRTTMVEATNLATTGS S PTVAKTTTTFNTLAGSLFT PLTTPGMST LA

SESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPF MV

PFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLR PE

ΚϋΒβΑΤΑνΟΑίσΤΗΕΡϋΡΕΟΕσΕΌΗΕΡΕΥΚΕΕβΝΕΤΝΟΙΟΕΕσΡΥΤΕϋΕΝβΕΥνΝΟΡΤ HR

SSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGS RK

ΓΝΤΜΕΞνΕζίσΕΕΚΡΕΓΚΝΤΞνΟΡΕΥεΟΟΗΕΤΕΕΡΡΕΚΟσΑΑΤσνϋΑΙΟΤΗΗΕΟΡΚΞΡσ LN

ΗΕΟΕΥΜΕΕεΚΕΤΝΏΙΕΕΕΟΡΥΤΕΌΗΝΞΕΥνΝσΓΤΗΟεενΞΤΤΞΤΡΟΤβΤνϋΕΡΤΞΟΤΡ SS

ΕΞΞΡΤΙΜΑΑΟΡΕΕνΡΡΤΕΝΡΤΙΤΝΕΟΥσΕΌΜσΗΡΟεΡΚΓΝΤΤΕΡνΕΟσΕΕσΡΙΓΚΝΤβ VG

PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGP YT

LDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTI TN

LKYEEDMHRPGSRKFNTTERVLQTLVGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGV DA

ΙΟΤΗΗΕιΌΡΚΞΡΟνΌΗΕζίΕΥνΐΕΕΒΟΕΤΝσίΚΕΕαΡΥΤΕΏΗΝΞΕΥνΝΟΡΤΗνΐΙΡνΡΤΞεΤ PG

TSTVDLGSGTPSSLPSPTSATAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERV

LQ . SLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWE LS

134

QLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSA GP

LLVPFTLNFTITNLQYEEDMHHPGSEKFNTTEEVLQGLLGPMFKNTSVGLLYSGCELT LL

ΕΡΕΚΝσΑΑΤσΜΌΑΙΟεΗΕΙ,ΏΡΚΞΡσΕΝΕΕΟΕΥΝΕΕεΟΕΤΗΟΙΚΕΙΧϊΡΥΤΕΌΕΝεΕΥνΝ GF

ΤΗΕδενΑΡΤδΤΡσΤΞΤνΟΕΘΤεσΤΡδεί,ΡδΡΤΤΑνΡΕΕνΡΕΤΕΝΡΤΙΤΝΕΟΥσΕΟΜΕΗ PG εΕΚΕΝΤΤΕΕνΕΟσΕΕσΡΕΕΚΝεδνσΡΕΥεΘΟΕΕΙδΕΕεΕΚΙΧΪΑΑΤΟνΌΑίεΤΗΗΕΝΡΟ SP σΕΠΕΕΟΕΥ^ΕΞΟΜΤΝσίΚΕΙΌΡΥΤΕΟΕΝδΕΥνΝΟΕΤΗΕΕεσΕΤΤΞΤΡΜΤΞΤνηΕσΤΞ GT

PSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHEPGSRKFNATEEVLQGLLSPIFKNS SV

ΟΡΙιΥΒσαΕΙΖΓβΙιΕΡΕΚϋΟΑΑΤσΜΟΑνΟΙ,ΥΗΡΝΡΚΕΡσί,ΟΕΕΟϋΥΜΕΙιβΟΙ/ΓΗΝΙΤΕΙΧϊ PY

SLDEDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFT IT

Ν1ιΗΥΕΕΝΜζ2ΗΡσ3ΕΚΕΝΤΤΕΕνΐιζ2σΐ_ίΕΚΡΙιΕΚΝΤ3νσΡΕΥ3σθΕΙιΤΙ.1ιΕΡΕΚζ2ΕΑΑΤσ VD

TICTHEVDPIGPGLDEEELYWELSQLTNSITELGPYTLDEDSLYVNGFNPWSSVPTTS TP

ΘΤΞΤνΗΕΑΤΞατΡβεΕΡσΗΤΑΡνΡΕΕΙΡΡΤΕΝΓΤΙΤΝΕΗΥΕΕΝΜΟΗΡσεΕΚΡΝΤΤΕΕν

LQ

GLLKPLFKSTSVGPLYSGCELTLLEPEKHGAATGVDAICTLELDPTGPGLDEEELYWE LS

QLTNSVTELGPYTLDEDSLYVNGFTHESSVPTTSIPGTSAVHLETSGTPASLPGHTAP GP

ΕΕνΡΕΤΕΝΕΤΙΤΝΕΟΥΕΕϋΜΕΗΡσΞΕΚΕΝΤΤΕΕνΕΟσΕΙιΚΡΕΕΚΞΤενΟΡΕΥΞσεΕΕΤ LL

ΕΡΕΚΕσΑΑΤσνϋΤΙΟΤΗΕΙιΟΡΙιΝΡσΕϋΕΕΟΙιΥννΕΙιβΚΙΖΓΕσίΙΕΙισΡΥΙιυΟΕσδΙιΥνΝ GF

THRNFVPITSTPGTSTVHLGTSETPSSLPEPIVPGPLLVPFTIjNFTITNLQYEEAMRH

PG

SEKFNTTEEVLQGLLEPLFKNTSIGPLYSSCELTLLEPEKDKAATEVDAICTHHPDPQ SP

ΟΕΝΕΕΟΕΥΜΕΕεΟΙιΤΗαΐΤΕΕσΡΥΤΕΟΕΟεΕΥνίΧΙΕΤΙΜεΡΙΡΤΤΞΤΡΟΤβΙλΤΝΕστε GI

ΡΡΞΕΡΕΤΤΑΤσΡΕΕνΡΕΤΕΝΕΤΙΤΝΕΟΥΕΕΝΜσΗΡσεΕΚΕΝΙΤΕενΕΟσίιΕΚΡΙιΕΚΞΤ SV

GPLYSGCELTLLRPEKDGVATEVDAICTHEPDPKIPGLDEQQLYWELSQLTHSITELG PY

TLDEDSLYVNGFTQESSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFT

135

II

ΝΕΟΥΕΕΠΜΗΕΡσΕΕΚΡΝΤΤΕΕνΕΟσί,ΕΜΡΕΡΚΝΤδνΕΕΕΥεΟΟΕΕΤΕΕΕΡΕΚΟσΑΑΤΕ VD

ΑνΟΤΗΕΡΌΡΚΕΡΟΙ,ΌΕΕΕΙίΥΚΚΕεΟΙ,ΤΗΟΙΤΕΙβΡΥΤΕΌΕΗΕυΥνΝΟΕΤΗΟΕΕΜΤΤΤΕ TP

DTSTMHLATSETPASLSGPTTASPLLVLFTINFTITNLEYEENMHHPGSEKFNTTEEV LQ

GLLEPVFKNTSVGPIjYSGCEIjTLLEPKKDGAATKVDAICTYEPDPKSPGLDEEQLYWE

LS

QLTHSITEIjGPYTLDEDSLiYVNGFTQESSVPTTSIPGTPTVDLGTSGTPVSKPGPSAA

SP

LLVLFTLNFTITNLEYEENMQHPGSEKFNTTEEVLQGLLESLFKSTSVGPLYSGCELT LL

ΕΡΕΚΌΟΤΑΤΟνΌΑΙΟΤΗΗΡΟΡΚΞΡΕΙ,ΌΕΕΟΙίΥΜΕΙιΕΟΙΖΓΗΝΙΤΕΒΟΡΥΑΙ,ΌΝΠΕΙίΕνΝ GF

THESSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNIjEYEENMWP

GS

ΕΚΕΝΤΤΕΕνΕΟΟΕΙιΕΡΕΕΚΝΤενσΡΙιΥεσΟΕΕΤΕΙιΕΡΕΚΟσΕΑΤσνΏΑΐσΤΗΕΡΏΡΤα PG

ΙιΠΕΕΟΕΥΙιΕΕδΟΕΤΗΕΙΤΕΕΟΡΥΤΙιΟΕΟΕΕΥνΝσΡΤΗΕΞδνΡΤΤδΤσννδΕΕΡΕΤΕΝΡ TI

ΝΝΕΕΥΜΑΟΜσςΡΟΞΕΚΡΝΙΤϋΝνΜΟΗΕΙιΞΡΕΡΟΕΞΞΕΟΑΕΥΤΟΟΕνίΑΕΕΞνΚΝσΑΕΤ EV

DLLCTYLQPLSGPGLPIKQVFHELSQQTHGITELGPYSLDKDSLYLNGYNEPGPDEPP TT

PKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLIj ΕΡ

ΕΕΟΚεδΜαΡΕΥΕαΟΟΕΙΞΕΕΡΕΚΌσΑΑΤσνυΤΤΟΤΥΗΡΟΡνσΡσΕΌΙΟΟΕΥΚΕΙ^ΟΕΤ HG

VTQLGFYVLDEDSLFINGYAPQNIjSIEGEYQINFHIVNWNLSNPDPTSSEYITLIjEDI

QD

ΚνΤΤΕΥΚσΞΟΕΗΏΤΡΕΕΟΕνΤΝΙιΤΜϋΞνΕνΤνΚΑΕΕβΞΝΙιϋΡΕΙιλΖΕΟνΕΙιΌΚΤΕΝΑβΡ HW

LGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQENK ΕΝ

IEDALNQLFENSSIKSYFSDCQVSTFESVPNEHHTGVDSLCNFSPLAEEVDEVAIYEE FL

ΕΜΤΕΝσΤΟΙιΟΝΡΤΙιϋΕΒβνυνίΧϊΥΒΡΝΕΝΕΡΙΖΓσΝΕϋΕΡΕνίΑνίΙιΙΟΕΑσΕΙισίιΙΤΟΙ) IC

GVLVTTEEEKKEGEYNVQQQC PGYYQ SHLDLEDLQ (SEQ ID NO:4)

136 (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_OO5823

Yamaguchi,N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci.

USA. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA. 93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); W02003101283 (Claim 14); (W02002102235 (Claim 13; Page 287-288); W02002101075 (Claim 4; Page 308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57); Cross-references: MIM:601051; NP_005814.2; NM_005823_l

622 aa

ΜΑΏΡΤΑΚΡΒΙΧ35θσΤΡΑΒΟ3ΙΛΡΕ13Ρ3ΙΧ31/νν0Ρ3ΕΤΒΑΟΕΤΟ0ΕΑΑΡΙ,ΌσνΒΑΝΡΡΝΙ

SS

ΙιΞΡΕ0ΕΙιΟΡΡ0ΑΕν3ΟΕ3ΤΕΕνΕΕΙιΑνΑΙ&gt;Αζ2ΚΝνκΐύ3ΤΕζ2ΕΕΟΕΑΗΚΙι3ΕΡΡΕΟΕΟΑΙι

PL

ΠίιΙ,ΙιΡΙιΝΡΟΑΡβσΡΟΑαΤΚΡΡεΕΙΤΚΑΝνΌΕΕΡΕσΑΡΕΕΟΕΕΕΡΑΑΕΑαννσνΕΟβΕΕβ

EA ΌνΕΑΕσΘΕΑαΌΕΡΘΕΡνΑΕδΑΕνΕΕΡΕΕνΕΟΡσΡΙ,ΌΟϋΟΟΕΑΑΕΑΑΕΟΟΘΟΡΡΥΟΡΡΕ TW

SVSTMDALEGLLPVLGQPIIESIPQGIVAAWEQESSEDPSWEQPEETILEPEFEEEVE

KT ΑΟΡεσκΚΑΕΕΙΌΕΕΕΙΡΥΚΚΝΕΕΕΑΟνϋΑΑΕΕΑΤΟΜΌΕνΝΑΙΡΓΤΥΕΰΕΌνΕΚΗΚΕΌΕ LY

ΡΰσΥΡΕ3νΐ0ΗΕΟΥΕΡΕΚΜ3ΡΕΟΙΕΚΜΝνΤ3ΕΕΤΕΚΑΕΕΕνΝΚαΗΕΜ5Ρ0νΑΤΕΙΌΕΡ

VK σΕσΟΙιΟΚΟΤΕϋΤΙ/ΓΑΡΥΡσΥΕεΞΕΒΡΕΕΕΒΞνΡΡΒΞΙΜΑνΕΡΟϋΙιΟΤΟΟΡΕΟΙιϋνΐιΥΡ KA

ELAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLETDAVLPLTVAE

VQ

ΚΕΕσΡΗνΕσΐιΚΑΕΕΕΗΗΡνΕΙΛ7ΙΕΕ2Ε0ΟϋΙ&gt;ΌΤΙΛ3ΕσΕ0θσΐΡΝΟΥΕνΕϋΕ3Μ0ΕΑΙι3

GT

PCLLGPGPVLTVLALLLASTLA (SEQ ID NO:5) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424,

137

J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999),

Feild, J.A., etal. (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582);

W02004022778 (Claim 2); EP1394274 (Example 11); W02002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19); W0200157188 (Claim 20; Page 329); W02004032842 (Example IV); W0200175177 (Claim 24; Page 139140);

Cross-references: MIM:604217; NP_006415.1; NM_006424_l

690 aa

MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIELLPSYSTATLI ΌΕΡΤΕνΏΌΡνίΝΙιΡΤΙύΟΠβσίΚΝεΕΕΌΤΚΟΚΙΙΛΖΡΡζίΟΐσΕΙιΙΕΙιΙΧϊΡΙιΥΡΡνΟΞΙ,ΌΙΙιΞΞ AFQLVGGKMAGQFFSNSSIMSNPLLGLVIGVLVTVLVQSSSTSTSIWSMVSSSLLTVRA AIPIIMGANIGTSITNTIVAIjMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHY ΕΕΙΙΤ0ΕΙλΖΕ3ΡΗΡΚΝσΕϋΑΡΟΕΕΚνΐΤΚΡΡΤΚΒΐν0ΕΌΚΚνΐ30ΙΑΜΝΟΕΚΑΚΝΚ3ΕνΚ IWCKTFTNKTQINVTVPSTANCTSPSLCWTDGIQNWTMKNVTYKENIAKCQHIFVNFHLP ΌΙιΑνΟΤΙΙ,ΙιΙΙιΞΙιΙινίίΟΟΟΕΙΜίνΚΙΙιΟΒνίιΚΟΟνΑΤνίΚΚΤΙΝΤΠΡΡΡΡΡΑΚΙιΤΟΥΕΑΙ LVGAGMTFIVQSSSVFTSALTPLIGIGVITIERAYPLTLGSNIGTTTTAILAALASPGNA LRSSL·QIAL·CHFFFNISGILI_JWYPIPFTRL·PIRMAKGL·GNISAKYRWFAVFYL·IIFFFL·I PLTVFGLSLAGWRVLVGVGVPWFIIILVLCLRLLQ SRC PRVLPKKLQNWNFLPLWMRSL· KPWDAWSKFTGCFQMRCCYCCRVCCRACCLLCGCPKCCRCSKCCEDLEEAQEGQDVPVK APETFDNITISREAQGEVPASDSKTECTAL (SEQ ID NO:6) (7) Serna 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878,

Nagase T., et al. (2000) DNA Res. 7 (2):143-150): W02004000997 (Claim 1);

W02003003984 (Claim 1); W0200206339 (Claim 1; Page 50); W0200188133 (Claim 1; Page 41-43,48-58); W02003054152 (Claim 20); W02003101400 (Claim 11);

Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;

HGNC: 10737;

1093 aa

MVIjAGPLAVSLIjIjPSIjTLLVSHLSSSQDVSSEPSSEQQLCAIjSKHPTVAFEDLQPWVS

138

NF

TYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWASSEDTRRSCQSKG

KT

EEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHN

ST

AVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLF

AY

FFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYY

NE

LQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPI

AN

PIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLW

DL νΟΑΚΌΤΕΥΗνΕΥίσΤΕΞσΤΙΕΚΑΕδΤΑεΡεΕΗσΟΥΕΕΕΙ,ΗνΕΡΡσΕΡΕΡΕΡεΕΕΙΕΗε

AR

ALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQ

NI

TACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAI HI

ANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNEN

TP

C PVPIFWASWGSWSKC S SNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGC PEVRRN

TP

WTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGSGSCDTDA

LV

EDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDA

AE

YQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEE

AL

CATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPV

IL

PASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQSQESTLVHPAT

PN

HLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQTNVYTTTYYPSPLNKHS

FR

PEASPGQRCFPNS (SEQ ID NO :7) (8) PSCA hlg (270005002Rik, C530008016Rik, RIKEN cDNA 270005002,

RIKEN cDNA 270005002 gene, Genbank accession no. AY358628);

139

US2003129192 (Claim 2); US2004044180 (Claim 12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5); W02003105758 (Claim 12); US2003206918 (Example 5); EP1347046 (Claim 1); W02003025148 (Claim 20);

Cross-references: GI:37182378; AAQ88991.1; AY358628_1

141 aa

MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEVME QSAGIMYRKSCASSTkACLIASAGYQSFCSPGKLNSVCISCCNTPLCNGPRPKKRGSSASA LRPGLRTTILFLKLALFSAHC (SEQ ID NO:8) (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177,34-39,1991; Ogawa Y., etal. 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., etal. J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., etal. J. Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al. Gene 228,43-49,1999; Strausberg R.L., et al. Proc. Natl. Acad. Sci. USA.

99, 16899-16903, 2002; Bourgeois C., etal. J. Clin. Endocrinol. Metab. 82, 31163123, 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. Eur. J. Hum. Genet. 5,180-185,1997; PuffenbergerE.G., etal. 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., etal. 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., etal. Mol. Med. 7, 115-124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206; W02004045516 (Claim 1); W02004048938 (Example 2); W02004040000 (Claim 151); W02003087768 (Claim 1); WO2003016475 (Claim 1); W02003016475 (Claim 1);

W0200261087 (Fig 1); W02003016494 (Fig 6); W02003025138 (Claim 12;

140

Page 144); W0200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; Fig 2);

W0200177172 (Claim 1; Page 297-299); US2003109676; US6518404 (Fig 3);

US5773223 (Claim la; Col 31-34); W02004001004;

442 aa

MQPPPSLCGEALiVALVLACGLSEIWGEEEGFPPDEATPLLQTAEIMTPPTKTLWPKGSNA SLAESLAPAEVPKGDETAGSPPETISPPPCQGPIEIKETFKYINTWSCLVFVLGIIGNS ΤΕΕΕΙΙΥΚΝΚΟΜΕΝσΡΝΙΕΙΑ3ΕΑΙΌΟΕΕΗΐνΐΟΙΡΙΝνΥΚΕΙΛ^^ΡΡσΑΕΜεκΐ.νΡΡΙ QKASVGITVLSLCALSIDEYEAVASWSEIKGIGVPKWTAVEI'VIjIWVVSWIjAVPEAIGF DIITMDYKGSYLEICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM LRKKSGMQIALNDHLKQEEEVAKTVFCLVLVFAIjCWIjPLiHIjSEIIjKLTLYNQNDPNECEL LSFLLVLDYIGINMASLNSCINPIALYLVSKEFKNCFKSCLCCWCQSFEEKQSLEEKQSC LKFKANDHGYDNFESSNKYSSS (SEQ ID NO :9) (10) MSG783 (RNF124, hypothetical protein FU20315, Genbank accession no. NM_017763);

W02003104275 (Claim 1); W02004046342 (Example 2); W02003042661 (Claim 12); W02003083074 (Claim 14; Page 61); W02003018621 (Claim 1); W02003024392 (Claim 2; Fig 93); WO200166689 (Example 6); Cross-references: LocusID:54894; NP_060233.2; NM_017763_l 783 aa

MSGGHQLQLAALWPWLLMATLQAGFGETGLVLAAAVESEESAEQKAIIEVIPLKMDPTGK ΕΝΕΤΕΕΟνΕΑβνΑΕΙΤΡΑΕσΚΕΜΟΒΗΡΕΥΕαΝΑεΠΌΌΝΕΕΡσΕΐείνΚΕΕεΡΕΕΑΡΕΡΟΕ SLASKAEMAGEEGASAVLFDITEDRAAAEQLQQPLGLTWPWLIWGNDAEKIjMEFVYKNQ KAHVEIELKEPPAWPDYDVWILMTWGTIFVIILASVLEIECEPEHSEPDPLQQETAWAI SQLATEEYQASCEQAEGEWPDSGSSCSSAPVCAICLEEFSEGQELEVISCLHEFHRNCVD PWLHQHETCPLCVFNITEGDSFSQSLGPSESYQEPGERLHLIEQHPGHAHYHLPAAYLLG PSESAVAEPPEPGPFLPSQEPGMGPEHHEFPEAAHPEAPGEQQELAGAQHPYAQGWGMSH ΕΟΒΤΒΟΗΡΑΑσΡνΡΕΕΕΑΕΡΡΌβΞσΒΟΕΒΥαΤΕΕΞΟΥΕΑΌΟΡΑβΌΞεεσΡσΗΟΞΒΒΏβνν NCTDISLQGVHGSSSTFCSSLSSDFDPLVYCSPKGDPQEVDMQPSVTSEPESLDSWPTG ETQVSSHVHYHEHEHHHYKKEFQWHGEKPGPETGVPQSEPPIPETQPQPEPPSPDQQVTG SNSAAPSGELSNPQCPEALPEPAPGPVDASSICPSTSSLFNLQKSSLSAEHPQEKEEGGP 3ΕΡΤΡσ3ΕΡ0ϋΑΤνΗΡΑ0ζ2ΙΕΡΗΥΤΡ3νΑΥΡνί3ΡΕΑΗΡΕΙθαΡΡσΐιΌΚΕΙ.Ι_ιΡΕΤΡΟΡσΥ3 NSQPVWLCLTPEQPLEPHPPGEGPSEWSSDTAEGRPCPYPHCQVLSAQPGSEEELEEIjCE QAV (SEQ ID NO :10)

141 (11) STEAK (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAK, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138,

Lab. Invest. 82 (11):1573-1582 (2002)); W02003087306; US2003064397 (Claim 1; Fig 1); WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; Fig4B); W02003104270 (Claim 11); W02003104270 (Claim 16); US2004005598 (Claim 22); W02003042661 (Claim 12); US2003060612 (Claim 12; Fig 10); WO200226822 (Claim 23; Fig 2); WO200216429 (Claim 12; Fig 10);

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

490 aa

MESISMMGSPKSLSETVLPNGINGIKDAEKVTVGVIGSGDFAKSLTIRLIRGGYHWIGS ΕΝΡΚΕΑβΕΓΕΡΗγνΌνΤΗΗΕϋΑΙ,ΤΚΤΝΙΙΡνΑΙΗΕΕΗΥΤΞΙιΝΌΕΕΗΙιΙ,νΟΚΙΙιΙΌνΒΝΝΜ ΕΙΝΟΥΡΕΕΝΑΕΥΕΑΞΕΕΡΌΞΕίνκσΡΝννδΑΝΑΕΟΕσΡΚΌΑεΕΟνΥΙΟΕΝΝΙΟΑΗΟΟνίΕ ΕΑΕΟΕΝΕΙΡΙΌΕσδΕΒΞΑΕΕΙΕΝΏΡΕΕΕΕΤΕννΕΟΡνννΑΙδΕΑΤΕΕΡΕΥΕΡνΕΟνίΗΡΥΑ ΕΝΟΟΕΌΡΥΚΙΡΙΕίνΝΚΤΕΡίνΑΙΤΕΕεΕνΥΕΑαΕΕΑΑΑΥΟΕΥΥσΤΚΥΕΕΡΡΡΗΕΕΤνίΕΟ CEKQLGLLSFFFAMVHVAYSLCLPMEESERYLFLNMAYQQVHANIENSWNEEEVWEIEMY Ι3ΡσΐΜ3ΕσΕΕ31ιΒΑνΤ3ΙΡ3ν3ΝΑΙ&gt;ΝΜΕΕΡ3ΡΙ03ΤΕαΥνΑΕΕΙ3ΤΕΗνΕΙΥσΜΚΕΑΕΕ EEYYEFYTPPNFVLALVLPSIVILGKIILFLPCISQKLKEIKKGWEKSQFLEEGIGGTIP HVSPEEVTVM (SEQ ID NO:11) (12) TrpM4 (BR22450, FU20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636 Xu,X.Z., et al. Proc. Natl. Acad. Sci. USA. 98 (19):10692-10697 (2001), Cell

109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003));

US2OO3143557 (Claim 4); W0200040614 (Claim 14; Page 100-103); W0200210382 (Claim 1; Fig 9A); W02003042661 (Claim 12); W0200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14; Fig 1A-D);

142

Cross-references: MIM:606936; NP_060106.2; NM_017636_l

1214 aa

MWPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVAMEDAFGAAWTV WDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPAAVYSLVTRTWGFRAPNLWS νΕΟΟΕσσΡνΕΟΤΝΕΟϋΕΕΡΡαυνΡΑΑΟΕΤσΑΝίντσσΕΗΤσίσΡΗνσνΑνΡϋΗΰΜΑΕΤσΟ TKWAMGVAPWGWRNRDTLINPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTH GCL.GGENRFRLRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMIjTRIENATQAQLPCLL νΑΟΒσσΑΑΟΟΕΑΕΤΕΕΟΤΕΑΡΟΒσσΑηΟΟΕΑηΠΡΙΡηΡΡΡΚΟΌΕΕνΕΟΑΟνΕΡΙΜΤΡΚΕΕ ΕΤνΥΞεΕΌΟΕΕΕΡΕΤίνΕΚΑΕνΚΑΟσΕΕΕΑεΑΥΕΌΕΕΡΕΑνΑνΜΓΡνΠΙΑΟΞΕΕΓΡσΟΙΟνί Ρ3ΡΗΕΕΑ3ΕΜΟΑΕΕΝϋΡΡΕΡνΡΕΕΙ3ΗΟΕ3ΙΧ3ΗΓΕΤΡΜΡΙ,Αζ)ΕΥ3ΑΑΡ3Ν3ΕΙΚΝΙιΙιϋζ)Α ΕΗεΑσΤΚΑΡΑΕΚσσΑΑΕΕΡΡΡΟνσΗνΕΡΜΕΕαΚΜΟΑΡΡΥΡΕσΟΑΝΟΡΗΡσοσΡσΕΞΜΥΕΕ εϋΚΑΤΕΡΕδΕΌΑσΕσΟΑΡΜΕΌΕΕΕνίΑΕΕΕΝΡΑΟΜΑΜΥΡνίΕΜσΕΝΑνΕΕΑΕΟΑεΕΕΕΡνΜΑ ΡΕΕΡΟΑΕΕΑΑΚΡΚΌΕΑΡΚΡΕσΜσνΌΕΡσΕΟΥΡΞΕΕνΡΑΑΡΕΕΕΚΡΟΡΕνίσΟΑΤαΕΟΕΑΜΟ ADARAFFAQDGVQSLLTQKWWGDMASTTPIWALVLAFFCPPLIYTRLITFRKSEEEPTRE ELEFDMDSVINGEGPVGTADPAEKTPLGVPRQSGRPGCCGGRCGGRRCLRRWFHFWGAPV ΤΙΡΜσΝννΕΥΕΕΡΕΕΕΡδΡνΕΕνοΡΟΡΑΡΡσΕΕΕΕΕΕΥΡΜΑΡΤΕΕΟΕΕΕΡΟσΕΕσσσσΕΕ Α3σσρσΡσΗΑ3Ε30ΡΕΡΕΥΕΑΌ3ΜΝΰΟΌΕνΑΕΤΟΡΕΕσνσθΡΕΤΡσΕΥΗΕσΡΤνΕεΐΌΡΜ VFTVRLLHIFTVNKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEGIjLRPRDSDFP SILRRVFYRPYLQIFGQIPQEDMDVALMEHSNC SSEPGFWAHPPGAQAGTCVSQYANWLV νΕΙίΙ,νίΕΕΙινΑΝΙΙιΕνΝΕΙιΙΑΜΕβΥΤΕΟΚνζίΟΝΞΌΕΥΜΚΑΟηΥηΕΐηΕΕΗΒΕΡΑΙ,ΑΡΡΕΙ VISHLRLLLRQLCRRPRSPQ PSS PALEHFRVYLSKEAERKLLTWESVHKENFLLARARDK RESDSERLKRTSQKWLALKQLGHIREYEQRIjKVLEREVQQCSRVLGWVAEALSRSAIjLP PGGPPPPDLPGSKD (SEQ ID NO:12) (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212, Ciccodicola,A., etal. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); W02003083041 (Example 1); W02003034984 (Claim 12); W0200288170 (Claim 2; Page 52-53); W02003024392 (Claim 2; Fig 58); W0200216413 (Claim 1; Page 94-95, 105); W0200222808 (Claim 2; Fig 1); US5854399 (Example 2; Col 17-18); US5792616 (Fig 2);

Cross-references: MIM:187395; NP_003203.1; NM_003212_l

188 aa

MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDSIWPQEEPAIR PRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPH

143

ΟΤΝΙιΡΚΚΟΞΙΧΤΚΌνίΗαΟΙίΕΟΕΡΟΑΕΙιΡΟΟΟαίι'νΜΟΕΗΙ,νΑΒΡΤΡΕΙ,ΡΡβΑΡΤΤΤΕΜΕνσΐ CLSIQSYY *

(SEQ ID NO :13) (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004,

Fujisakuetal. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J.J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl.

Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., etal. Mol. Immunol.

35,1025-1031, 1998; Weis J.J., et al. Proc. Natl. Acad. Sci. USA. 83, 5639-5643, 1986; Sinha S.K., et al. (1993) J. Immunol. 150, 5311-5320; W02004045520 (Example 4); US2004005538 (Example 1);

W02003062401 (Claim 9); W02004045520 (Example 4); WO9102536 (Fig 9.1-9.9); W02004020595 (Claim 1);

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

1033 aa

MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGE KSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKT ΝΓΞΜΝσΝΚδννίεΟΑΝΝΜΝσΡΤΡΕΡΤΟνενΡΡΕΕΟΡΑΕΡΜΙΗΝσΗΗΤΕΕΝνσΕΙΑΡσΕΕνΤ YSCESGYLLVGEKIINCLSSGKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANF FCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPHiNGRHIGNSLANVSYGSI νΤΥΤσΟΡΟΡΕΕΟνΝΡΙΙ,ΐσΕδΤΙιΡσΤνΏδΟΚΤστνβσΡΑΡΕΟΕΕδΤεΑνΟΟΡΗΡΟΙΕΡσΡ MVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEPSAPVCEKECQAPPNILNGQ KEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLT KPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNGAHTG SSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLIjAV QCSHVHIANGYKISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKE ΤΟΟΗνΡΰΕΙ,ΟΕΕΡΑΟΕΕνΕΕνΝΤεΟΟΙΧϊΥΟΙίΤΟΗΑΥΟΜσΟΟΑΕΝσίΜΕΚΚΙΡΙιΟΚνίΗΟΗ PPPVIVNGKHTGMMAENFLYGNEVSYECDQGFYLLGEKKLQCRSDSKGHGSWSGPSPQCL RSPPVTRCPNPEVKHGYKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGV PTCIKKAFIGCPPPPKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTW /·

SQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAWTLECEDGYMLEGSPQSQCQSDHQ WNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRERNYYTDTSQKEAFHLEA REVYSVDPYNPAS (SEQ ID NO :14)

144 (15) CD79b (CD79B, CD79P, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626 or 11038674, Proc. Natl. Acad. Sci. USA. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller etal. (1992) Eur. J. Immunol. 22 (6): 1621-1625); W02004016225 (claim 2, Fig 140); W02003087768, US2004101874 (claim 1, page 102); W02003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); US5644033; W02003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Fig 17A/B); W0200055351 (claim 11, pages 1145-1146); Cross-references: MIM: 147245; NP_000617.1; NM_000626_l

229 aa ΜΑΡΙΛΙιΞΡνΡΒΗΜΜνΑΙίΕΙιΙιΙιΒΑΕΡνΡΑΑΕβΕΟηΥηΝΡΚΟβΑΟβΕίνίΟβΡΚΡΙΑΚΚηΟΕΤ νΚΜΗσΥΜΝβΑΞσΝνβΜΕΜΚΰΕΜΟΕΝΡΟΟΕΚΕΕΚσΗΜΕΕδΟΝΕβΒΑΤΒΤΙΟσίΗΡΕΏΝσίΥ ΡΟΟΟΚΟΝΝΤεΕνΥΟΟΟσΤΕΕΗνΜαΕΒΤΕΑΟΕΚΟΚΝΤυΚΌΘΙIMIQTLLIILFIIVPIFLL LDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO:15) (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764, Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. USA. 98 (17):9772-9777 (2001), Xu,M.J., et al. (2001) Biochem. Biophys. Res. Commun. 280 (3):768775; W02004016225 (Claim 2); W02003077836; W0200138490 (Claim 5; Fig 18D-1-18D-2); W02003097803 (Claim 12); W02003089624 (Claim 25); Cross-references: MIM:606509; NP_110391.2; NM_030764_l

508 aa MLLWSLLVIFDAVTEQADSLTLVAPSSVFEGDSIVLKCQGEQNWKIQKMAYHKDNKELSV FKKFSDFLIQSAVLSDSGNYFCSTKGQLFIjWDKTSNIVKIKVQELFQRPVIjTASSFQPIE σσΡν3ΙιΚΟΕΤΡ1ι3Ρ0ηΐιθν0Ιι0ΕΟΡΡΡΕΝζ2νΐ.σ3σνν335ΡΕΙ;0Ι3Α·\Μ3ΕΟΤσ3ΥΜΟΚΑΕ TVTHRIRKQSLQSQIHVQRIPISNVSLEIRAPGGQVTEGQKIiILIjCSVAGGTGNVTFSWY REATGTSMGKKTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKWNIPVRIPVSRP VLTLRSPGAQAAVGDLLELHCEALRGSPPILYQFYHEDVTLGNSSAPSGGGASFNLSLTA ' ’ ΕΗ3ΟΝΥ30ΕΑΝΝΟΙΧ3Α003ΕΑνΡν3Ι3ΟΡΙ)ΟΥΡΗΏΙιΜΤΑθνΐΛΛ31ιΓθνΐΧϊΕΤθνΑϋίιΥΑ

145

LFHKISGESSATNEPRGASRPNPQEFTYSSPTPDMEELQPVYVNVGSVDVDWYSQVWSM

QQPESSANIRTLLENKDSQVIYSSVKKS (SEQ ID NO :16) (17) HER2 (ErbB2, Genbank accession no. Ml 1730, Coussens L., et al. Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature 319, 230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. USA. 82, 6497-6501,1985; Swiercz J.M., et al. J. Cell Biol. 165, 869-880, 2004; Kuhns J.J., etal. 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; W02004048938 (Example 2); W02004027049 (Fig II); W02004009622; W02003081210; W02003089904 (Claim 9); W02003016475 (Claim 1); US2003118592; W02003008537 (Claim 1); W02003055439 (Claim 29; Fig 1A-B); W02003025228 (Claim 37; Fig 5C); WO200222636 (Example 13; Page 95-107); W0200212341 (Claim 68; Fig 7); WO200213847 (Page 71-74); W0200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46); W0200141787 (Page 15); W0200044899 (Claim 52; Fig 7); W0200020579 (Claim 3; Fig 2); US5869445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); W02004043361 (Claim 7); W02004022709; W0200100244 (Example 3; Fig 4);

Accession: P04626; EMBL; Ml 1767; AAA358O8.1. EMBL; Ml 1761; AAA358O8.1.

1255 aa

MEL·AAL·CRWGLLL7VLLPPGAASTQVCTGTDMKLRL·PASPETHL·DMLRHLYQGCQWQGNL· ELTYLPTNASLSFIjQDIQEVQGYVLIAHNQVRQVPLQRLjRIVRGTQLFEDNYAIjAVLDNG DPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPIjPTDCCHEQC AAGCTGPKHSDCLACLHFNHSGICELHCPAIjVTyNTDTFESMPNPEGRYTFGASCVTACP YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSAN ίςΕΡΑσΟΚΚΙΕσδΕΑΕΙιΡΕδΕΙΧαηΡΑδΝΤΑΡΙιΟΡΕΟΕΟνΡΕΤΙιΕΕΙτσΥΙιΥΙδΑνίΡΟδΙιΡ ΟΕδνΕΟΝΕΟνίΕσΕΙΕΗΝσΑΥδΕΤΕΟσΕσίδΜΙΌΕΕδΕΡΕΕσδσΕΑΕΙΗΗΝΤΗΙ,εΡνΗΤν PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQEC VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC ΡδΟνΚΡΟΕδΥΜΡΙΝΚΕ’ΡϋΕΕΟΑΟΟΡσΡΙΝΌΤΗδσνϋΙ,ΟΟΚσεΡΑΕΟΕΑδΡΙΖΓδΙΙδΑννσ ΙΕΕνννΊύσννΕΟΙΕΙΚΡΡΰΟΚΙΕΚΥΤΜΡΕΙιΕΟΕΤΕΙινΕΡΕΤΡδσΑΜΡΝΟΑΟΜΡΙΕΚΕΤΕΕ

146

RKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSP γνεΕΙιΙβΙΟΒΤΞΤνΟΙ,νΤΟΕΜΡΥσΟΙιΙιΟΗνΕΕΝηαΕΕσΞΟϋΙιΙ,ΝΜΟΜΟΙΑΚΟΜεΥΙιΕΟνΕ Ι&gt;νΗΕΏΕΑΑΕΝνΕνΚ3ΡΝΗνΚΙΤΌΕσΕΑΕΙ;ΕΌΙΟΕΤΕΥΗΑΙΧ50ΚνΡΙΚΝΜΑΕΕ3ΙΕΕΕΕΕΤ HQSDVWSYGWVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM IDSECRPRFRELVSEFSRMARDPQRFWIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDA ΕΕΥΕνΡΟΟσΡΡΟΡΟΡΑΡσΑσσΜνΗΗΕΗΕδΒεΤΕδΟσΟΠΕΤΕσΕΕΡβΕΕΕΑΡΕΞΡΕΑΡΞΕσ ΑσΞΌνΡΟσΟΕσΜσΑΑΚσΕΟΞΕΡΤΗΏΡδΡΕΟΕΥΞΕϋΡΤνΡΕΡδΕΤΌΟΥνΑΡΕΤΟεΡΰΡΕΥν NQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFi_lFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID NO:17) (18) NCA (CEACAM6, Genbank accession no. Ml8728);

Barnett T., etal Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys.

Res. Commun. 150, 89-96, 1988; StrausbergR.L., etal. Proc. Natl. Acad. Sci.

USA. 99:16899-16903, 2002; W02004063709; EP1439393 (Claim 7);

W02004044178 (Example 4); W02004031238; W02003042661 (Claim 12);

WO200278524 (Example 2); WO200286443 (Claim 27; Page 427);

W0200260317 (Claim 2);

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

344 aa

ΜΰΡΡΞΑΡΡΟΕΙ,ΗνΡΜΚΕνίιΙ,ΤΑΞΙιΕΤΕΜΝΡΡΊ’ΤΑΚΙιΤΙΕΒΤΡΕΝνΑΕΟΚΕνί,ΙιΙιΑΗΝυΡΟ

NRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTGFY

TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWV

NGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASTINRSDPVTLNVLYGPDVP

TISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQ AHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI (SEQ ID NO :18) (19) MDP (DPEP1, Genbank accession no. BC017023,

Proc. Natl. Acad. Sci. USA. 99 (26):16899-16903 (2002));

W02003016475 (Claim 1); WO200264798 (Claim 33; Page 85-87);

JP05003790 (Fig 6-8); WO9946284 (Fig 9);

Cross-references: MIM:179780; AAH17023.1; BC017023_l

411 aa

MWSGWWL·WPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQL·L·DMFNNRL·QDERANL·TT

147

LAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVRRTLEQMDVVHRMCRMYPETFLYVT βδΑσίΚΟΑΡΚΕσΚνΑδΙιίσνΕσβΗεΐϋΒδΙιΟνίιΡΑυΥΟΙΧϊΜΗΥΙ/ΓΙΖΓΗδαΝΤ^ΑΌΝΪΐΙίν DTGDSEPQSQGLSPFGQRWKELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYS VCASRRNVPDDVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLDHIKEVAGARAVG

ΡθσθΡΙΧ;νΡΡνΡΕσΐ_ιΕϋν3ΚΥΡΟΙιΙΑΕΕΙιΡΡΝΜΤΕΑΕνκσΑΙ.ΑΟΝΙίΙιΡνΡΕΑνΕζ)Α3Ν1ΖΓ

ΟΑΡΕΕΕΡΙΡΙ,ΟΟΙ,σσβΟΡΤΗΥσΥΒΒσΑββΙ.ΗΡΗΝσί,ΙιΙΛΒΙ,ΑΡΙ,νΐ,ΟΙ,δΙ.Ι, (SEQ ID NO :19) (20) IL20Ra (IL20Ra, ZCYTOR7, Genbank accession no. 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; DumoutierL., etal. J. Immunol. 167, 3545-3549, 2001; ParrishNovak J., et al. J. Biol. Chem. 277,47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry 42:12617-12624; Sheikh F., etal. (2004) J.

Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); W02003029262 (Page 74-75); W02003002717 (Claim 2;

Page 63); WO200222153 (Page 45-47); US2002042366 (Page 20-21);

W0200146261 (Page 57-59); W0200146232 (Page 63-65);

WO9837193 (Claim 1; Page 55-59);

Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971;

AAF01320.1.

553 aa

MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGIjPKPANITFLSINMKNVLQWTPPE GLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKC SKWAESGRFYPFLETQIGPPEVALTTDEKSISWLTAPEKWKRNPEDLPVSMQQIYSNLK YNVSVLNTKSNRTWSQCVTNHTL·VLTWLEPNTL·YCVHVESFVPGPPRRAQPSEKQCARTL·

KDQSSEFKAKIIFWYVLPISITVFLFSVMGYSIYRYIHVGKEKHPANLILIYGNEFDKRF

FVPAEKIVINFITLNISDDSKISHQDMSLLGKS SDVS SLNDPQ PSGNLRPPQEEEEVKHL GYASHLMEIFCDSEENTEGTSFTQQESLSRTIPPDKTVIEYEYDVRTTDICAGPEEQELS Ιυζ)ΕΕν3Τζ&gt;σΤΕΙιΕ3ζ)ΑΑΕΑνΐ.ΟΡζ)ΤΙιζ)Υ3ΥΤΡζ)Ιιζ)ΟΙΌΡΒΑζ)ΕΗΤΟ3ΕΕαΡΕΕΕΡ3ΤΤΙυν

ΟΝηΡ0ΤΟΡΙ£!ΙΡ3Ι,33ΡΓ)0Ο3ΕσθΕΡ3ΕσθσΐΧ3ΕΕσΐ.Ε3ΡΙ,ΥΕΕΡΑΡηΡΡΡσΕΝΕΤΥΙ,Μ0

FMEEWGLYVQMEN

CSEQ ID NO :20)

148 (21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)

Gaiy S.C., et al. Gene 256,139-147, 2000; Clark H.F., et al. Genome

Res. 13, 2265-2270, 2003; Strausberg R.L., etal. Proc. Natl. Acad. Sci.

USA. 99, 16899-16903, 2002; US2003186372 (Claim 11);

US2OO3186373 (Claim 11); US2003119131 (Claim 1; Fig 52);

US2003119122 (Claim 1; Fig 52); US2003119126 (Claim 1);

US2003119121 (Claim 1; Fig 52); US2003119129 (Claim 1);

US2003119130 (Claim 1); US2OO3119128 (Claim 1; Fig 52);

US2003119125 (Claim 1); W02003016475 (Claim 1); W0200202634 (Claim 1);

911 aa

ΜΑΟυΐ,ΡΙιΕΑΑΙινίιΑΟΑΡΑΑΙΑΟνΕΕβϋβδΕηΕΑΡΕ'νΕΙΑΟΟΑΡΙιΟσνίισσΑΙ/ΓΙΡΟΗνΗ

ΥΕΕΡΡΡδΕΚΑνΒΟΞΡΗνΚΝΤΡΕΕΕαΕΕΑΕνΕνΑΗΘνΚνκνΝΕΑΥΗΡΗνΑΕΡΑΥΡΑεΕΤΟν SLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGWFLYREGSARYAFSFSGAQEACA ΕίσΑΗΙΑΤΡΕΟΕΥΑΑΥΕσΟΥΕΟΟΌΑσ^εΏΟΤνΕΥΡΙΟΤΡΕΕΑσΥαΌΜϋσΡΡανΚΝΥσνν ΠΡΌΌΕΥΌνΥεΥΑΕΠΕΝσΕΕΡΕσΏΡΡΕΚΕΤΕΕΕΑΕΑΥσΟΕΕσΑΕΙΑΤΤΟΟΕΥΑΑνωσσΌΌΗ CSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPS AIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSST PEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALW AWPSELSSPGPEASLPTEPAAQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPT ΕΤΕΡΤΡΕΕΕΝΕΑβΡεΡΒΤΙΛΖΕΑΕΕνΟΕΑΤσΟΡΕΕΞΟνΡΕΟΕΒΕΕΤΟεδΕΟΑΡβΕΕΡΑΤΕΑ PEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAWPASGDCVPSPCHNGGT CLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYG AHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLS σΕΝΟννΜνΝΗΟΟσΟΝδΌνΡΟΝΥΗΕεΥΤΟΚΜσΕνΞΟσΡΡΡΕΕΡΕΑΟνΡσΕΡΕΕΕΥΕνϋΤνΕ ΕΥΕΟΕΕσΕΑΟΕΝΕΡΕΙΕΟΟΕΝΟΕΜΕΑΡΟΙδΟνΡΕΕΡΑΕΑΕΗΡΕΕΟΡΕΟΕΟΟΕΙιΙισΕΜΚΑΕ LIPPSSPMPGP (SEQ ID NO:21) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.

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)); W02003042661 (Claim 12); W0200053216 (Claim 1;

149

Page 41); W02004065576 (Claim 1); W02004020583 (Claim 9);

W02003004529 (Page 128-132); W0200053216 (Claim 1; Page 42); Cross-references: MIM:600997; NP_004433.2; NM_004442_l 987 aa

ΜΑ1,ΡΕΙ_ισΑΑ1ιΙ.Ιι1&gt;ΡΙ,ΙιΑΑνΕΕΤΙιΜΟ3ΤΤΑΤΑΕΙΧ3ΝΜνΗΡΡ3θννΕΕν3ΟΥΟΕΝΜΝΤΙΕΤΥ0 VCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSIPSVPGSCKETFNDYYYEADF DSATKTFPNWMENPWVKVDTIAADESFSQVDLGGRVMKINTEVRSFGPVSRSGFYLAFQD YGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLY CNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGA ΤΝανεΕΝσΥΥΕΑΌΕΟΡΕΟΜΡΟΤΤΙΡεΑΡΰΑνίεΒνΝΕΤΞΕΜΕΕΜΤΡΡΕΌεαΟΕΕΟΕνΥΝΙ ICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAVNGVTDQSPF SPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKELSE YNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEK LPLIIGSSAAGLVFLIAVWIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDP FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVC SGHLKLPGKREIFVAIKTLKSGY TEKQRRDFLSEASIMGQFDHPNVIHLEGWTKSTPVMIITEFMENGSLDSFLRQNDGQFT VIQLVGMLRGIAAGMKYLADMNYVHRDLAARNIIj'VNSNLVCKVSDFGLSRFLEDDTSDPT YTSALGGKIPIRWTAPEAIQYRKFTSASDVWSYGIVMWEVMSYGERPYWDMTNQDVINAI EQDYRLPPPMDCPSALHQLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSG INLPLLDRTIPDYTSFNTVDEWLEAIKMGQYKESFANAGFTSFDWSQMMMEDILRVGVT LAGHQKKILNSIQVMRAQMNQIQSVEV (SEQ ID NO:22) (23) ASLG659 (B7h, Genbank accession no. AX092328)

US20040101899 (Claim 2); W02003104399 (Claim 11); W02004000221 (Fig 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Fig 60); W02002102235 (Claim 13; Page 299); US2003091580 (Example 2); W0200210187 (Claim 6; Fig 10); W0200194641 (Claim 12; Fig 7b); W0200202624 (Claim 13; Fig 1A-1B); US2002034749 (Claim 54; Page 45-46); W0200206317 (Example 2; Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469); W0200202587 (Example 1; Fig 1);

W0200140269 (Example 3; Pages 190-192); W0200036107 (Example 2; Page 205-207); W02004053079 (Claim 12); W02003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318; . _t..

282 aa

150

ΜΑβΙίσΟΙΙιΕΤίβΙΙβΙΙΙΙΙΑΟΑΙΑΙ,ΙίσΡσίβσΡΗβΙΤνΤΤνΑβΑσΝίσΕΟΟΙΙιβαΤΕΕΡ

DIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV

QLTDAGTYKCYIITSKGKKNANIjEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTW

WASQVDQGANFSEVSNTSFELNSENVTMKWSVLYNVTINNTYSCMIENDIAKATGDIKV

ΤΕΘΕΙΚΚΕΞΗΙ,ΟίιΙ,ΝεΚΑΘΙιΟνεΞΕΕΑΙΘΜΑΙιΙ,ΡΙ,ΘΡΥΙιΜΙιΚ (SEQ ID NO :23) (24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.

AJ297436)

Reiter R.E., etal. Proc. Natl. Acad. Sci. USA. 95, 1735-1740, 1998; Gu

Z., etal. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res.

Commun. (2000) 275(3):783-788; W02004022709; EPl394274 (Example 11); US2004018553 (Claim 17); W02003008537 (Claim 1);

WO200281646 (Claim 1; Page 164); W02003003906 (Claim 10; Page

288); W0200140309 (Example 1; Fig 17); US2001055751 (Example 1;

Fig lb); W0200032752 (Claim 18; Fig 1); WO9851805 (Claim 17;

Page 97); WO9851824 (Claim 10; Page 94); W09840403 (Claim 2; Fig

IB);

Accession: 043653; EMBL; AF043498; AAC39607.1.

123 aa

ΜΚΑν^ΑΣΕΜΑαΐ,ΑΒΰΡΟΤΑΕΙ.αΥδαΚΑΟνΞΝΕϋαΕΟνΕΝεΤΟΙΌΕΟΟΜΤΑΡΙΡΑναΕηΤ

VISKGC SLNC VDDSQDYYVGKKNITCCDTDLCNASGAHALQ PAAAI 1ιΑΙ,1ιΡΑΙΧ3ΙιΒΠ&lt;;Ρ

GQL (SEQ ID NO :24) (25) GEDA (Genbank accession No. AY260763);

AAP14954 lipoma HMGIC fusion-partner-like protein /pid=AAP14954.1 Homo sapiens

Species: Homo sapiens (human)

W02003054152 (Claim 20); W02003000842 (Claim 1); W02003023013 (Example 3, Claim 20); US2003194704 (Claim 45);

151

Cross-references: GI:30102449; AAP14954.1; AY260763_l

236 aa

MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVENSEAIGVLWAIFTICFAIVNWCFIQPYW ίσοσνοΤΡΟΑσΥΕσΕΕΗΥΟΙΟΝσΕΞΕΕΙιΤΟΕσΞΕΤΟΕΞΊΈΡΞσΑΕΚΑΑΞΕΕΙΟΕΞΜΜΕΙΙ ACIICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKEMCGEKTDKYT ΕσΑΟενΚΜΑΥΙΕΑΙϊσίΕϋΑΕΙΕδΡΕΑΓνΕσΝΚΟΟεΕΜΑΕΕΕΚΑΕΝΚνΕΕεΟΥδΕΕ (SEQ ID NO:25) (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. NP_443177.1);

NP_443177 BAFF receptor /pid=NP_443177.1 - Homo sapiens

Thompson,J.S., etal. Science 293 (5537), 2108-2111 (2001); W02004058309;

W02004011611; W02003045422 (Example; Page 32-33); W02003014294 (Claim 35; Fig 6B); W02003035846 (Claim 70; Page 615-616); WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); W0200224909 (Example 3; Fig 3);

Cross-references: MIM:606269; NP_443177.1; NM_052945_l

184 aa

ΜΕΕΟΡΕΞΕΕΟΕΏΑΡΑΡΤΡΟνΡΑΕΟΓΏΕΕνΕΗΟνΑΟΟΙιΕΕΤΡΕΡΚΡΑΟΑΒΞΡΑΡΕΤΑΙιΟΡΟ

ΕΞνσΑσΑσΕΑΑΙ,ΡΙιΡΟΙιΙιΕΟΑΡΑΕΕσΕΑΙΛΖΕΑΙΛΖΙινΟΕνΞΙ^ΕΕΕΟΕΕΕΕσΑβΒΑΕΑΡΟαΟ KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG PEQQ (SEQ ID NO:26) (27) CD22 (B-cell receptor CD22-B isoform, Genbank accession No. NP001762.1);

Stamenkovic,I. and Seed,B., Nature 345 (6270), 74-77 (1990); US2003157113;

US2003118592; W02003062401 (Claim 9); W02003072036 (Claim 1; Fig 1);

WO200278524 (Example 2);

Cross-references: MIM:107266; NP_001762.1; NM_001771_l

847 aa

ΜΗΕΙΧΪΡνίΕΕΕΕνΕΕΥΕΑΓεηβΞΚνΐνΕΕΗΡΕΤΕΥΑΝΕΟΑΟννΐΙΡΟΤΥΕΑΕΟσΟΕΕΞΕΙΙ,ΕΗ

152

ΝΡΕΥΝΚΝΤεΚΕΠσΤΗΒΥΕΒΤΚΟβΚνΡΒΕΟΚΗνΟΡΕΘΌΚΝΚΝΟΤΒείΗΡνΗΕΝΠΒΘΟΕσΒΗ ΜΕδΚΤΕΚΜΜΕΕΙΗΕΝνΞΕΕΡΡΡΡΗΙΟΕΡΡΕΙΟΕΞΟΕνΤΙΖΓΟΙ,ΙιΝΡΞΟΥΟΥΡΙΟΙιΟνΐΙιΕΕΟ νΡΜΗΟΑΑνΤΞΤΞΕΤΙΚενΡΤΗδΕΕΚΡεΡςΜΒΗΗΘΚίνΤΟΟΕΟΌΑϋΟΚΡΕεΝΌΤνΟΕΝνΚΗ TPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPL PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPK KVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNT TIACARCNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQ FFWEKNGRLLGKESQLJflFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPHHSQKLRLEPVKVQHSGAYWCQ σΤΝΞνσΚΟΕΞΡΕΞΤΕΤνΥΥΞΡΕΤΙΟΕΕνΑναΕσΞσΕΑΙΕΙΕΑΙΟΟΕΚΕΟΕΕΝΚΕΤΟεΟΰΟ ΕΟΕΝΞΞαΟΞΡΡνΕΝΚΚνΕΕΑΡΕΞΕΟΡΗΞΕΟΟΥΝΡΜΜΕΌσΐΞΥΤΤΕΕΕΡΕΜΝΙΡΕΤσΌΑΕΞ SEMQRPPRTCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENV DYVILKH (SEQ ID NO:27) (28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation) PROTEIN SEQUENCE Full mpggpgv...dvqlekp (1..226; 226 aa), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19ql3.2, Genbank accession No. NP_001774.1;

W02003088808, US20030228319; W02003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, Fig 16); WO9207574 (Fig 1); US5644033; Baetal. (1992) J. Immunol. 148(5):1526-1531; Mueller etal. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto etal. (1994) Immunogenetics 40(4):287-295; Preud’homme etal. (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(ll):3457-3464;

226 aa

ΜΡΟσΡσνΕΟΑΕΡΑΤΙΡΣΕΡΙ,Ι^ΑνΥΙ,σΡσΟΟΑΕΝΜΗΚνΡΑΒΕΜνΞΙΧΪΕηΑΗΡΟΟΡΗΝΒΞΝ NANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCG TYLRVRQPPPRPFLDMGEGTKNRIITAEGII LLFCAWPGTLIjLFRKRWQNEKIjGLDAGD EYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP '

153 (SEQ ID NO:28) (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia) PROTEIN SEQUENCE Full mnypltl...atslttf (1..372; 372 aa), pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: llq23.3, Genbank accession No. NP_001707.1;

W02004040000; W02004015426; US2003105292 (Example 2); US6555339 (Example 2); W0200261087 (Fig 1); W0200157188 (Claim 20, page 269); W0200172830 (pages 12-13); W0200022129 (Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38); US5440021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, Fig 5); Dobner et al. (1992) Eur. J. Immunol. 22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779;

372 aa

ΜΝΥΡΙιΊΊ,ΕΜΏΙ,ΕΝΙ,ΕΏΕΕΜΕΙ,ΟΕΙΛΝΥΝΏΤεΐινΕΝΗΕΟΡΑΤΕΰΡΙιΜΑΞΕΚΑνΕνΡνΑΥΞΕ ΙΕΙιΙιΘνίΟΝνίινΕνίΕΕΗΗΗΟΤΚΞΒΤΕΤΕΙιΕΗΕΑνΑΏΙιΙιΕνΕΙΕΡΕΑνΑΕΟΞνονννΕΟΤΕ Ι,ΟΚΤνίΑΕΗΚνΝΕΥΟεεΕΕΕΑΟΙΑνΌΗΥΕΑίνΗΑνΗΑΥΕΗΕΗΕΕΞΙΗΙΤεΘΤίννΕνΟΕΕΙ, ΑΙιΡΕΙΕΕΑΚνδΟΟΗΗΝΝδΙιΡΚεΤΕεΟΕΝΰΑΕΤΗΑΜΕΤΞΕΕΙιΥΗνΑαΕΕΙ,ΡΜΙινΜΟΝσΥνσ ννΗΕΕΕΟΑΟΕΕΡΰΚΟΚΑνκνΑΙΕνΤΞΙΕΕΕσΝΞΡΥΗΐνίΕΕΌΤΕΑΚΕΚΑνΏΝΤΟΚΕΝΘεΕ PVAITMCEFLGLT^HCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSL· SESENATSLTTF (SEQ ID NO :29) (30) HLA-DOB (Beta subunit of MHC class Π molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes) PROTEIN SEQUENCE Full mgsgwvp...vllpqsc (1..273; 273 aa, pl: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1;

Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics 29(6):411-413; Beck etal. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et

154 al. (1987) J. Biol. Chem. 262:8759-8766; Beck etal. (1996) J. Mol. Biol. 255:113; Naruse et al. (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13, Fig 15); US6153408 (Col 35-38); US5976551 (col 168-170); US6011146 (col 145146); Kasahara et al. (1989) Immunogenetics 30(l):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119;

273 aa

MGSGWVPWWALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKVQFWRFIFNL EEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFTVGRK VQ PEVTVYPERT PLLHQHNLLHC SVTGFYPGDIKIKWFLNGQEERAGVMSTGPIRNGDWT ΕΟΤννΜΙιΕΜΤΡΕΙίσΗνΥΤΟΙ.νϋΗεΞίιΙ.ΞΡνΞνΕΚΒΑΟΞΕΥβνίΕΚΜΙ.βσίΑΑΡΙιΙΧϊΙ,ΙΡΙ.Ε VGIVIQLRAQKGYVRTQMSGNEVSRAVLLPQSC (SEQ ID NO:30) (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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability) PROTEIN SEQUENCE Full mgqagck...lephrst (1..422; 422 aa), pl: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17pl3.3, Genbank accession No. NP_002552.2;

Le etal. (1997) FEBS Lett. 418(1-2): 195-199; W02004047749; W02003072035 (claim 10); Touchman etal. (2000) Genome Res. 10:165-173; W0200222660 (claim 20); W02003093444 (claim 1); W02003087768 (claim 1);

W02003029277 (page 82);

422 aa

Μσζ)ΑσθΚσΐισΐι3υθΥΚΤΕΚΥνΐΑΚΝΚΚνθΙιΙ.ΥΡΙιΙ;0Α3ΙΙιΑΥΙιννΜΛΓΕΙΚΚαΥ0ΟλΓΟΤ SLQSAVITKVKGVAFTNTSDLGQRIWDVADYVIPAQGENVFFWTNLIVTPNQRQNVCAE NEGIPDGACSKDSDCHAGEAVTAGNGVKTGRCLRRENLARGTCEIFAWCPLETSSRPEEP FLKEAEDFTIFIKNHIRFPKFNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRW AGSDFQDIALEGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARY YRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKKYEEVRGLEDS ΞΟΕΑΕΟΕΑΒσΐΌΙ^ΕΟΙ,ΤεΟΡΟΙΛΟΜΡΕΟςΕΙ,ΟΕΡΡΕΑΚΡσΒΞεΟΚσΝσενΟΡΟΙ,ΕΕΡΗΡ

ST ' '

155 (SEQ ID NO :31) (32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full maeaity.-.tafrfpd (1..359; 359 aa), pl: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9pl3.3, Genbank accession No. NP_001773.1;

W02004042346 (claim 65); W02003026493 (pages 51-52,57-58); W0200075655 (pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:1689916903;

359 aa

MAEAITYADLRFVKAPLKKSISSELGQDPGADDDGEITYENVQVPAVLGVPSSLASSVLG ΟΚΑΑνκεΕΟΡΤΑεΚΕΑντεΡΑνσΕΙΙ.ΡεΕΤΤΟΕΕΥΙ,ΙιΕσίιΙ.ΙΖΓσΕΙιΕΟνΤΑΐαΕΟνΕΥΙιΟ νεΟΟΕΟΟΤΝΗνΕΕνΤΝεΞΕΗΟΟΕΗΙ,ΚΙΤΟΕΘΟεΤ^ΕΟΕΟσεΗΚΕΙ,ΑΟεΟΕΑΕΟνΕΟΚΑΗΟΑ AEGQLQACQADRQKTKETIjQSEEQQRRALEQKIjSNMENEIjKPFFTCGSADTCCPSGWIMH ΟΚεΟΕΥΙΒΕΤΒΚΝΜΟΕΞΟΚΟΟΕΤΕβεΚΕΑΤΕΞΕΙΥΡΟεΗεΥΥΕΕΝΒΕΕΡΝΟσΞσΝβΥνΠΌ LSSNKDWKLTDDTQETETYAQSSKCNKVHKTWSWWTLESESCESSLPYICEMTAFEFPD (SEQ ID NO:32) (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis) PROTEIN SEQUENCE Full mafdvsc...rwkyqhi (1..661; 661 aa), pl: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5ql2, Genbank accession No. NP_005573.1;

US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822;

W02003083047; WO9744452 (claim 8, pages 57-61); W0200012130 (pages 2426);

661 aa

MAFDVSCFFWWLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEFLEF βΡΝΠιΡΊΊΗΝΕΤΡΒΕϋΜΝΙιΤΡΙιΟΙΖΓΕΟΟΤΝνίΙΗΕΟΤΡΟΒΗΗΟΙιΒ'ΓΙινί/ΓΟΝΡΕΙΕ’ΜΑΕΤε · ΙΝΟΡΚΌΙιΚΗΕΡΕΙΟΤΟΙΞΝΙ,ΕΡΙΡνΗΝΙιΕΝΕΕΞΙίΥΙ,ΟΞΝΗΙΞΞΙΚΡΡΚΟΡΡΑΕΝΕΚνίιΟΡ QNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIEIjGAFDSTVFQSIjNFGGTPNLiSVIFN

156 αβΟΝΒΤΤΰεΒΜΣσΤΡΕΌΙΌΌΕΌΙδΞΆΜΏΚαΕαΕΜΒνΕδΏΝΒΟΕΗΕΚΒΌΙΞΞΤΤΡΟΟΓΤζ)!; 0ΕΕΟΕΤΑΤΗ1,ΚΟΙιΡ3ΟΜΚΟΙιΝυΐιΚΚΕνΐι3νΝΗΕΐΧ2Ιι00Ι3ΑΑΝΕΡ3Ι&gt;ΤΗ1&gt;ΥΙΡΟΝνκΚΙιΗ ΕΟνΟΟΕΕΚΕαΝΕΰΤΕΟΕδΗΝΏΙΕΑβΌααδΕΟΕΚΝΕεΗΕΟΤΠΝΕΞΗΝΕΡΕΟΕΟδΟΑΡΚΕεΡ ΟΕΕΏΙιΟΙιΑΕΤΕΙιΗΙΝΑΡΟΞΡΓΟΝΙιΗΕΕΟνΕΝΙιΤΥΟΕΙ,ΟΤΒΝΟΗΙιΙιΑΟΙιΡνί,ΡΗΙιΝΙ,ΚΟΝΗ ΕΟΌΘΤΙΤΚΤΝΕΕΟΤνσδΕΕνΕΙΕΞδαΘΕΕείΟΟΟΑΕΗεΕΟΚΜΒΗνϋΕδΗΝεΕΤαΟδΙΌδΕ ΞΗΕΚσΐΥΕΝΙιΑΑΝΒΙΝΙΙΞΡΕΙιΙιΡΙΙιΒΟΟΒΤΙΝΙιΞΗΝΡΙ,ϋΟΤΟΞΝΙΗΕΙΖΓΚΥΚΕΝΙιΗΚΙιΕ ΟβΕΕΤΤΟΑΝΡΡβΕΕσνΚΙιβϋνκυβαΟΙΤΑΙΟΙΡΡϋΐνΕΙιϋΙιΑΙΙιΙιΕΡΑνΚΥΙιΙύηΜΚΥΟΗ I (SEQ ID NO:33) (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation) PROTEIN SEQUENCE Full mlprlll...vdyedam (1..429; 429 aa), pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: Iq21-lq22, Genbank accession No. NP_443170.1;

W02003077836; W0200138490 (claim 6, Fig 18E-1-18-E-2); Davis etal. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; W02003089624 (claim 8);

EPl347046 (claim 1); W02003089624 (claim 7);

429 aa

ΜΏΡΗΕΙιΕΙ,ΙΟΑΡΙιεΕΡΑΕΕΕΙιΙΑδΡεΗΡΤΕσδΡλΖΤΕΤΟΚΜΡΕΕΟΞδΟΑΟΕΟΕΟΕΕΕΟΤΚΑ LGPGWSSSPKLQIAAMWKEDTGSYWCEAQTMASKVLRSRRSQINVHRVPVADVSLETQPP σαονΜΕΟΟΕΙ,νΕΙΟενΑΜσΤΟΟΙΤΕΕΜΥΚσΑνσΕΝΕΟΞΚΤΟΕεΕΤΑΕΥΕΙΡβνΕΕεΟΑΕΟ YYCVAENGYGPSPSGLVSITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILY WFYHEDITLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGIjGAQRSEAVTLNFTVPTGA RSNHLTSGVIEGLIjSTLGPATVALLFCYGLKRKIGRRSARDPIjRSLPSPLPQEFTYLNSP TPGQLQPIYENVNWSGDEVYSLAYYNQPEQESVAAETLGTHMEDKVSLDIYSRLRKANI TDVDYEDAM (SEQ ID NO :34) (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies) PROTEIN SEQUENCE Full mllwvil...assaphr (1..977; 977 aa), pl:

157

6.88 MW: 106468 TM: 1 [P] Gene Chromosome: lq21, Genbank accession No.

NP_112571.1;

W02003024392 (claim 2, Fig 97); Nakayama et al. (2000) Biochem. Biophys.

Res. Commun. 277(1):124-127; W02003077836; W0200138490 (claim 3, Fig 18B-1-18B-2);

977 aa

ΜΙ,ΙιΚνίΕΙινίιΑΡνΒΟΟΕΑΕΤΡΕΡΙΙΕΙιΟΡΡΜΤΤνΕΟαΕΕνΤΙΖΓΟΚβΕΕΕΥβΡΟΚΤΚΙπίΥΗΕ YLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSW· LRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNT νΚΙΟνΟΕΡΕΤΕΡνΕΚΑββΡΟΡΙβσΝΡνΤΙΖΓαΕΤΟΕβυΕΕβΟνΡΙ/ΕΡΕΡΕΕΟΟΟΤβσΕσίίβ LSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFE GTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNG LGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEAQRGSLPILYQFHHEDAA LERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSITVPVSHPVLTLSSA ΕΑΙιΤΡΕσΑΤνΤΙιΗΟΕνΟΕσΒΡΟΙΙ,ΥΟΡΥΗΕΟΜΡΙΛίΒΒβΤΡβνσΕνΒΡβΡβΙΖΓΕΟΗεΟΝΥΥ ΟΤΑΟΝΟΡαΡΟΕΕΕννΕΏΡνΤνΡνΕΕΡΙΕΤΕΕνΡΕΑΟΑννΟΌΕΕΕΕΗΟΕΑΡΕΟΞΡΡΙΕΥΜΓ ΥΗΕΟνΤΕσβββΑΡΒΟσΕΑΒΡΝΕβΙ/ΓΑΕΗβσΝΥβΟΕΑΝΝβΙ,νΑΟΗΒΟΤΙΒΙ,ΒνίνΡνβΕΡΙ ΕΤΡΕΑΡΕΑΟΑννσηΕΕΕΕΗαΕΑΒΕσεδΡΙΙΖΥΝΕΥΗΕΌνΤΕσΚΙΕΑΡΞΟαΟΑΞΕΝΒεΕΤΤΕ HSGIYSCEADNGPEAQRSEMVTLKVAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSP LIIjYRFFHEDVTIjGNRSSPSGGASLNLSLiTAEHSGNYSCEADNGL&gt;GAQRSETVTLYITGIj TANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYH NVPAWEELQPVYTNANPRGENWYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKVA STPVSGSLFLASSAPHR (SEQ ID NO :35)

See also: W004/045516 (03 Jun 2004); W003/000113 (03 Jan

2003); W002/016429 (28 Feb 2002); WO02/16581 (28 Feb 2002); WO03/024392 (27 Mar 2003); W004/016225 (26 Feb 2004); W001/40309 (07 Jun 2001), and U.S. Provisional patent application Serial No. 60/520842 “COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN”, filed 17 Nov 2003; all of which are incorporated herein by reference in their entirety.

In an embodiment, the Ligand-Linker-Drug Conjugate has Formula

Illa, where the Ligand is an antibody Ab including one that binds at least one.of

CD30, CD40, CD70, Lewis Y antigen, w=0, y=0, and D has Formula lb.

158

Exemplary Conjugates of Formula Illa include where R¹⁷ is -(CH₂)5-. Also included are such Conjugates of Formula Illa in which D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Formula Illa containing about 3 to about 8, in one aspect, about 3 to about 5 Drug moieties D, that is, Conjugates of Formula la wherein p is a value in the range about 3-8, for example about 3-5. Conjugates containing combinations of the structural features noted in this paragraph are also contemplated as within the scope of the compounds of the invention.

In another embodiment, the Ligand-Linker-Drug Conjugate has Formula Illa, where Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Y antigen, w=l, y=0, and D has Formula lb. Included are such Conjugates of Formula Illa in which R¹⁷ is -(CH₂)₅-. Also included are such Conjugates of Formula Illa in which W is -Val-Cit-, and/or where D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Formula Illa containing about 3 to about 8, preferably about 3 to about 5 Drug moieties D, that is, Conjugates of Formula la wherein p is a value in the range of about 3-8, preferably about 3-5. Conjugates containing combinations of the structural features noted in this paragraph are also exemplary.

In an embodiment, the Ligand-Linker-Drug Conjugate has Formula Illa, where the Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Y antigen, w=l, y=l, and D has Formula lb. Included are Conjugates of Formula Illa in which R¹⁷ is -(CH₂)s-. Also included are such Conjugates of Formula Illa where: W is -Val-Cit-; Y has Formula X; D has the structure of Compound 2 in Example 3 and esters thereof; p is about 3 to about 8, preferably about 3 to about 5 Drug moieties D. Conjugates containing combinations of the structural features noted in this paragraph are also contemplated within the scope of the compounds of the invention.

A further embodiment is an antibody drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody that binds one of the tumor-associated antigens (1)-(35) noted above (the “TAA Compound”).

159

Another embodiment is the TAA Compound or pharmaceutically acceptable salt or solvate thereof that is in isolated and purified form.

Another embodiment is a method for killing or inhibiting the multiplication of a tumor cell or cancer cell comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to kill or inhibit the multiplication of a tumor cell or cancer cell.

Another embodiment is a method for treating cancer comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat cancer, alone or together with an effective amount of an additional anticancer agent.

Another embodiment is a method for treating an autoimmune disease, comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat an autoimmune disease.

The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

4.5.1 PRODUCTION OF RECOMBINANT ANTIBODIES

Antibodies of the invention can be produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression.

Recombinant expression of antibodies, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the

160 synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides, e.g., by PCR.

Alternatively, a nucleic acid molecule encoding an antibody can be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody can be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by, e.g., PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

If an antibody that specifically recognizes a particular antigen is not commercially available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin), antibodies specific for a particular antigen can be generated by any method known in the art, for example, by immunizing a patient, or suitable animal model such as a rabbit or mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 7796). Alternatively, a clone encoding at least the Fab portion of the antibody can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it can be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., International Publication No. WO 86/05807; WO 89/01036; and U.S. Patent No. 5122464). Vectors'containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid

161 encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).

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

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to the F(ab’)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab’)2 fragments.

162

Once a nucleic acid sequence encoding an antibody has been obtained, the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY).

An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

The host cells used to express the recombinant antibody can be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2).

A variety of host-expression vector systems can be utilized to express the immunoglobulin antibodies. Such host-expression systems represent vehicles by which the coding sequences of the antibody can be produced and subsequently purified, but also represent cells that can, when transformed^ or transfected with the appropriate nucleotide coding sequences, express an antibody

163 immunoglobulin molecule in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, vectors that direct the expression of high levels of fusion protein products that are readily purified might be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye &amp; Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke &amp; Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX Vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed*to include thrombin or factor Xa protease &gt;

164 cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa califomica nuclear polyhedrosis virus (AcNPV) or the analogous virus from Drosophila Melanogaster is used as a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) results in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan &amp; Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen to modulate the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for

165 the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express an antibody can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody. Such engineered cell lines can be particularly useful in screening and evaluation of tumor antigens that interact directly or indirectly with the antibody.

A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &amp; Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: DHFR, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA™· 77:357; O’Hare etal., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which

166 confers resistance to mycophenolic acid (Mulligan &amp; Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley &amp; Sons, NY.; Colberre-Garapin et al., 1981,7. Mol. Biol. 150:1).

The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers that enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used to encode both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The

167 coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.

Once the antibody has been recombinantly expressed, it can be purified using any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

In yet another exemplary embodiment, the antibody is a monoclonal antibody.

In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an autoimmune disease, an infectious organism, or other disease state.

PRODUCTION OF ANTIBODIES

The production of antibodies will be illustrated with reference to anti-CD30 antibodies but it will be apparent for those skilled in the art that antibodies to other members of the TNF receptor family can be produced and modified in a similar manner. The use of CD30 for the production of antibodies is exemplary only and not intended to be limiting.

The CD30 antigen to be used for production of antibodies may be, e.g., a soluble form of the extracellular domain of CD30 or a portion thereof, containing the desired epitope. Alternatively, cells expressing CD30 at their cell surface (e.g., L540 (Hodgkin’s lymphoma derived cell line with a T cell phenotype) and L428 (Hodgkin’s lymphoma derived cell line with a B cell phenotype)) can be used to generate antibodies. Other forms of CD30 useful for generating antibodies will be apparent to those skilled in the art.

In another exemplary embodiment, the ErbB2 antigen to be used for production of antibodies may be, e.g., a soluble form of the extracellular

168 domain of ErbB2 or a portion thereof, containing the desired epitope. Alternatively, cells expressing ErbB2 at their cell surface (e.g., NIH-3T3 cells transformed to overexpress ErbB2; or a carcinoma cell line such as SK-BR-3 cells, see Stancovski etal. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991))can be used to generate antibodies. Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art.

(i) Polyclonal antibodies

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), Nhydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R¹N=C=NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 pg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

(it) Monoclonal antibodies ......

169

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier monoclonal indicates the character of the antibody as not being a mixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4816567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described

170 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)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody·protein, to obtain the synthesis of monoclonal antibodies in the · '' recombinant host cells. Review articles on recombinant expression in bacteria of

171

DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4816567; and Morrison, et al. (1984) Proc. Natl Acad. Sci. USA 81:6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a nonimmunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

(iii) Humanized antibodies

A humanized antibody may have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken

172 from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called best-fit method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

In another embodiment, the antibodies may be humanized with retention of high affinity for the antigen and other favorable biological properties. Humanized antibodies may be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate

173 immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Various forms of the humanized antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGl antibody.

The Examples describe production of an exemplary humanized anti-ErbB2 antibody. The humanized antibody may, for example, comprise nonhuman hypervariable region residues incorporated into a human variable heavy domain and may further comprise a framework region (FR) substitution at a position selected from the group consisting of 69H, 71H and 73H utilizing the variable domain numbering system set forth in Kabat et al., 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. Another Example describes preparation of purified trastuzumab antibody from the HERCEPTIN® formulation.

(iv) Human antibodies

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (Jh) gene in chimeric and germ-line mutant mice

174 results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits etal., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of Vgene segments can be used for phage display. Clackson et al., Nature, 352.-624628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5565332 and 5573905. As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Patents Nos. 5567610 and 5229275). Human anti-CD30 antibodies are described in U.S. Patent Application Serial No. 10/338,366.

175 (v) Antibody fragments

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from 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., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Patent No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

(vi) Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the CD30 protein. Alternatively, an anti-CD30 arm may be combined with an arm which binds to a Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the CD30-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD30.

Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different

176 antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991). According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

177

According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab j₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby etal., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

178

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from die Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The diabody technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (Vh) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

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

(vii) Other amino acid sequence modifications

Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final

179 construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of the antibody that are favored locations for mutagenesis is called alanine scanning mutagenesis as described by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxylterminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.

180

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally-occurring residues are divided into 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, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phagedisplayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and

181 the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cellmediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. AntiCancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent No. 5739277, for example. As used herein, the term salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG₂, IgG₃, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

(viii) Glycosylation Variants

Antibodies in the ADC of the invention may be glycosylated at conserved positions in their constant regions (Jefferis and Lund, (1997).Chem.

Immunol. 65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32). The

182 oligosaccharide side chains of the immunoglobulins affect the protein’s function (Boyd et al., (1996) Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem. 29:4175-4180), and the intramolecular interaction between portions of the glycoprotein which can affect the conformation and presented threedimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. For example, it has been reported that in agalactosylated IgG, the oligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminal Nacetylglucosamine residues become available to bind mannose binding protein (Malhotra et al., (1995) Nature Med. 1:237-243). Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinant humanized murine monoclonal IgGl antibody which recognizes the CDw52 antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO) cells resulted in a complete reduction in complement mediated lysis (CMCL) (Boyd et al., (1996) Mol. Immunol. 32:1311-1318), while selective removal of sialic acid residues using neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-Νacetylglucosaminyltransferase ΙΠ (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umanaetal. (1999) Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or Olinked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or

183 xylose to a hydroxyamino acid, most commonly serine or threonine, although 5hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which the glycosylation pattern of an antibody is altered. By altering is meant deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition of glycosylation (glycosylation pattern), the extent of glycosylation, etc.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlying nucleic acid sequence. These methods include, but are not limited to, isolation from a natural source (in the case of naturally-occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody. ’

The glycosylation (including glycosylation pattern) of antibodies may also be altered without altering the amino acid sequence or the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g., antibodies, as potential therapeutics is rarely the native cell, significant variations in the glycosylation pattern of the antibodies can be expected. See, e.g., Hse et al., (1997) J. Biol. Chem. 272:9062-9070. In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media formulation; culture density, oxygenation, pH, purification schemes and the like. Various methods

184 have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Patent Nos. 5047335; 5510261; 5278299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered, e.g., make defective in processing certain 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 compositional analysis, sequential enzymatic digestion, and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, without limitation, enzymatic treatment (commonly performed using peptide-N-glycosidase F/endo^-galactosidase), elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.

SCREENING FOR ANTIBODY-DRUG CONJUGATES (ADC)

Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADC) that have potential as prophylactic or therapeutic treatments of diseases or disorders involving overexpression of proteins including Lewis Y, CD30, CD40, and CD70. Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADC) that have potential as prophylactic or therapeutic treatments of diseases or disorders involving overexpression of HER2 (US6632979). Screening for a useful ADC may involve administering candidate ADC over a range of doses to the transgenic animal, and assaying at various time points for the effect(s) of the ADC on the disease or disorder being evaluated. Alternatively, or additionally, the drug can be

185 administered prior to or simultaneously with exposure to an inducer of the disease, if applicable. Candidate ADC may be screened serially and individually, or in parallel under medium or high-throughput screening format. The rate at which ADC may be scrrened for utility for prophylactic or therapeutic treatments of diseases or disorders is limited only by the rate of synthesis or screening methodology, including detecting/measuring/analysis of data.

One embodiment is a screening method comprising (a) transplanting cells from a stable renal cell cancer cell line into a non-human animal, (b) administering an ADC drug candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the formation of tumors from the transplanted cell line.

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

Another embodiment is a screening method comprising (a) contacting cells from a stable Hodgkin’s disease cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to induce cell death. In one embodiment the ability of the ADC candidate to induce apoptosis is evaluated.

One embodiment is a screening method comprising (a) transplanting cells from a stable cancer cell line into a non-human animal, (b) administering an ADC drug candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the formation of tumors from the transplanted cell line. The invention also concerns a method of screening ADC candidates for the treatment of a disease or disorder characterized by the overexpression of HERZ comprising (a) contacting cells from a stable breast cancer cell line with a drug candidate and (b) evaluating the ability of the ADC candidate to inhibit the growth of the stable cell line.

Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with an ADC drug candidate and (b)

186 evaluating the ability of the ADC candidate to block ligand activation of HER2. In one embodiment the ability of the ADC candidate to block heregulin binding is evaluated. In another embodiment the ability of the ADC candidate to block ligand-stimulated tyrosine phosphorylation is evaluated. ;

Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to induce cell death. In one embodiment the ability of the ADC candidate to induce apoptosis is evaluated.

Another embodiment is a screening method comprising (a) administering an ADC drug candidate to a transgenic non-human mammal that overexpresses in its mammary gland cells a native human HER2 protein or a fragment thereof, wherein such transgenic mammal has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland, and develops a mammary tumor not responding or poorly responding to anti-HER2 antibody treatment, or to a non-human mammal bearing a tumor transplanted from said transgenic non-human mammal; and (b) evaluating the effect of the ADC candidate on the target disease or disorder. Without limitations, the disease or disorder may be a HER2-overexpressing cancer, such as breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic and bladder cancer. The cancer preferably is breast cancer which expressed HER2 in at least about 500,000 copies per cell, more preferably at least about 2,000,000 copies per cell. ADC drug candidates may, for example, be evaluated for their ability to induce cell death and/or apoptosis, using assay methods well known in the art and described hereinafter.

In one embodiment, candidate ADC are screened by being administered to the transgenic animal over a range of doses, and evaluating the animal's physiological response to the compounds over time. Administration may be oral·, or by suitablednjection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the

187 compound in conjunction with co-factors that would enhance the efficacy of the compound. If cell lines derived from the subject transgenic animals are used to screen for compounds useful in treating various disorders, the test compounds are added to the cell culture medium at an appropriate time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply the compound of interest to the culture medium in conjunction with co-factors that would enhance the efficacy of the compound.

Thus, provided herein are assays for identifying ADC which specifically target and bind a target protein, the presence of which is correlated with abnormal cellular function, and in the pathogenesis of cellular proliferation and/or differentiation that is causally related to the development of tumors.

To identify an ADC which blocks ligand activation of an ErbB (e.g., ErbB2) receptor, the ability of the compound to block ErbB ligand binding to cells expressing the ErbB (ErbB2) receptor (e.g., in conjugation with another ErbB receptor with which the ErbB receptor of interest forms an ErbB heterooligomer) may be determined. For example, cells isolated from the transgenic animal overexpressing HER2 and transfected to express another ErbB receptor (with which HER2 forms hetero-ol igomer) may be incubated, i.e. culturing, with the ADC and then exposed to labeled ErbB ligand. The ability of the compound to block ligand binding to the ErbB receptor in the ErbB hetero-oligomer may then be evaluated.

For example, inhibition of heregulin (HRG) binding to breast tumor cell lines, overexpressing HER2 and established from the transgenic nonhuman mammals (e.g., mice) herein, by the candidate ADC may be performed using monolayer cultures on ice in a 24-well-plate format. Anti-ErbB2 monoclonal antibodies may be added to each well and incubated for 30 minutes. ¹²⁵1-labeled rHRGpi 177-224 (25,000 cpm) may then be added, and the incubation may be continued for 4 to 16 hours. Dose response curves may be prepared and an IC50 value (cytotoxic activity) may be calculated for the compound of interest.

188

Alternatively, or additionally, the ability of an ADC to block ErbB ligand-stimulated tyrosine phosphorylation of an ErbB receptor present in an ErbB hetero-oligomer may be assessed. For example, cell lines established from the transgenic animals herein may be incubated with a test ADC and then assayed for ErbB ligand-dependent tyrosine phosphorylation activity using an antiphosphotyrosine monoclonal antibody (which is optionally conjugated with a detectable label). The kinase receptor activation assay described in U.S. Patent No. 5766863 is also available for determining ErbB receptor activation and blocking of that activity by the compound.

In one embodiment, one may screen for ADC which inhibit HRG stimulation of pl80 tyrosine phosphorylation in MCF7 cells essentially as described below. For example, a cell line established from a HER2-transgenic animal may be plated in 24-well plates and the compound may be added to each well and incubated for 30 minutes at room temperature; then rHRGP 1177-244 may be added to each well to a final concentration of 0.2 nM, and the incubation may be continued for about 8 minutes. Media may be aspirated from each well, and reactions may be stopped by the addition of 100 μΐ of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μΐ) may be electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane. Antiphosphotyrosine (at 1 gg/ml) immunoblots may be developed, and the intensity of the predominant reactive band at M_r -180,000 may be quantified by reflectance densitometry. An alternate method to evaluate inhibition of receptor phosphorylation is the KIRA (kinase receptor activation) assay of Sadick et al. (1998) Jour, of Pharm. and Biomed. Anal. Some of the well established monoclonal antibodies against HER2 that are known to inhibit HRG stimulation of pl80 tyrosine phosphorylation can be used as positive control in this assay. A dose-response curve for inhibition of HRG stimulation of pl 80 tyrosine phosphorylation as determined by reflectance densitometry may be prepared and an IC50 for the compound of interest may be calculated. E

189

One may also assess the growth inhibitory effects of a test ADC on cell lines derived from a HER2-transgenic animal, e.g., essentially as described in Schaefer et al. (1997) Oncogene 15:1385-1394. According to this assay, the cells may be treated with a test compound at various concentrations for 4 days and stained with crystal violet or the redox dye Alamar Blue. Incubation with the compound may show a growth inhibitory effect on this cell line similar to that displayed by monoclonal antibody 2C4 on MDA-MB-175 cells (Schaefer et al., supra). In a further embodiment, exogenous HRG will not significantly reverse this inhibition.

To identify growth inhibitory compounds that specifically target an antigen of interest, one may screen for compounds which inhibit the growth of cancer cells overexpressing antigen of interest derived from transgenic animals, the assay described in U.S. Patent No. 5677171 can be performed. According to this assay, cancer cells overexpressing the antigen of interst are grown in a 1:1 mixture of Fl2 and DMEM medium supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin. The cells are plated at 20,000 cells in a 35 mm cell culture dish (2 mls/35mm dish) and the test compound is added at various concentrations. After six days, the number of cells, compared to untreated cells is counted using an electronic COULTER™ cell counter. Those compounds which inhibit cell growth by about 20-100% or about 50-100% may be selected as growth inhibitory compounds.

To select for compounds which induce cell death, loss of membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD uptake may be assessed relative to control. The PI uptake assay uses cells isolated from the tumor tissueof interest of a transgenic animal. According to this assay, the cells are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. The cells are seeded at a density of 3 x 10⁶ per dish in 100 x 20 mm dishes and allowed to attach overnight. The medium is then removed and replaced with fresh medium alone or medium containing various concentrations of the

190 compound. The cells are incubated for a 3-day time period. Following each treatment, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutes at 4 °C, the pellet resuspended in 3 ml cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped 12 x 75 mm tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 pg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those compounds which induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing compounds.

In order to select for compounds which induce apoptosis, an annexin binding assay using cells established from the tumor tissue of interest of the transgenic animal is performed. The cells are cultured and seeded in dishes as discussed in the preceding paragraph. The medium is then removed and replaced with fresh medium alone or medium containing 10 pg/ml of the antibody drug conjugate (ADC). Following a three-day incubation period, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged, resuspended in Ca²⁺ binding buffer and aliquoted into tubes as discussed above for the cell death assay. Tubes then receive labeled annexin (e.g., annexin VFITC) (1 gg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those compounds which induce statistically significant levels of annexin binding relative to control are selected as apoptosis-inducing compounds.

IN VITRO CELL PROLIFERATION ASSAYS

Generally, the cytotoxic or cytostatic activity of an antibody drug conjugate (ADC) is measured by: exposing mammalian cells having receptor proteins to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays were used to measure viability (proliferation),

191 cytotoxicity, and induction of apoptosis (caspase activation) of the ADC of the invention.

The in vitro potency of antibody drug conjugates was measured by a cell proliferation assay (Example 18, Figures 7-10). The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (U.S. Patent Nos. 5583024; 5674713 and 5700670). This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells (Crouch etal. (1993) J. Immunol. Meth. 160:81-88, U.S. Patent No. 6602677). The CellTiter-Glo® Assay was conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing. The cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.

The homogeneous add-mix-measure format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The CellTiter-Glo® Assay generates a glow-type luminescent signal, produced by the luciferase reaction, which has a half-life generally greater thah five hours, depending on cell type and medium used. Viable cells are reflected in relative luminescence 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 reagent injectors and provides flexibility for continuous or batch mode processing of multiple plates. This cell proliferation assay can be used with

192 various multiwell formats, e.g., 96 or 384 well format. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is presented as relative light units (RLU), measured over time.

Luciferase

ATP + Luciferin + O₂---------►- Oxyluciferin + AMP + PPi + CO₂ + light

Mg⁺²

The anti-proliferative effects of antibody drug conjugates were measured by the cell proliferation, in vitro cell killing assay above against four different breast tumor cell lines (Figures 7-10). IC50 values were established for SK-BR-3 and BT-474 which are known to over express HER2 receptor protein. Table 2a shows the potency (IC50) measurements of exemplary antibody drug conjugates in the cell proliferation assay against SK-BR-3 cells. Table 2b shows the potency (IC50) measurements of exemplary antibody drug conjugates in the cell proliferation assay against BT-474 cells.

Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab; Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; Trastuzumab-MC-MMAF, 4.1 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; and Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab did not inhibit the proliferation of MCF-7 cells (Figure 9).

Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; TrastuzumabMC-vc-PAB-MMAF, 3.7 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab; Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; and Trastuzumab-MC-MMAF, 4.1 MMAF/Ab did not inhibit the proliferation of MDA-MB-468 cells (Figure 10).

MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor protein. The anti-H]ER2 antibody drug conjugates of the invention therefore show selectivity for inhibition of cells which express HER2.

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Table 2a

SK-BR-3 cells

<td> Antibody Drug Conjugate H = trastuzumab linked via a cysteine [cys] except where noted</td><td> IC₅₀ (^g ADC/ml)</td>

<td> H-MC-MMAF, 4.1 MMAF/Ab</td><td> 0.008</td>

<td> H-MC-MMAF, 4.8 MMAF/Ab</td><td> 0.002</td>

<td> H-MC-vc-PAB-MMAE,</td><td> 0.007</td>

<td> H-MC-vc-PAB-MMAE</td><td> 0.015</td>

<td> H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab</td><td> 0.0035 - 0.01</td>

<td> H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab</td><td> 0.006 - 0.007</td>

<td> H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab</td><td> 0.006</td>

<td> H-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab</td><td> 0.0035</td>

<td> H-MC-MMAF, 4.1 MMAF/Ab</td><td> 0.0035</td>

<td> H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab</td><td> 0.010</td>

<td> H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab</td><td> 0.007</td>

<td> H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab</td><td> 0.015</td>

<td> H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.</td><td> 0.010</td>

<td> H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab</td><td> 0.0025</td>

<td> H-MC-MMAE, 8.8 MMAE/Ab</td><td> 0.018</td>

<td> H-MC- MMAE, 4.6 MMAE/Ab</td><td> 0.05</td>

<td> H-MC-(L)val-(L)cit-PAB-MMAE, 8.7 MMAE/Ab</td><td> 0.0003</td>

<td> H-MC-(D)val-(D)cit-PAB-MMAE, 8.2 MMAE/Ab</td><td> 0.02</td>

<td> H-MC-(D)val-(L)cit-PAB-MMAE, 8.4 MMAE/Ab</td><td> 0.0015</td>

<td> H-MC-(D)val-(L)cit-PAB-MMAE, 3.2 MMAE/Ab</td><td> 0.003</td>

<td> H-Trastuzumab</td><td> 0.083</td>

<td> H-vc-MMAE, linked via a lysine [lys]</td><td> 0.002</td>

<td> H-phe-lys-MMAE, linked via a lysine [lys]</td><td> 0.0015</td>

<td> 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab</td><td> 0.004 · - ·</td>

<td> Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab</td><td> 0.01</td>

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<td> 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab</td><td> 0.01</td>

<td> 4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab</td><td> 0.02</td>

<td> Anti-TF Fab-MC-vc-PAB-MMAE’</td><td> -</td>

Table 2b BT474 cells

<td> Antibody Drug Conjugate H = trastuzumab linked via a cysteine [cys]</td><td> IC50 (μ8 ADC/ml)</td>

<td> H-MC-MMAF, 4.1 MMAF/Ab</td><td> 0.008</td>

<td> H-MC-MMAF, 4.8 MMAF/Ab</td><td> 0.002</td>

<td> H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab</td><td> 0.015</td>

<td> H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab</td><td> 0.02 - 0.05</td>

<td> H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab</td><td> 0.01</td>

<td> H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab</td><td> 0.01</td>

<td> H-MC-vc-PAB-MMAE, 3.3 MMAE/Ab</td><td> 0.02</td>

<td> H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.</td><td> 0.02</td>

<td> H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab</td><td> 0.015</td>

<td> H-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab</td><td> 0.010</td>

<td> H-MC-MMAF, 4.1 MMAF/Ab</td><td> 0.00015</td>

<td> H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab</td><td> 0.0025</td>

<td> H-MC-MMAE, 8.8 MMAE/Ab</td><td> 0.04</td>

<td> H-MC- MMAE, 4.6 MMAE/Ab</td><td> 0.07</td>

<td> 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab</td><td> 0.008</td>

<td> Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab</td><td> 0.01</td>

<td> 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab</td><td> 0.015</td>

<td> 4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab</td><td> 0.04</td>

<td> Anti-TF Fab-MC-vc-PAB-MMAE’</td><td> -</td>

H = trastuzumab

7C2 = anti-HER2 murine antibody which binds a different epitope than trastuzumab.

. Fc8 = mutant that does not bind to FcRn

195

Hg = “Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore nonglycosylated.)

Anti-TF Fab = anti-tissue factor antibody fragment * activity against MDA-MB-468 cells

In a surprising and unexpected discovery, the in vitro cell proliferation activity results of the ADC in Tables 2a and 2b show generally that ADC with a low average number of drug moieties per antibody showed efficacy, e.g., IC50 &lt; 0.1 pg ADC/ml. The results suggest that at least for trastuzumab ADC, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5.

IN VIVO PLASMA CLEARANCE AND STABILITY

Pharmacokinetic plasma clearance and stability of ADC were investigated in rats and cynomolgus monkeys. Plasma concentration was measured over time. Table 2c shows pharmacokinetic data of antibody drug conjugates and other dosed samples in rats. Rats are a non-specific model for ErbB receptor antibodies, since the rat is not known to express HER2 receptor proteins.

Table 2c Pharmacokinetics in Rats

H = trastuzumab linked via a cysteine [cys] except where noted mg/kg dose except where noted

<td> Sample dose mg/kg</td><td> AUCinf day* pg/mL</td><td> CL mL/day/kg</td><td> Cmax pg/mL</td><td> T‘/2 Term, days</td><td> % Conj.</td>

<td> H-MC-vc-PAB-MMAE (Total Ab</td><td> 78.6</td><td> 26.3</td><td> 39.5</td><td> 5.80</td><td> 40.6</td>

<td> H-MC-vc-PAB-MMAE (Conj.)</td><td> 31.1</td><td> 64.4</td><td> 33.2</td><td> 3.00</td><td></td>

<td> H-MC-vc-PAB-MMAF (Total Ab)</td><td> 170</td><td> 12.0</td><td> 47.9</td><td> 8.4</td><td> 50.0</td>

<td> H-MC-vc-PAB-MMAF (Conj.)</td><td> 83.9</td><td> 24.0</td><td> 44.7</td><td> 4.01</td><td></td>

<td> H-MC-MMAE (Total Ab)</td><td> 279</td><td> 18.9</td><td> 79.6</td><td> 7.65</td><td> 33</td>

196

<td> H-MC-MMAE (Conj.) 5 mg/kg</td><td> 90.6</td><td> 62.9</td><td> 62.9</td><td> 4.46</td><td></td>

<td> H-MC-MMAF (Total Ab)</td><td> 299</td><td> 6.74</td><td> 49.1</td><td> 11.6</td><td> 37</td>

<td> H-MC-MMAF (Conj.)</td><td> 110</td><td> 18.26</td><td> 50.2</td><td> 4.54</td><td></td>

<td> H-MC-vc-MMAF, wo/PAB, (Total Ab)</td><td> 306</td><td> 6.6</td><td> 78.7</td><td> 11.9</td><td> 19.6</td>

<td> H-MC-vc-MMAF, wo/PAB, (Conj.)</td><td> 59.9</td><td> 33.4</td><td> 82.8</td><td> 2.1</td><td></td>

<td> H-Me-vc-PAB-MMAF (Total Ab)</td><td> 186</td><td> 10.8</td><td> 46.9</td><td> 8.3</td><td> 45.3</td>

<td> H-Me-vc-PAB-MMAF (Conj.)</td><td> 84.0</td><td> 23.8</td><td> 49.6</td><td> 4.3</td><td></td>

<td> H-Me-vc-PAB-MMAE (Total Ab)</td><td> 135</td><td> 15.0</td><td> 44.9</td><td> 11.2</td><td> 23.8</td>

<td> H-Me-vc-PAB-MMAE (Conj.)</td><td> 31.9</td><td> 63.8</td><td> 45.2</td><td> 3.0</td><td></td>

<td> H-MC-vc-MMAF, wo/PAB, (Total Ab)</td><td> 306</td><td> 6.6</td><td> 78.7</td><td> 11.9</td><td> 19.6</td>

<td> H-MC-vc-MMAF, wo/PAB, (Conj.)</td><td> 59.9</td><td> 33.4</td><td> 82.8</td><td> 2.1</td><td></td>

<td> H-MC-(D)val-(L)cit-PABMMAE (Total Ab)</td><td> 107</td><td> 19.2</td><td> 30.6</td><td> 9.6</td><td> 38.1</td>

<td> H-MC-(D)val-(L)cit-PAB- MMAE (Conj.)</td><td> 40</td><td> 50.4</td><td> 33.7</td><td> 3.98</td><td></td>

<td> H-MC-(Me)-vc-PAB-MMAE, Total Ab</td><td> 135.1</td><td> 15.0</td><td> 44.9</td><td> 11.2</td><td> 23.8</td>

<td> H-MC-(Me)-vc-PAB-MMAE, Conj.</td><td> 31.9</td><td> 63.8</td><td> 45.2</td><td> 2.96</td><td></td>

<td> H-MC-(D)val-(D)cit-PABMMAE, Total Ab</td><td> 88.2</td><td> 22.8</td><td> 33.8</td><td> 10.5</td><td> 38.3</td>

<td> H-MC-(D)val-(D)cit-PABMMAE, Conj.</td><td> 33.6</td><td> 59.8</td><td> 36.0</td><td> 4.43</td><td></td>

<td> H-MC-vc-PAB-MMAE, Total Ab</td><td> 78.6</td><td> 26.3</td><td> 39.5</td><td> 5.8</td><td> 40.6</td>

<td> H-MC-vc-PAB-MMAE, Conj. H linked to MC by lysine [lys]</td><td> 31.1</td><td> 64.4</td><td> 33.2</td><td> 3.00</td><td></td>

<td> MMAF 200gg/kg</td><td> 0.99</td><td> 204</td><td> 280</td><td> 0.224</td><td> -</td>

\9Ί

<td colspan="2"></td><td></td><td></td><td></td><td></td>

<td> MMAE 206 gg/kg</td><td> 3.71</td><td> 62.6</td><td> 649</td><td> 0.743</td><td></td>

<td> HER F(ab’)₂-MC-vc-MMAE, Total Ab</td><td> 9.3</td><td> 217</td><td> 34.4</td><td> 0.35</td><td> 95</td>

<td> HER F(ab’)₂-MC-vc-MMAE, Conj.</td><td> 8.8</td><td> 227</td><td> 36.9</td><td> 0.29</td><td></td>

<td> 4D5-H-Fab-MC-vc-MMAF, Total Ab</td><td> 43.8</td><td> 46.2</td><td> 38.5</td><td> 1.49</td><td> 68</td>

<td> 4D5-H-Fab-MC-vc-MMAF, Conj.</td><td> 29.9</td><td> 68.1</td><td> 34.1</td><td> 1.12</td><td></td>

<td> 4D5-H-Fab-MC-vc-MMAE, Total Ab</td><td> 71.5</td><td> 70.3</td><td> 108</td><td> 1.18</td><td> 59</td>

<td> 4D5-H-Fab-MC-vc-MMAE, Conj.</td><td> 42.2</td><td> 118.9</td><td> 114</td><td> 0.74</td><td></td>

<td> 4D5-H-Fab</td><td> 93.4</td><td> 53.9</td><td> 133</td><td> 1.08</td><td> -</td>

<td> H-MC-vc-PAB-MMAF, Total Ab</td><td> 170</td><td> 12.03</td><td> 47.9</td><td> 8.44</td><td> 49.5</td>

<td> H-MC-vc-PAB-MMAF, Conj.</td><td> 83.9</td><td> 23.96</td><td> 44.7</td><td> 4.01</td><td></td>

<td> H-MC-vc-PAB-MMAFDMAEA, Total Ab</td><td> 211</td><td> 9.8</td><td> 39.8</td><td> 8.53</td><td> 34.3</td>

<td> H-MC-vc-PAB-MMAFDMAEA, Conj.</td><td> 71.5</td><td> 28.2</td><td> 38.8</td><td> 3.64</td><td></td>

<td> H-MC-vc-PAB-MMAF-TEG, Total Ab</td><td> 209</td><td> 9.75</td><td> 53.2</td><td> 8.32</td><td> 29.7</td>

<td> H-MC-vc-PAB-MMAF-TEG, Conj.</td><td> 63.4</td><td> 31.8</td><td> 34.9</td><td> 4.36</td><td></td>

AUC inf is the area under the plasma concentration-time curve from time of dosing to infinity and is a measure of the total exposure to the measured entity (drug, ADC). CL is defined as the volume of plasma cleared of the measured entity in unit time and is expressed by normalizing to body weight. Tl/2 term is the half-life of the drug in the body measured during its elimination phase. The % Conj. term is the relative amount of ADC compared to total

198 antibody detected, by separate ELISA immunoaffinity tests (“Analytical Methods for Biotechnology Products”, Ferraiolo et al, p85-98 in Pharmacokinetics of Drugs (1994) P.G. Welling and L.P. Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110, Springer-Verlag. The % Conj. calculation is simply ALJCinf of ADC AUCinf total Ab, and is a general indicator of linker stability, although other factors and mechanisms may be in effect.

Figure 11 shows a graph of a plasma concentration clearance study after administration of the antibody drug conjugates: H-MC-vc-PAB-MMAFTEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats. Concentrations of total antibody and ADC were measured over time.

Figure 12 shows a graph of a two stage plasma concentration clearance study where ADC was administered at different dosages and concentrations of total antibody and ADC were measured over time.

IN VIVO EFFICACY

The in vivo efficacy of the ADC of the invention was measured by a high expressing HER2 transgenic explant mouse model. An allograft was propagated from the Fo5 mmtv transgenic mouse which does not respond to, or responds poorly to, HERCEPTIN® therapy. Subjects were treated once with ADC and monitored over 3-6 weeks to measure the time to tumor doubling, log cell kill, and tumor shrinkage. Follow up dose-response and multi-dose experiments were conducted.

Tumors arise readily in transgenic mice that express a mutationally activated form of neu, the rat homolog of HER2, but the HER2 that is overexpressed in breast cancers is not mutated and tumor formation is much less robust in transgenic mice that overexpress nonmutated HER2 (Webster et al. (1994) Semin. Cancer Biol. 5:69-76).

To improve tumor formation with nonmutated HER2, transgenic mice were produced using a HER2 cDNA plasmid in which an upstream ATG was deleted in order to prevent initiation of translation at such upstream ATG codons, which would otherwise reduce the frequency of translation initiation from

199 the downstream authentic initiation codon of HER2 (for example, see Child et al. (1999) J. Biol. Chem. 274: 24335-24341). Additionally, a chimeric intron was added to the 5’ end, which should also enhance the level of expression as reported earlier (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 was derived from a Promega vector, pCIneo mammalian expression vector (bp 890-1022). The cDNA 3’-end is flanked by human growth hormone exons 4 and 5, and polyadenylation sequences. Moreover, FVB mice were used because this strain is more susceptible to tumor development. The promoter from MMTV-LTR was used to ensure tissue-specific HER2 expression in the mammary gland. Animals were fed the AIN 76A diet in order to increase susceptibility to tumor formation (Rao et al. (1997) Breast Cancer Res. and Treatment 45:149-158).

Table 2d Tumor measurements in allograft mouse model - MMTV-HER2 Fo5 Mammary Tumor, athymic nude mice single dose at day 1 (T = 0) except where noted H = trastuzumab linked via a cysteine [cys] except where noted

<td> Sample Drugs per antibody</td><td> Dose</td><td> Ti</td><td> PR</td><td> CR</td><td> Tumor doubling time (days)</td><td> Mean log cell kill</td>

<td> Vehicle</td><td></td><td></td><td></td><td></td><td> 2-5</td><td> 0</td>

<td> H-MC-vc-PAB-MMAE 8.7 MMAE/Ab</td><td> 1250 gg/m²</td><td> 5/5</td><td> 4/7</td><td> 0/7</td><td> 18</td><td> 1.5</td>

<td> H-MC-vc-PAB-MMAF 3.8 MMAF/Ab</td><td> 555 gg/m²</td><td> 2/5</td><td> 2/7</td><td> 5/7</td><td> 69</td><td> 6.6</td>

<td> H-MC(Me)-vc-PABMMAF</td><td></td><td></td><td></td><td></td><td> &gt;50</td><td> 6.4</td>

<td> H-MC-MMAF 4.8 MMAF/Ab</td><td> 9.2 mg/kg Ab 550 gg/m²atO, 7, 14 and 21 days</td><td> 7/7</td><td> 6/7</td><td> 0/7</td><td> 63</td><td> 9</td>

<td> H-MC-MMAF 4.8 MMAF/Ab</td><td> 14 mg/kg Ab 840 gg/m²at 0, 7, 14 and 21 days</td><td> 5/5</td><td> 5/7</td><td> 2/7</td><td> &gt;63</td><td></td>

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<td> H-MC-vc-PAB-MMAF 5.9 MMAF/Ab</td><td> 3.5 mg/kg Ab 300 gg/m²atO, 21, and 42 days</td><td> 5/6</td><td> 1/7</td><td> 3/7</td><td> &gt;36</td><td></td>

<td> H-MC-vc-PAB-MMAF 5.9 MMAF/Ab</td><td> 4.9 mg/kg Ab 425 gg/m²atO, 21, and 42 days</td><td> 4/7</td><td> 2/7</td><td> 5/7</td><td> &gt;90</td><td></td>

<td> H-MC-vc-PAB-MMAF 5.9 MMAF/Ab</td><td> 6.4 mg/kg Ab 550 gg/m²at 0, 21, and 42 days</td><td> 3/6</td><td> 1/7</td><td> 6/7</td><td> &gt;90</td><td></td>

<td> H-(L)val-(L)cit-MMAE 8.7 MMAE/Ab</td><td> 10 mg/kg</td><td> in</td><td> 1/7</td><td> 0/7</td><td> 15.2</td><td> 1.1</td>

<td> H-MC-MMAE 4.6 MMAE/Ab</td><td> 10 mg/kg</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 4</td><td> 0.1</td>

<td> H-(D)val-(D)citMMAE 4.2 MMAE/Ab</td><td> 10 mg/kg</td><td> in</td><td> 0/7</td><td> 0/7</td><td> 3</td><td></td>

<td> H-(D)val-(L)cit-MMAE 3.2 MMAE/Ab</td><td> 13 mg/kg</td><td> HI</td><td> 0/7</td><td> 0/7</td><td> 9</td><td> 0.6</td>

<td> H-MC(Me)-vc-MMAE 3.0 MMAE/Ab</td><td> 13 mg/kg</td><td> Hl</td><td> 3/7</td><td> 0/7</td><td> 17</td><td> 1.2</td>

<td> H-(L)val-(D)cit-MMAE 3.5 MMAE/Ab</td><td> 12 mg/kg</td><td> Hl</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0.2</td>

<td> H-vc-MMAE 8.7 MMAE/Ab</td><td> 10 mg/kg</td><td> Hl</td><td></td><td></td><td> 17</td><td></td>

<td> H-cys-vc-MMAF 3.8 MMAF/Ab</td><td> 1 mg/kg</td><td> Hl</td><td></td><td></td><td> 3</td><td></td>

<td> H-cys-vc-MMAF 3.8 MMAF/Ab</td><td> 3 mg/kg</td><td> Hl</td><td></td><td></td><td> &gt;17</td><td></td>

<td> H-cys-vc-MMAF 3.8 MMAF/Ab</td><td> 10 mg/kg</td><td> 4/7</td><td> 4/7</td><td> 3/7</td><td> &gt;17</td><td></td>

<td> H-MC-vc-MMAF-TEG 4 MMAF/Ab</td><td> 10 mg/kg</td><td> 3/6</td><td> 1/7</td><td> 6/7</td><td> 81</td><td> 7.8</td>

<td> H-MC-vc-MMAF-TEG 4 MMAF/Ab</td><td> 10 mg/kg q3wk x 3</td><td> 0/5</td><td> 0/7</td><td> 7/7</td><td> 81</td><td> 7.9</td>

<td> H-vc-MMAF (lot 1)</td><td> 10 mg/kg</td><td> 4/6</td><td> 2/8</td><td> 5/8</td><td></td><td></td>

<td> H-vc-MMAF (lot 2)</td><td> 10 mg/kg</td><td> 7/8</td><td> 1/8</td><td> 1/8</td><td></td><td></td>

<td> H-MC-MMAF</td><td> 10 mg/kg 550 gg/m²</td><td> 8/8</td><td> 1/8</td><td> 0/8</td><td> 18</td><td></td>

<td> H-(Me)-vc-MMAF</td><td> 10 mg/kg</td><td> 3/7</td><td> 2/8</td><td> 5/8</td><td></td><td></td>

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<td> H-vc-MMAE 7.5 MMAE/Ab</td><td> 3.7 mg/kg at 0, 7, 14, 21, 28 days</td><td> 6/6</td><td> 0/7</td><td> 1/7</td><td> 17</td><td> 2.3</td>

<td> H-vc-MMAE 7.5 MMAE/Ab</td><td> 7.5 mg/kg at 0, 7, 14, 21, 28 days</td><td> 5/7</td><td> 3/7</td><td> 3/7</td><td> 69</td><td> 10</td>

<td> anti IL8-vc-MMAE 7.5 MMAE/Ab</td><td> 7.5 mg/kg at 0, 7, 14, 21, 28 days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0.5</td>

<td> anti IL8-VC-MMAE 7.5 MMAE/Ab</td><td> 3.7 mg/kg at 0, 7, 14, 21, 28 days</td><td> 6/6</td><td> 0/7</td><td> 0/7</td><td> 3</td><td> 0.2</td>

<td> H-fk-MMAE 7.5 MMAE/Ab</td><td> 7.5 mg/kg at 0, 7, 14, 21, 28 days</td><td> ΊΠ</td><td> 1/7</td><td> 0/7</td><td> 31</td><td> 4.4</td>

<td> H-fk-MMAE 7.5 MMAE/Ab</td><td> 3.7 mg/kg at 0, 7, 14, 21, 28 days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 8.3</td><td> 0.9</td>

<td> anti DL8-fk-MMAE 7.5 MMAE/Ab</td><td> 7.5 mg/kg at 0, 7, 14, 21, 28 days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 6</td><td> 0.5</td>

<td> anti IL8-fk-MMAE 7.5 MMAE/Ab</td><td> 3.7 mg/kg at 0, 7, 14, 21, 28 days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 3</td><td> 0.1</td>

<td> Trastuzumab</td><td> 7.5 mg/kg at 0, 7, 14, 21, 28 days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0.4</td>

<td> H-vc-MMAE 8.7 MMAE/Ab</td><td> 10 mg/kg 1250 gg/m²</td><td> 6/6</td><td> 3/6</td><td> 0/6</td><td> 15</td><td> 1.3</td>

<td> H-vc-MMAE</td><td> 10 mg/kg 1250 gg/m²at 0, 7, and 14 days</td><td> ΊΠ</td><td> 5/7</td><td></td><td> &gt;19</td><td></td>

<td> H-vc-MMAE</td><td> 3 mg/kg at 0, 7, and 14 days</td><td> 7/7</td><td></td><td></td><td> 8</td><td></td>

<td> H-vc-MMAE</td><td> 1 mg/kg at 0, 7, and 14 days</td><td> 7/7</td><td></td><td></td><td> 7</td><td></td>

<td> H-vc-MMAF</td><td> 10 mg/kg</td><td> 8/8</td><td> 5/8</td><td></td><td> &gt;21</td><td></td>

<td> H-vc-MMAF</td><td> 10 mg/kg at 0, 7, and 14 days</td><td> 4/7</td><td> 4/7</td><td> 3/7</td><td> &gt;21</td><td></td>

<td> H-vc-MMAF</td><td> 3 mg/kg at 0, 7, and 14</td><td> 7/7</td><td></td><td></td><td> 6 ‘</td><td></td>

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<td></td><td> days</td><td></td><td></td><td></td><td></td><td></td>

<td> H-vc-MMAF</td><td> 1 mg/kg at 0, 7, and 14 days</td><td> 8/8</td><td></td><td></td><td> 4</td><td></td>

<td> Trastuzumab .</td><td> 10 mg/kg at 0 and 7 days</td><td> 8/8</td><td></td><td></td><td> 3</td><td></td>

<td> Hg-MC-vc-PABMMAF 4.1 MMAF/Ab</td><td> 10 mg/kg at 0 days</td><td> 6/7</td><td> 3/8</td><td> 5/8</td><td> 56</td><td> 5.1</td>

<td> Fc8-MC-vc-PABMMAF 4.4 MMAF/Ab</td><td> 10 mg/kg at Odays</td><td> 7/7</td><td> 6/8</td><td> 0/8</td><td> 25</td><td> 2.1</td>

<td> 7C2-MC-vc-PABMMAF 4 MMAF/Ab</td><td> 10 mg/kg at 0 days</td><td> 5/6</td><td> 6/8</td><td> 1/8</td><td> 41</td><td> 3.7</td>

<td> H-MC-vc-PAB-MMAF 5.9 MMAF/Ab</td><td> 10 mg/kg at 0 days</td><td> 3/8</td><td> 3/8</td><td> 5/8</td><td> 62</td><td> 5.7</td>

<td> 2H9-MC-vc-PABMMAE</td><td></td><td> 9/9</td><td></td><td></td><td> &gt;14 days</td><td></td>

<td> 2H9-MC-vc-PABMMAF</td><td></td><td> 9/9</td><td></td><td></td><td> &gt;14 days</td><td></td>

<td> llDlO-vc-PAB-MMAE</td><td></td><td> 9/9</td><td></td><td></td><td> &gt;14 days</td><td></td>

<td> 1 lDlO-vc-PAB-MMAF</td><td></td><td> 9/9</td><td></td><td></td><td> 11 days</td><td></td>

7C2 = anti-HER2 murine antibody which binds a different epitope than trastuzumab.

Fc8 = mutant that does not bind to FcRn

Hg = “Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore nonglycosylated.)

2H9 = Anti-EphB2R

11D1O = Anti-0772P

The term Ti is the number of animals in the study group with tumor at T = 0 -s- total animals in group. The term PR is the number of animals attaining partial remission of tumor 4- animals with tumor at T = 0 in group. The term CR is the number of animals attaining complete remission of tumor 4- animals with tumor at T = 0 in group. The term Log cell kill is the time in days for the tumor volume to double - the time in days for the control tumor volume to double divided by 3.32 X time for tumor volume to double in control animals (dosed with Vehicle). The log-cell-kill calculation takes into account tumor growth delay

203 resulting from treatment and tumor volume doubling time of the control group. Anti-tumor activity of ADC is classified with log-cell-kill values of:

++++ &gt;3.4 (highly active) +++ = 2.5-3.4 ++ = 1.7-2.4 + = 1.0-1.6 inactive = 0

Figure 13 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 gg/m²) and Trastuzumab-MC-vc-PAB-MMAF (555 gg/m²). (H = Trastuzumab). The growth of tumors was retarded by treatment with ADC as compared to control (Vehicle) level of growth. Figure 14 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660 gg/m²) of Trastuzumab-MC-MMAE and 1250 gg/m²Trastuzumab-MC-vc-PAB-MMAE. Figure 15 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed with 650 gg/m² Trastuzumab-MC-MMAF. Table 2d and Figures

13-15 show that the ADC have strong anti-tumor activity in the allograft of a HER2 positive tumor (Fo5) that originally arose in an MMTV-HER2 transgenic mouse. The antibody alone (e.g., Trastuzumab) does not have significant antitumor activity in this model (Erickson et al. U.S. Patent No. 6632979). As illustrated in Figures 13-15, the growth of the tumors was retarded by treatment with ADC as compared to control (Vehicle) level of growth.

In a surprising and unexpected discovery, the in vivo anti-tumor activity results of the ADC in Table 2d show generally that ADC with a low average number of drug moieties per antibody showed efficacy, e.g., tumor doubling time &gt; 15 days and mean log cell kill &gt; 1.0. Figure 16 shows that for the antibody drug conjugate, trastuzumab-MC-vc-PAB-MMAF, the mean tumor volume diminished and did not progress where the MMAF: trastuzumab ratio was 2 and 4, whereas tumor progressed at a ratio of 5.9 and 6, but at a rate lower than

204

Vehicle (buffer). The rate of tumor progression in this mouse xenograft model was about the same, i.e. 3 days, for Vehicle and trastuzumab. The results suggest that at least for trastuzumab ADC, the optimal ratio of drug moieties per antibody may be less than about 8, and may be about 2 to about 4.

4.5.5 RODENT TOXICITY

Antibody drug conjugates and an ADC-minus control, “Vehicle”, were evaluated in an acute toxicity rat model. Toxicity of ADC was investigated by treatment of male and female Sprague-Dawley rats with the ADC and subsequent inspection and analysis of the effects on various organs. Gross observations included changes in body weights and signs of lesions and bleeding. Clinical pathology parameters (serum chemistry and hematology), histopathology, and necropsy were conducted on dosed animals.

It is considered that weight loss, or weight change relative to animals dosed only with Vehicle, in animals after dosing with ADC is a gross and general indicator of systemic or localized toxicity. Figures 17-19 show the effects of various ADC and control (Vehicle) after dosing on rat body weight.

Hepatotoxicity was measured by elevated liver enzymes, increased numbers of mitotic and apoptotic figures and hepatocyte necrosis. Hematolymphoid toxicity was observed by depletion of leukocytes, primarily granuloctyes (neutrophils), and/or platelets, and lymphoid organ involvement, i.e. atrophy or apoptotic activity. Toxicity was also noted by gastrointestinal tract lesions such as increased numbers of mitotic and apoptotic figures and degenerative enterocolitis.

Enzymes indicative of liver injury that were studied include:

AST (aspartate aminotransferase)

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

-Liver:Plasma ratio of 7000:1

-Tl/2: 17 hrs

ALT (alanine aminotransferase)

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

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-Liver:Plasma ratio of 3000:1

-Tl/2:42 hrs; diurnal variation

GGT (g-glutamyl transferase)

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

-Poor predictor of liver injury; commonly elevated in bile duct disorders

The toxicity profiles of trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF were studied in female Sprague-Dawley rats (Example 19). The humanized trastuzumab antibody does not bind appreciably to rat tissue, and any toxicity would be considered non-specific. Variants at dose levels of 840 and 2105 ug/m² MMAF were compared to trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug/m².

Animals in groups 1, 2, 3,4, 6, and 7 (Vehicle, 9.94 &amp; 24.90 mg/kg trastuzumab-MC-val-cit-MMAF, 10.69 mg/kg trastuzumab-MC(Me)-valcit-PAB-MMAF, and 10.17 &amp; 25.50 mg/kg trastuzumab-MC-MMAF, respectively) gained weight during the study. Animals in groups 5 and 8 (26.78 mg/kg trastuzumab-MC(Me)-val-cit-PAB-MMAF and 21.85 mg/kg trastuzumabMC-val-cit-PAB-MMAF, respectively) lost weight during the study. On Study Day 5, the change in body weights of animals in groups 2, 6 and 7 were not significantly different from group 1 animals. The change in body weights of animals in groups 3, 4, 5 and 8 were statistically different from group 1 animals (Example 19).

Rats treated with trastuzumab-MC-MMAF (groups 6 and 7) were indistinguishable from vehicle-treated control animals at both dose levels; i.e. this conjugate showed a superior safety profile in this model. Rats treated with trastuzumab-MC-val-cit-MMAF (without the self-immolative PAB moiety; groups 2 and 3) showed dose-dependent changes typical for MMAF conjugates; the extent of the changes was less compared with a full length MC-val-cit-PABMMAF conjugate (group 8). The platelet counts on day 5 were at approximately

206

30% of baseline values in animals of group 3 (high dose trastuzumab-MC-val-citMMAF) compared with 15% in animals of group 8 (high dose trastuzumab-MCval-cit-PAB-MMAF). Elevation of liver enzymes AST and ALT, of bilirubin and the extent of thrombocytopenia was most evident in animals treated with trastuzumab-MC(Me)-val-cit-PAB-MMAF (groups 4 and 5) in a dose-dependent fashion; animals of group 5 (high dose group) showed on day 5 levels of ALT of approximately lOx the baseline value and platelets were reduced by approximately 90% at the time of necropsy.

Female Sprague Dawley Rats were also dosed at high levels (Example 19, High Dose study: Groups 2, 3,4) with trastuzumab-MC-MMAF, and Vehicle control (Group 1). Mild toxicity signals were observed, including a dose-dependent elevation of liver enzymes ALT, AST and GGT. On day 5 animals in the highest dose group showed a 2-fold elevation of ALT and a 5-fold elevation of AST; GGT is also elevated (6U/L). Enzyme levels show a trend towards normalization on day 12. There was a mild granulocytosis in all three dose groups on day 5, the platelet count remained essentially unchanged in all animals. Morphological changes were mild; animals treated at the 4210gg/m²dose level (Group 2) showed unremarkable histology of liver, spleen, thymus, intestines and bone marrow. Mildly increased apoptotic and mitotic activity was observed in thymus and liver, respectively in animals treated at the 5500gg/m²dose level (Group 3). The bone marrow was normocellular, but showed evidence of granulocytic hyperplasia, which is consistent with the absolute granulocytosis observed in the peripheral blood counts in these animals. Animals at the highest dose in group 4 showed qualitatively the same features; the mitotic activity in the liver appears somewhat increased compared to animals in Group 3. Also, extramedullary hematopoiesis was seen in spleen and liver.

EphB2R is a type 1 TM tyrosine kinase receptor with close homology between mouse and human, and is over-expressed in colorectal cancer cells. 2H9 is an antibody against EphB2R. The naked antibody has no effect on tumor growth, but 2H9-val-cit-MMAE killed EphB2R expressing cells and showed efficacy in a mouse xenograft model using CXF1103 human colon tumors

207 (Mao etal (2004) Cancer Res. 64:781-788). 2H9 and 7C2 are both mouse IgGl anti-HER2 antibodies. The toxicity profiles of 2H9-MC-val-cit-PAB-MMAF (3.7 MMAF/Ab), 7C2-MC-val-cit-PAB-MMAF (4 MMAF/Ab), and trastuzumab-MCval-cit-PAB-MMAF (5.9 MMAF/Ab) were compared. The differences in the structure of each immunoconjugate or the drug portion of the immunoconjugate may affect the pharmacokinetics and ultimately the safety profile. The humanized trastuzumab antibody does not bind appreciably to rat tissue, and any toxicity would be considered non-specific.

CYNOMOLGUS MONKEY TOXICITY/SAFETY

Similar to the rat toxicity/safety study, cynomolgus monkeys were treated with ADC followed by liver enzyme measurements, and inspection and analysis of the effects on various organs. Gross observations included changes in body weights and signs of lesions and bleeding. Clinical pathology parameters (serum chemistry and hematology), histopathology, and necropsy were conducted on dosed animals (Example 19).

The antibody drug conjugate, H-MC-vc-PAB-MMAE (H = trastuzumab linked through cysteine) showed no evidence of liver toxicity at any of the dose levels tested. Peripheral blood granulocytes showed depletion after a single dose of 1100mg/m2 with complete recovery 14 days post-dose. The antibody drug conjugate H-MC-vc-PAB-MMAF showed elevation of liver enzymes at 550 (transient) and 880 mg/m2 dose level, no evidence of granulocytopenia, and a dose-dependent, transient (groups 2 &amp; 3) decline of platelets.

SYNTHESIS OF THE COMPOUNDS OF THE INVENTION

The Exemplary Compounds and Exemplary Conjugates can be made using the synthetic procedures outlined below in Schemes 5-16. As described in more detail below, the Exemplary Compounds or Exemplary Conjugates can be conveniently prepared using a Linker having a reactive site for binding to the Drug and Ligand. In one aspect, a Linker has a reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a

208

Ligand, such as but not limited to an antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody attachment.

In another embodiment, a Linker has a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.

Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for a Linker because they can react with secondary amino groups of a Drug to form an amide linkage. Also useful as a reactive site is a carbonate functional group on a Linker, such as but not limited to p-nitrophenyl carbonate, which can react with an amino group of a Drug, such as but not limited to N-methyl valine, to form a carbamate linkage. Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and

K. Liibke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.

The synthesis of an illustrative Stretcher having an electrophilic maleimide group is illustrated below in Schemes 8-9. General synthetic methods useful for the synthesis of a Linker are described in Scheme 10. Scheme 11 shows the construction of a Linker unit having a val-cit group, an electrophilic

209 maleimide group and a PAB self-immolative Spacer group. Scheme 12 depicts the synthesis of a Linker having a phe-lys group, an electrophilic maleimide group, with and without the PAB self-immolative Spacer group. Scheme 13 presents a general outline for the synthesis of a Drug-Linker Compound, while Scheme 14 presents an alternate route for preparing a Drug-Linker Compound. Scheme 15 depicts the synthesis of a branched linker containing a BHMS group. Scheme 16 outlines the attachment of an antibody to a Drug-Linker Compound to form a Drug-Linker-Antibody Conjugate, and Scheme 14 illustrates the synthesis of Drug-Linker-Antibody Conjugates having, for example but not limited to, 2 or 4 drugs per Antibody.

As described in more detail below, the Exemplary Conjugates are conveniently prepared using a Linker having two or more Reactive Sites for binding to the Drug and a Ligand. In one aspect, a Linker has a Reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a Ligand, such as an antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody attachment.

In another embodiment, a Linker has a Reactive site which has a nucleophilic group that is reactive to an electrophilic group present on a Ligand, such as an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker. .......

210

4.6.1 DRUG MOIETY SYNTHESIS

Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Liibke, “The Peptides”, volume 1, pp 76-136,1965, Academic Press) that is well known in the field of peptide chemistry.

The auristatin/dolastatin drug moieties may be prepared according to the general methods of: U.S. Patent No. 5635483; U.S. Patent No. 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) AntiCancer Drug Design 13:243-277; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863.

In one embodiment, a Drug is prepared by combining about a stoichiometric equivalent of a dipeptide and a tripeptide, preferably in a one-pot reaction under suitable condensation conditions. This approach is illustrated in Schemes 5-7, below.

Scheme 5 illustrates the synthesis of an N-terminal tripeptide unit F which is a useful intermediate for the synthesis of the drug compounds of Formula lb.

211

Scheme 5

<img file="IN520DEN2012A_D0074.tif" />

r₃

PG^OR r₂ o bb

When PG = Z:

H₂,10% Pd/C, EtOH

<img file="IN520DEN2012A_D0075.tif" />

DEPC, Et₃N

<img file="IN520DEN2012A_D0076.tif" />

As illustrated in Scheme 5, a protected amino acid A (where PG represents an amine protecting group, R⁴ is selected from hydrogen, Ci-Cg alkyl, C3-Cg carbocycle, -O-(Ci-Cg alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3C8 heterocycle, alkyl-(C3-Cg heterocycle) wherein R⁵ is selected from H and methyl; or R⁴ and R⁵ join, have the formula -(CR^aR^b)n- wherein R^a and R^b are independently selected from hydrogen, Ci-C8 alkyl and C3-Cg carbocycle and n is selected from 2, 3,4, 5 and 6, and form a ring with the carbon atom to which they are attached) is coupled to ί-butyl ester B (where R⁶ is selected from -H and -CiC8 alkyl; and R⁷ is selected from hydrogen, Ci-Cg alkyl, C₃-Cg carbocycle, -O(Ci-Cg alkyl), -aryl, alkyl-aryl, aIkyl-(C₃-Cg carbocycle), C₃-Cg heterocycle and alkyl-(C₃-Cg heterocycle)) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (see, for example, Miyazaki,

K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).

Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley &amp; Sons. Exemplary protected amino acids A are

212

PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PGaminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PGphenylalanine, PG-phenylglycine, and PG-terFbutylglycine. Z is an exemplary protecting group. Fmoc is another exemplary protecting group. An exemplary tbutyl ester B is dolaisoleuine Fbutyl ester.

The dipeptide C can be purified, e.g., using chromatography, and subsequently deprotected, e.g., using H₂ and 10% Pd-C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine D readily forms a peptide bond with an amino acid BB (wherein R¹ is selected from -H, -Ci-Cs alkyl and -C3-C8 carbocycle; and R² is selected from -H and -Ci-Cs alkyl; or R¹ and R² join, have the formula -(CR^aR^b)nwherein R^a and R^b are independently selected from -H, -Ci-Cs alkyl and -C3-C8 carbocycle and n is selected from 2, 3,4, 5 and 6, and form a ring with the nitrogen atom to which they are attached; and R³ is selected from hydrogen, Ci-Cs alkyl, C3-C8 carbocycle, -O-(Ci-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C₈carbocycle), C3-C8 heterocycle and alkyl-(C3-Cs heterocycle)). N, V-Dialkyl amino acids are exemplary amino acids for BB, such as commercially available N,N-dimethyl valine. Other Α,/V-dialkyl amino acids can be prepared by reductive bis -alkylation using known procedures (see, e.g., Bowman, R.E, Stroud, H.H J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two exemplary amino acids BB useful for the synthesis ofW-monoalkyl derivatives. The amine D and the amino acid BB react to provide the tripeptide E using coupling reagent DEPC with triethylamine as the base. The C-terminus protecting group of E is subsequently deprotected using HC1 to provide the tripeptide compound of formula F.

Illustrative DEPC coupling methodology and the PyBrop coupling methodology shown in Scheme 5 are outlined below in General Procedure A and General Procedure B, respectively. Illustrative methodology for the deprotection of a Z-protected amine via catalytic hydrogenation is outlined below in General Procedure C.

213

General Procedure A: Peptide synthesis using DEPC. The Nprotected or N, N-disubstituted amino acid or peptide D (1.0 eq.) and an amine BB (1.1 eq.) are diluted with an aprotic organic solvent, such as dichloromethane (0.1 to 0.5 M). An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by DEPC (1.1 eq.). The resulting solution is stirred, preferably under argon, for up to 12 hours while being monitored by HPLC or TLC. The solvent is removed in vacuo at room temperature, and the crude product is purified using, for example, HPLC or flash column chromatography (silica gel column). Relevant fractions are combined and concentrated in vacuo to afford tripeptide E which is dried under vacuum overnight.

General procedure B: Peptide synthesis using PyBrop. The amino acid B (1.0 eq.), optionally having a carboxyl protecting group, is diluted with an aprotic organic solvent such as dichloromethane or DME to provide a solution of a concentration between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, or Z-protected amino acid A (1.1 eq.) is added as a solid in one portion, then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction is monitored by TLC or HPLC, followed by a workup procedure similar to that described in General Procedure A.

General procedure C: Z-removal via catalytic hydrogenation. Z-protected amino acid or peptide C is diluted with ethanol to provide a solution of a concentration between 0.5 and 1.0 mM in a suitable vessel, such as a thickwalled round bottom flask. 10% palladium on carbon is added (5-10% w/w) and the reaction mixture is placed under a hydrogen atmosphere. Reaction progress is monitored using HPLC and is generally complete within 1-2 h. The reaction mixture is filtered through a pre-washed pad of celite and the celite is again washed with a polar organic solvent, such as methanol after filtration. The eluent solution is concentrated in vacuo to afford a residue which is diluted with an organic solvent, preferably toluene. The organic solvent is then removed in vacuo to afford the deprotected amine C. ·

214

Scheme 6 shows a method useful for making a C-terminal dipeptide of formula K and a method for coupling the dipeptide of formula K with the tripeptide of formula F to make drug compounds of Formula lb.

Scheme 6

<img file="IN520DEN2012A_D0077.tif" />

HCl/EtOAc

<img file="IN520DEN2012A_D0078.tif" />

<img file="IN520DEN2012A_D0079.tif" />

^R3 h ? ^R?

R₂ O ^R4 Rj R6 Rg O

F

DEPC, Et₃N

<img file="IN520DEN2012A_D0080.tif" />

When PG = Fmoc:

DEA/CH₂C1₂

<img file="IN520DEN2012A_D0081.tif" />

The dipeptide K can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, G.R., et al. Synthesis, 1996, 719-725), with an amine of formula H using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Scheme 5.

The dipeptide of formula K can then be coupled with a tripeptide of formula F using General Procedure D to make the Fmoc-protected drug

215 compounds of formula L which can be subsequently deprotected using General Procedure E in order to provide the drug compounds of formula (lb).

General procedure D: Drug synthesis. A mixture of dipeptide

K (1.0 eq.) and tripeptide F (1 eq.) is diluted with an aprotic organic solvent, such as dichloromethane, to form a 0.1M solution, then a strong acid, such as trifluoroacetic acid (1/2 v/v) is added and the resulting mixture is stirred under a nitrogen atmosphere for two hours at 0°C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is azeotropically dried twice, preferably using toluene. The resulting residue is dried under high vacuum for 12 h and then diluted with and aprotic organic solvent, such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical functionality on the residue. The reaction mixture is monitored by either TLC or HPLC and upon completion, the reaction is subjected to a workup procedure similar or identical to that described in General Procedure A.

General procedure E: Fmoc-removal using diethylamine. An Fmoc-protected Drug L is diluted with an aprotic organic solvent such as dichloromethane and to the resulting solution is added diethylamine (½ v/v). Reaction progress is monitored by TLC or HPLC and is typically complete within 2 h. The reaction mixture is concentrated in vacuo and the resulting residue is azeotropically dried, preferably using toluene, then dried under high vacuum to afford Drug lb having a deprotected amino group.

Scheme 7 shows a method useful for making MMAF derivatives of Formula (lb).

216

Scheme 7

<img file="IN520DEN2012A_D0082.tif" />

<img file="IN520DEN2012A_D0083.tif" />

HCl/dioxane

<img file="IN520DEN2012A_D0084.tif" />

<img file="IN520DEN2012A_D0085.tif" />

The dipeptide O can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, G.R., et al. Synthesis, 1996, 719-725), with a protected amino acid of formula M using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Schemes 5 and 6.

The dipeptide of formula O can then be coupled with a tripeptide of formula F using General Procedure D to make the Fmoc-protected MMAF

217 compounds of formula P which can be subsequently deprotected using General Procedure E in order to provide the MMAF drug compounds of formula (lb).

Thus, the above methods are useful for making Drugs that can be used in the present invention.

4.6.2 DRUG LINKER SYNTHESIS

To prepare a Drug-Linker Compound of the present invention, the

Drug is reacted with a reactive site on the Linker. In general, the Linker can have the structure:

Reactive Site 2 —Aa-W_w—Y_y-— Reactive Site 1 when both a Spacer unit (-Y-) and a Stretcher unit (-A-) are present. Alternately, the Linker can have the structure:

Reactive Site 2 —A_a- W_w

Reactive Site 1 when the Spacer unit (-Y-) is absent.

The Linker can also have the structure:

Reactive Site 2 —W_wReactive Site 1 when both the Stretcher unit (-A-) and the Spacer unit (-Y-) are absent.

The Linker can also have the structure:

Reactive Site 2 ---Aa — Reactive Site 1 when both the Amino Acid unit (W) and the Spacer Unit (Y) are absent.

In general, a suitable Linker has an Amino Acid unit linked to an optional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 is present at the terminus of the Spacer and Reactive site 2 is present at the terminus of the

218

Stretcher. If a Spacer unit is not present, then Reactive site 1 is present at the Cterminus of the Amino Acid unit.

In an exemplary embodiment of the invention, Reactive Site No. 1 is reactive to a nitrogen atom of the Drug, and Reactive Site No. 2 is reactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactive to different functional groups.

In one aspect of the invention, Reactive Site No. 1 is

-COOH.

In another aspect of the invention, Reactive Site No. 1 is

<img file="IN520DEN2012A_D0086.tif" />

In still another aspect of the invention, Reactive Site No. 1 is a pnitrophenyl carbonate having the formula

<img file="IN520DEN2012A_D0087.tif" />

In one aspect of the invention, Reactive Site No. 2 is a thiolaccepting group. Suitable thiol-accepting groups include haloacetamide groups having the formula

<img file="IN520DEN2012A_D0088.tif" />

wherein X represents a leaving group, preferably O-mesyl, Otosyl, -Cl, -Br, or -I; or a maleimide group having the formula

219

<img file="IN520DEN2012A_D0089.tif" />

Useful Linkers can be obtained via commercial sources, such as

Molecular Biosciences Inc.(Boulder, CO), or prepared as summarized in Schemes

8-10 below.

<img file="IN520DEN2012A_D0090.tif" />

<img file="IN520DEN2012A_D0091.tif" />

<img file="IN520DEN2012A_D0092.tif" />

wherein X is -CH₂- or -CH₂OCH₂-; and n is an integer ranging either from 0-10 when X is -CH₂-; or 1-10 when X is -CH₂OCH₂-.

The method shown in Scheme 9 combines maleimide with a glycol under Mitsunobu conditions to make a polyethylene glycol maleimide Stretcher

220 (see for example, Walker, M.A. J. Org. Chem. 1995, 60,5352-5), followed by installation of a p-nitrophenyl carbonate Reactive Site group.

Scheme 9

<img file="IN520DEN2012A_D0093.tif" />

THF

<img file="IN520DEN2012A_D0094.tif" />

wherein E is -CH2- or -CH2OCH2-; and e is an integer ranging from 0-8;

Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can be prepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7, 180-186.

Scheme 10 illustrates a general synthesis of an illustrative Linker unit containing a maleimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.

221

Scheme 10

<img file="IN520DEN2012A_D0095.tif" />

1. NaHCO₃, DME/H₂O ___________________ Fmoc

2. EEDQ, h₂n-Q-^_0H

<img file="IN520DEN2012A_D0096.tif" />

1. diethylamine, CH₂CI₂

2. R, DIEA, CH₂CI₂

3. bis(4nitrophenyl)carbonate

DIEA, CH₂CI₂

1. NaHCO₃, dme/h₂o

2. diethylamine, CH₂CI₂

3. compond R, DMF

<img file="IN520DEN2012A_D0097.tif" />

OMe

O Ri 0

R¹=benzyl; R²=(CH2)4NHMtr (U) R¹=isopropyl; R²=(CH2)₃NHCONH₂ (V)

<img file="IN520DEN2012A_D0098.tif" />

wherein Q is -Ci-Cg alkyl, -O-(Ci-Cg alkyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and n is an integer ranging from 0-10.

Useful Stretchers may be incorporated into a Linker using the commercially available intermediates from Molecular Biosciences (Boulder, CO) described below by utilizing known techniques of organic synthesis. Stretchers of formula (Illa) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit as depicted in Schemes 11 and 12:

<img file="IN520DEN2012A_D0099.tif" />

where n is an integer ranging from 1-10 and T is -H or -SO₃Na;

222

<img file="IN520DEN2012A_D0100.tif" />

Stretcher units of formula (Illb) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:

223 ο ο.

<img file="IN520DEN2012A_D0101.tif" />

<img file="IN520DEN2012A_D0102.tif" />

<img file="IN520DEN2012A_D0103.tif" />

<img file="IN520DEN2012A_D0104.tif" />

Stretcher units of formula (IV) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:

<img file="IN520DEN2012A_D0105.tif" />

224

<img file="IN520DEN2012A_D0106.tif" />

Stretcher units of formula (Va) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:

<img file="IN520DEN2012A_D0107.tif" />

; and

Other useful Stretchers may be synthesized according to known procedures. Aminooxy Stretchers of the formula shown below can be prepared by treating alkyl halides with N-Boc-hydroxylamine according to procedures described in Jones, D.S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11), 4441-4447.

NH₂-O-R^l7-C(O)wherein -R¹⁷- is selected from -Ci-Cio alkylene-, -C3-C8 carbocyclo-, -O-(Ci-C₈alkyl)-, -arylene-, -C1-C10 alkylene-arylene-, -arylene-Ci-Cio alkylene-, -C1-C10 alkylene-(C₃-C₈ carbocyclo)-, -(C₃-C₈ carbocyclo)-Ci-Cio alkylene-, -C₃-C₈heterocyclo-, -Ci-Cioalkylene-(C₃-C₈ heterocyclo)-, -(C₃-C₈ heterocyclo)-Ci-Cio alkylene-, -(ClfcCEkOjr, -(CtLCtLOjr-CIL-; and r is an integer ranging from 110;

225

<img file="IN520DEN2012A_D0108.tif" />

Isothiocyanate Stretchers of the formula shown below may be prepared from isothiocyanatocarboxylic acid chlorides as described inAngew. Chem., 1975, 87(14):517.

S=C=N-R¹⁷-C(O wherein -R¹⁷- is as described herein.

Scheme 11 shows a method for obtaining of a val-cit dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyl selfimmolative Spacer.

Scheme 11

<img file="IN520DEN2012A_D0109.tif" />

DME, THF, H_ZO citrulline (1.05 eq.)

NaHCO₃(1.05 eq.)

<img file="IN520DEN2012A_D0110.tif" />

θπι

<img file="IN520DEN2012A_D0111.tif" />

<img file="IN520DEN2012A_D0112.tif" />

p-nitrophenyl-OCOO-p-nitrophenyl

DIEA (1.5 eq.), DMF

EEDQ(1.5 eq.) CH₂CI₂-MeOH (2:1)

<img file="IN520DEN2012A_D0113.tif" />

<img file="IN520DEN2012A_D0114.tif" />

p-nitrophenyl-OCOO-p-nitrophenyl

DIEA (1.5 eq.), DMF

<img file="IN520DEN2012A_D0115.tif" />

wherein Q is -Ci-C₈ alkyl, -O-(Ci-C₈ alkyl), -halogen, -nitro or cyano; and m is an integer ranging from 0-4.

226

Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide

Linker unit having a maleimide Stretcher unit and a p-aminobenzyl selfimmolative Spacer unit. Starting material AD (lys(Mtr)) is commercially available (Bachem, Torrance, CA) or can be prepared according to Dubowchik, et al. Tetrahedron Letters (1997) 38:5257-60.

Scheme 12

<img file="IN520DEN2012A_D0116.tif" />

<img file="IN520DEN2012A_D0117.tif" />

p-nitrophenyl-OCOO-p-nitrophenyl (2.0 eq.)

DIEA(1.5eq.), DMF

P

AH R=$—

O

<img file="IN520DEN2012A_D0118.tif" />

<img file="IN520DEN2012A_D0119.tif" />

wherein Q is -Ci-Cg alkyl, -O-(Ci-C₈ alkyl), -halogen, -nitro or cyano; and m is an integer ranging from 0-4.

227

As shown in Scheme 13, a Linker can be reacted with an amino group of a Drug Compound of Formula (lb) to form a Drug-Linker Compound that contains an amide or carbamate group, linking the Drug unit to the Linker unit. When Reactive Site No. 1 is a carboxylic acid group, as in Linker AJ, the coupling reaction can be performed using HATU or PyBrop and an appropriate amine base, resulting in a Drug-Linker Compound AK, containing an amide bond between the Drug unit and the Linker unit. When Reactive Site No. 1 is a carbonate, as in Linker AL, the Linker can be coupled to the Drug using HOBt in a mixture of DMF/pyridine to provide a Drug-Linker Compound AM, containing a carbamate bond between the Drug unit and the Linker unit

Alternately, when Reactive Site No. 1 is a good leaving group, such as in Linker AN, the Linker can be coupled with an amine group of a Drug via a nucleophilic substitution process to provide a Drug-Linker Compound having an amine linkage (AO) between the Drug unit and the Linker unit.

Illustrative methods useful for linking a Drug to a Ligand to form a Drug-Linker Compound are depicted in Scheme 13 and are outlined in General Procedures G-H.

Scheme 13

HATU

<td> Drug</td><td> + Linker-COOH ---------</td><td> -► Drug-NH-C(O)-Linker</td>

<td> (lb)</td><td> AJ</td><td> AK</td>

<td></td><td> 0</td><td> 0</td>

<td rowspan="2"> Drug +</td><td rowspan="2"> Linker-O^-C—O—NO₂</td><td> HOBt ||</td>

<td> Drug-Mil·· Ό O-Linker</td>

<td> (lb)</td><td> AL</td><td> AM</td>

<td rowspan="2"> Drug (lb)</td><td> base + T.inker-X</td><td> Drug—N-Linker</td>

<td> AN</td><td> AO</td>

General Procedure G: Amide formation using HATU. A Drug (lb) (1.0 eq.) and an N-protected Linker containing a carboxylic acid Reactive site

228 (1.0 eq.) are diluted with a suitable organic solvent, such as dichloromethane, and the resulting solution is treated with HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.). The reaction mixture is allowed to stir under an inert atmosphere, preferably argon, for 6h, during which time the reaction mixture is monitored using HPLC. The reaction mixture is concentrated and the resulting residue is purified using HPLC to yield the amide of formula AK.

Procedure H: Carbamate formation using HOBt. A mixture of a Linker AL having a p-nitrophenyl carbonate Reactive site (1.1 eq.) and Drug (lb) (1.0 eq.) are diluted with an aprotic organic solvent, such as DMF, to provide a solution having a concentration of 50-100 mM, and the resulting solution is treated with HOBt (2.0 eq.) and placed under an inert atmosphere, preferably argon. The reaction mixture is allowed to stir for 15 min, then an organic base, such as pyridine (1/4 v/v), is added and the reaction progress is monitored using HPLC. The Linker is typically consumed within 16 h. The reaction mixture is then concentrated in vacuo and the resulting residue is purified using, for example, HPLC to yield the carbamate AM.

An alternate method of preparing Drug-Linker Compounds is outlined in Scheme 14. Using the method of Scheme 14, the Drug is attached to a partial Linker unit (ZA, for example), which does not have a Stretcher unit attached. This provides intermediate AP, which has an Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc group is then removed and the resulting amine intermediate AQ is then attached to a Stretcher unit via a coupling reaction catalyzed using PyBrop or DEPC. The construction of Drug-Linker Compounds containing either a bromoacetamide Stretcher AR or a PEG maleimide Stretcher AS is illustrated in Scheme 14.

229

Scheme 14 h₃C ^rN h₃c

Z + Drug (lb)

HOBt pyridine

<img file="IN520DEN2012A_D0120.tif" />

<img file="IN520DEN2012A_D0121.tif" />

<img file="IN520DEN2012A_D0122.tif" />

<img file="IN520DEN2012A_D0123.tif" />

^RN

<img file="IN520DEN2012A_D0124.tif" />

OH wherein Q is -Q-Cg alkyl, -O-(Ci-C₈ alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.

Methodology useful for the preparation of a Linker unit containing a branched spacer is shown in Scheme 15.

230

Scheme 15

<img file="IN520DEN2012A_D0125.tif" />

<img file="IN520DEN2012A_D0126.tif" />

1. 1 M HCI, THF

2. Raney Ni, hydrazine

MeOH-THF

<img file="IN520DEN2012A_D0127.tif" />

<img file="IN520DEN2012A_D0128.tif" />

Scheme 15 illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit. The synthesis of the BHMS intermediate (AW) has been improved from previous literature procedures (see International Publication No, WO 9813059 to Firestone et al., and Crozet, M.P.; Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. (1985) 26:5133-5134) and utilizes as starting materials,

231 commercially available diethyl (4-nitrobenzyl)phosphonate (AT) and commercially available 2,2-dimethyl-l,3-dioxan-5-one (AU). Linkers AY and BA can be prepared from intermediate AW using the methodology described in Scheme 9.

4.6.3 DENDRITIC LINKERS

The linker may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to a Ligand, such as but not limited to an antibody (Sun et al. (2002) Bioorganic &amp; Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic &amp; Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which is related to the potency of the DrugLinker-Ligand Conjugate. Thus, where a cysteine engineered antibody bears only one reactive cytsteine thiol group, a multitude of drug moieties may be attached through a dendritic linker.

The following exemplary embodiments of dendritic linker reagents allow up to nine nucleophilic drug moiety reagents to be conjugated by reaction with the chloroethyl nitrogen mustard functional groups:

<img file="IN520DEN2012A_D0129.tif" />

<img file="IN520DEN2012A_D0130.tif" />

<img file="IN520DEN2012A_D0131.tif" />

232 ο

<img file="IN520DEN2012A_D0132.tif" />

CH₂OCH₂CH2CNHCH2CX3 or

Ο

II

CH2OCH2CH2CNHCH2CY3

4.6.4 CONJUGATION OF DRUG MOIETIES TO ANTIBODIES

Scheme 16 illustrates methodology useful for making Drug-LinkerLigand conjugates having about 2 to about 4 drugs per antibody. An antibody is treated with a reducing agent, such as dithiothreitol (DTT) to reduce some or all of the cysteine disulfide residues to form highly nucleophilic cysteine thiol groups (CH2SH). The partially reduced antibody thus reacts with drug-linker compounds, or linker reagents, with electrophilic functional groups such as maleimide or ahalo carbonyl, according to the conjugation method at page 766 of Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773.

Scheme 16

Drug-Linker

Antibody -------► Partially Reduced Antibody —Compound—Drug-Linker-Ligand Conjugate with Reduced Drug Load

For example, an antibody, e.g., AC 10, dissolved in 500 mM sodium borate and

500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 °C for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with ImM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice. The drug linker, e.g., MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and water at known concentration, is added to the chilled

233 reduced antibody in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and the ADC, e.g., AC 10MC-vc-PAB-MMAE, is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 gm filters under sterile conditions, and frozen for storage.

A variety of antibody drug conjugates (ADC) were prepared, with a variety of linkers, and the drug moieties, MMAE and MMAF. The following table is an exemplary group of ADC which were prepared following the protocol of Example 27, and characterized by HPLC and drug loading assay.

<td> Target (antigen)</td><td> ADC</td><td> isolated amount (mg)</td><td> drug/Ab ratio</td>

<td> 0772P</td><td> 16E12-MC-VC-PAB-MMAE</td><td> 1.75</td><td> 4</td>

<td> 0772P</td><td> 1 lDlO-MC-vc-PAB-MMAE</td><td> 46.8</td><td> 4.4</td>

<td> 0772P</td><td> 1 lDlO-MC-vc-PAB-MMAF</td><td> 54.5</td><td> 3.8</td>

<td> Brevican</td><td> Brevican-MC-MMAF</td><td> 2</td><td> 6</td>

<td> Brevican</td><td> Brevican-MC-vc-MMAF</td><td> 2</td><td> 6</td>

<td> Brevican</td><td> Brevican-MC-vc-PAB-MMAF</td><td> 1.4</td><td> 6</td>

<td> CD21</td><td> CD21 -MC-vc-PAB-MMAE</td><td> 38.1</td><td> 4.3</td>

<td> CD21</td><td> CD21 -MC-vc-PAB-MMAF</td><td> 43</td><td> 4.1</td>

<td> CRIPTO</td><td> 11F4-MC-VC-PAB-MMAF</td><td> 6</td><td> 4.8</td>

<td> CRIPTO</td><td> 25G8-MC-vc-PAB-MMAF</td><td> 7.4</td><td> 4.7</td>

<td> E16</td><td> 12G12-MC-vc-PAB-MMAE</td><td> 2.3</td><td> 4.6</td>

<td> E16</td><td> 3B5-MC-vc-PAB-MMAE</td><td> 2.9</td><td> 4.6</td>

<td> E16</td><td> 12B9-MC-vc-PAB-MMAE</td><td> 1.4</td><td> 3.8</td>

234

<td> E16</td><td> 12B9-MC-vc-PAB-MMAE</td><td> 5.1</td><td> 4</td>

<td> E16</td><td> 12G12-MC-vc-PAB-MMAE</td><td> 3</td><td> 4.6</td>

<td> E16</td><td> 3B5-MC-vc-PAB-MMAE</td><td> 4.8</td><td> 4.1</td>

<td> E16</td><td> 3B5-MC-vc-PAB-MMAF</td><td> 24.7</td><td> 4.4</td>

<td> EphB2R</td><td> 2H9-MC-VC-PAB-MMAE</td><td> 29.9</td><td> 7.1</td>

<td> EphB2R</td><td> 2H9-MC-fk-PAB-MMAE</td><td> 25</td><td> 7.5</td>

<td> EphB2R</td><td> 2H9-MC-vc-PAB-MMAE</td><td> 175</td><td> 4.1</td>

<td> EphB2R</td><td> 2H9-MC-vc-PAB-MMAF</td><td> 150</td><td> 3.8</td>

<td> EphB2R</td><td> 2H9-MC-vc-PAB-MMAF</td><td> 120</td><td> 3.7</td>

<td> EphB2R</td><td> 2H9-MC-vc-PAB-MMAE</td><td> 10.7</td><td> 4.4</td>

<td> IL-20Ra</td><td> IL20Ra-fk-MMAE</td><td> 26</td><td> 6.7</td>

<td> IL-20Ra</td><td> IL20Ra-vc-MMAE</td><td> 27</td><td> 7.3</td>

<td> EphB2</td><td> IL8-MC-VC-PAB-MMAE</td><td> 251</td><td> 3.7</td>

<td> MDP</td><td> MDP-vc-MMAE</td><td> 32</td><td></td>

<td> MPF</td><td> 19C3-vc-MMAE</td><td> 1.44</td><td> 6.5</td>

<td> MPF</td><td> 7D9-vc-MMAE</td><td> 4.3</td><td> 3.8</td>

<td> MPF</td><td> 19C3-vc-MMAE</td><td> 7.9</td><td> 3</td>

<td> MPF</td><td> 7D9-MC-vc-PAB-MMAF</td><td> 5</td><td> 4.3</td>

<td> Napi3b</td><td> 10Hl-vc-MMAE</td><td> 4.5</td><td> 4.6</td>

<td> Napi3b</td><td> 4C9-vc-MMAE</td><td> 3.0</td><td> 5.4</td>

<td> Napi3b</td><td> lOHl-vc-MMAE</td><td> 4.5</td><td> 4.8</td>

<td> Napi3b</td><td> lOHl-vc-MMAF</td><td> 6.5</td><td> 4</td>

235

<td> NCA</td><td> 3E6-MC-fk-PAB-MMAE</td><td> 49.6</td><td> 5.4</td>

<td> NCA</td><td> 3E6-MC-vc-PAB-MMAE</td><td> 56.2</td><td> 6.4</td>

<td> PSCA</td><td> PSCA-fk-MMAE</td><td> 51.7</td><td> 8.9</td>

<td> PSCA</td><td> PSCA-vc-MMAE</td><td> 61.1</td><td> 8.6</td>

<td> Napi3b</td><td> lOHl-MC-vc-PAB-MMAE</td><td> 75</td><td> 4.2</td>

<td> Napi3b</td><td> 1OH1 -MC-vc-PAB-MMAF</td><td> 95</td><td> 4.4</td>

<td> Napi3b</td><td> 10H1-MC-MMAF</td><td> 92</td><td> 4</td>

<td> EphB2R</td><td> 2H9-MC-vc-PAB-MMAE</td><td> 79</td><td> 5</td>

<td> EphB2R</td><td> 2H9-MC-MMAF</td><td> 92</td><td> 4.9</td>

<td> 0772P</td><td> 1 1D1O(Fc chimera)-MC-vc-PAB- MMAE</td><td> 79</td><td> 4.3</td>

<td> 0772P</td><td> 1 1D1O(Fc chimera)-MC-vc-PAB- MMAF</td><td> 70</td><td> 4.5</td>

<td> 0772P</td><td> 1 1D1O(Fc chimera)-MC-MMAF</td><td> 23</td><td> 4.5</td>

<td> Brevican</td><td> 6D2-MC-vc-PAB-MMAF</td><td> 0.3</td><td> 4.5</td>

<td> Brevican</td><td> 6D2-MC-MMAF</td><td> 0.36</td><td> 4.5</td>

<td> EphB2R</td><td> 2H9(Fc chimera)-MC-vc-PAB- MMAE</td><td> 1983</td><td> 4.3</td>

<td> E16</td><td> 12B9-MC-vc-PAB-MMAE</td><td> 14.1</td><td> 4.6</td>

<td> E16</td><td> 12B9-MC-vc-PAB-MMAF</td><td> 16.4</td><td> 4.5</td>

<td> E16</td><td> 12G12-MC-vc-PAB-MMAE</td><td> 10.5</td><td> 4.1</td>

<td> E16</td><td> 12G12-MC-vc-PAB-MMAF</td><td> 10.2</td><td> 3.8</td>

<td> E16</td><td> 3B5-MC-vc-PAB-MMAE</td><td> 58.6</td><td> 3.8</td>

236

<td> E16</td><td> 3B5-MC-vc-PAB-MMAF</td><td> 8</td><td> 3.1</td>

<td> 0772P</td><td> 1 lD10(Fc chimera)-MC-vc-PABMMAE</td><td> 340</td><td> 3.9</td>

<td> Steapl</td><td> (Steap 1 -92)-MC-vc-PAB -MMAE</td><td> 3.5</td><td> 4</td>

<td> Steapl</td><td> (Steapl-92)-MC-vc-PAB-MMAF</td><td> 4.7</td><td> 4</td>

<td> Steapl</td><td> (Steap 1 -120)-MC-vc-PAB - MMAE</td><td> 2</td><td> 4</td>

<td> Steapl</td><td> (Steap 1 -120)-MC-vc-PAB-MMAF</td><td> 2.3</td><td> 4</td>

<td> E16</td><td> 3B5-MC-vc-PAB-MMAF</td><td> 52.2</td><td> 4.5</td>

COMPOSITIONS AND METHODS OF ADMINISTRATION

In other embodiments, described is a composition including an effective amount of an Exemplary Compound and/or Exemplary Conjugate and a pharmaceutically acceptable carrier or vehicle. For convenience, the Drug units and Drug-Linker Compounds can be referred to as Exemplary Compounds, while Drug-Ligand Conjugates and Drug-Linker-Ligand Conjugates can be referred to as Exemplary Conjugates. The compositions are suitable for veterinary or human administration.

The present compositions can be in any form that allows for the composition to be administered to a patient. For example, the composition can be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. In one aspect, the compositions are administered parenterally. In yet another aspect, the Exemplary Compounds and/or the Exemplary Conjugates or compositions are administered intravenously.

237

Pharmaceutical compositions can be formulated so as to allow an Exemplary Compound and/or Exemplary Conjugate to be bioavailable upon administration of the composition to a patient. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of an Exemplary Compound and/or Exemplary Conjugate in aerosol form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the Exemplary Compound or Exemplary Conjugate, the manner of administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous or particulate, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a¹ flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.

238

When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. Physiological saline is an exemplary adjuvant. An injectable composition is preferably sterile.

The amount of the Exemplary Compound and/or Exemplary Conjugate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient’s circumstances.

239

The compositions comprise an effective amount of an Exemplary Compound and/or Exemplary Conjugate such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of an Exemplary Compound and/or Exemplary Conjugate by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. In one aspect, oral compositions can comprise from about 4% to about 50% of the Exemplary Compound and/or Exemplary Conjugate by weight of the composition. In yet another aspect, present compositions are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the Exemplary Compound and/or Exemplary Conjugate.

For intravenous administration, the composition can comprise from about 0.01 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugate per kg of the animal’s body weight. In one aspect, the composition can include from about 1 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugate per kg of the animal’s body weight. In another aspect, the amount administered will be in the range from about 0.1 to about 25 mg/kg of body weight of the Exemplary Compound and/or Exemplary Conjugate.

Generally, the dosage of an Exemplary Compound and/or Exemplary Conjugate administered to a patient is typically about 0.01 mg/kg to about 2000 mg/kg of the animal’s body weight. In one aspect, the dosage administered to a patient is between about 0.01 mg/kg to about 10 mg/kg of the animal’s body weight, in another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 250 mg/kg of the animal’s body weight, in yet another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 20 mg/kg of the animal’s body weight, in yet another aspect the dosage administered is between about 0.1 mg/kg to about 10 mg/kg of the animal’s body weight, and in yet another aspect, the dosage administered is between about 1 mg/kg to about 10 mg/kg of the animal’s body weight.

The Exemplary Compounds and/or Exemplary Conjugate or compositions can be administered by any convenient route, for example by

240 infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer an Exemplary Compound and/or Exemplary Conjugate or composition. In certain embodiments, more than one Exemplary Compound and/or Exemplary Conjugate or composition is administered to a patient.

In specific embodiments, it can be desirable to administer one or more Exemplary Compounds and/or Exemplary Conjugate or compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or preneoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.

In certain embodiments, it can be desirable to introduce one or more Exemplary Compounds and/or Exemplary Conjugate or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.

In yet another embodiment, the Exemplary Compounds and/or Exemplary Conjugate or compositions can be delivered in a controlled release system, such as but not limited to, a pump or various polymeric materials can be used. In yet another embodiment, a controlled-release system can be placed in

241 proximity of the target of the Exemplary Compounds and/or Exemplary Conjugate or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, with which an Exemplary Compound and/or Exemplary Conjugate is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a patient, the Exemplary Compound and/or Exemplary Conjugate or compositions and pharmaceutically acceptable carriers are sterile. Water is an exemplary carrier when the Exemplary Compounds and/or Exemplary Conjugates are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.

In an embodiment,the Exemplary Compounds and/or Exemplary Conjugates are formulated in accordance with routine procedures as a

242 pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where an Exemplary Compound and/or Exemplary Conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the Exemplary Compound and/or Exemplary Conjugate is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of Wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A

243 time-delay material such as glycerol monostearate or glycerol stearate can also be used.

The compositions can be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment or gel base. If intended for transdermal administration, the composition can be in the form of a transdermal patch or an iontophoresis device. Topical formulations can comprise a concentration of an Exemplary Compound and/or Exemplary Conjugate of from about 0.05% to about 50% w/v (weight per unit volume of composition), in another aspect, from 0.1% to 10% w/v.

The composition can be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the Exemplary Compound and/or Exemplary Conjugate.

The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients.

Whether in solid, liquid or gaseous form, the present compositions can include a pharmacological agent used in the treatment of cancer, an autoimmune disease or an infectious disease.

THERAPEUTIC USES OF THE EXEMPLARY CONJUGATES

The Exemplary Compounds and/or Exemplary Conjugates are i . ... K-3 . . ' useful for treating cancer, an autoimmune disease or an infectious disease in a patient.

244

4.8.1 TREATMENT OF CANCER

The Exemplary Compounds and/or Exemplary Conjugates are useful for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The Exemplary Compounds and/or Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of animal cancers. The Drug-LinkerLigand Conjugates can be used to deliver a Drug or Drug unit to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the Ligand unit of an Exemplary Conjugate binds to or associates with a cancer-cell or a tumorcell-associated antigen, and the Exemplary Conjugate can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, one or more specific peptide sequences within the Linker unit are hydrolytically cleaved by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of a Drug or a Drug-Linker Compound. The released Drug or Drug-Linker Compound is then free to migrate within the cell and induce cytotoxic or cytostatic activities. In an alternative embodiment, the Drug or Drug unit is cleaved from the Exemplary Conjugate outside the tumor cell or cancer cell, and the Drug or Drug-Linker Compound subsequently penetrates the cell.

In one embodiment, the Ligand unit binds to the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.

The specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated. For example, Exemplary Conjugates having a BR96 Ligand

245 unit can be useful for treating antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Exemplary Conjugates having an AntiCD30 or an anti-CD40 Ligand unit can be useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with

Exemplary Conjugates include, but are not limited to, those disclosed in Table 3.

TABLE 3

Solid tumors, including but not limited to:

fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing’s tumor leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer kidney cancer pancreatic cancer bone cancer breast cancer ovarian cancer prostate cancer esophogeal cancer stomach cancer oral cancer nasal cancer throat cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma

246 papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms’ tumor cervical cancer uterine cancer testicular cancer small cell lung carcinoma bladder carcinoma lung cancer epithelial carcinoma glioma glioblastoma multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningioma skin cancer melanoma neuroblastoma retinoblastoma blood-borne cancers, including but not limited to: acute lymphoblastic leukemia “ALL” acute lymphoblastic B-cell leukemia acute lymphoblastic T-cell leukemia acute myeloblastic leukemia “AML” acute promyelocytic leukemia “APL” acute monoblastic leukemia acute erythroleukemic leukemia acute megakaryoblastic leukemia acute myelomonocytic leukemia

247 acute nonlymphocyctic leukemia acute undifferentiated leukemia chronic myelocytic leukemia “CML” chronic lymphocytic leukemia “CLL” hairy cell leukemia multiple myeloma acute and chronic leukemias:

lymphoblastic myelogenous lymphocytic myelocytic leukemias

Lymphomas:

Hodgkin’s disease non-Hodgkin’s Lymphoma

Multiple myeloma

Waldenstrom’s macroglobulinemia

Heavy chain disease

Polycythemia vera

The Exemplary Conjugates provide conjugation-specific tumor or cancer targeting, thus reducing general toxicity of these compounds. The Linker units stabilize the Exemplary Conjugates in blood, yet are cleavable by tumorspecific proteases within the cell, liberating a Drug.

MULTI-MODALITY THERAPY FOR CANCER

Cancers, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of an Exemplary Conjugate and/or an Exemplary Compound.

In other embodiments, methods for treating or preventing cancer are provided, including administering to a patient in need thereof an effective amount of an Exemplary Conjugate and a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found

248 to be refractory. The Exemplary Conjugates can be administered to a patient that has also undergone surgery as treatment for the cancer.

In one embodiment, the additional method of treatment is radiation therapy.

In a specific embodiment, the Exemplary Conjugate is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of an Exemplary Conjugates, in one aspect at least an hour, five hours, 12 hours, a day, a week, a month, in further aspects several months (e.g., up to three months), prior or subsequent to administration of an Exemplary Conjugate.

A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents listed in Table 4 can be administered. With respect to radiation, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.

Additionally, methods of treatment of cancer with an Exemplary Compound and/or Exemplary Conjugate are provided as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.

The Exemplary Compounds and/or Exemplary Conjugates can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain · cancers, including, but not limited to leukemias and lymphomas, such treatment

249 involving autologous stem cell transplants. This can involve a multi-step process in which the animal’s autologous hematopoietic stem cells are harvested and purged of all cancer cells, the animal’s remaining bone-marrow cell population is then eradicated via the administration of a high dose of an Exemplary Compound and/or Exemplary Conjugate with or without accompanying high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the animal recovers.

MULTI-DRUG THERAPY FOR CANCER

Methods for treating cancer including administering to a patient in need thereof an effective amount of an Exemplary Conjugate and another therapeutic agent that is an anti-cancer agent are disclosed. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In one aspect, the anti-cancer agent includes, but is not limited to, a drug listed in Table 4.

TABLE 4

<td> Alkylating agents</td><td></td>

<td> Nitrogen mustards:</td><td> cyclophosphamide ifosfamide trofosfamide chlorambucil melphalan</td>

<td> Nitrosoureas:</td><td> carmustine (BCNU) lomustine (CCNU)</td>

<td> Alkylsulphonates</td><td> busulfan treosulfan</td>

<td> Triazenes:</td><td> decarbazine</td>

<td> Platinum containing compounds:</td><td> cisplatin carboplatin</td>

250

<td> Plant Alkaloids</td><td></td>

<td> Vinca alkaloids:</td><td> vincristine vinblastine vindesine vinorelbine</td>

<td> Taxoids:</td><td> paclitaxel docetaxol</td>

<td> DNA Topoisomerase Inhibitors</td><td></td>

<td> Epipodophy Ilins:</td><td> etoposide teniposide topotecan 9-aminocamptothecin camptothecin crisnatol</td>

<td> mitomycins:</td><td> mitomycin C</td>

<td> Anti-metabolites</td><td></td>

<td> Anti-folates:</td><td></td>

<td> DHFR inhibitors:</td><td> methotrexate trimetrexate</td>

<td> IMP dehydrogenase Inhibitors:</td><td> mycophenolic acid tiazofurin ribavirin EICAR</td>

<td> Ribonucleotide reductase Inhibitors:</td><td> hydroxyurea deferoxamine</td>

<td> Pyrimidine analogs:</td><td></td>

<td> Uracil analogs</td><td> 5-Fluorouracil</td>

<td></td><td> floxuridine doxifluridine ratitrexed</td>

<td> Cytosine analogs</td><td> cytarabine (ara C) cytosine arabinoside fludarabine</td>

<td> Purine analogs:</td><td> mercaptopurine thioguanine</td>

<td> Hormonal therapies:</td><td></td>

<td> Receptor antagonists:</td><td> •</td>

<td> Anti-estrogen</td><td> tamoxifen raloxifene megestrol</td>

<td> LHRH agonists:</td><td> goscrclin leuprolide acetate</td>

<td> Anti-androgens:</td><td> flutamide bicalutamide</td>

<td> Retinoids/Deltoids</td><td></td>

251

<td> Vitamin D3 analogs:</td><td> EB 1089 CB 1093 KH 1060</td>

<td> Photodynamic therapies:</td><td> vertoporfin (BPD-MA) phthalocyanine photosensitizer Pc4 demethoxy-hypocrellin A (2BA-2-DMHA)</td>

<td> Cytokines:</td><td> Interferon- a Interferon- γ tumor necrosis factor</td>

<td> Others:</td><td> Gemcitabine Velcade Revamid Thai amid</td>

<td> Isoprenylation inhibitors:</td><td> Lovastatin</td>

<td> Dopaminergic neurotoxins:</td><td> l-methyl-4-phenylpyridinium ion</td>

<td> Cell cycle inhibitors:</td><td> staurosporine</td>

<td> Actinomycins:</td><td> Actinomycin D</td>

<td></td><td> Dactinomycin</td>

<td> Bleomycins:</td><td> bleomycin A2 bleomycin B2 peplomycin</td>

<td> Anthracyclines:</td><td> daunorubicin Doxorubicin (adriamycin) idarubicin epirubicin pirarubicin zorubicin mtoxantrone</td>

<td> MDR inhibitors:</td><td> Verapamil</td>

<td> Ca^z+ATPase inhibitors:</td><td> Thapsigargin</td>

4.8.4 TREATMENT OF AUTOIMMUNE DISEASES

The Exemplary Conjugates are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease. The Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of an autoimmune disease in a patient. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the

252

Exemplary Conjugate is then taken up inside a target-cell through receptormediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic or cytostatic activities. In an alternative embodiment, the Drug is cleaved from the Exemplary Conjugate outside the target cell, and the Drug subsequently penetrates the cell.

In one embodiment, the Ligand unit binds to an autoimmune antigen. Inone aspect, the antigen is on the surface of a cell involved in an autoimmune condition.

In another embodiment, the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.

In one embodiment, the Ligand binds to activated lymphocytes that are associated with the autoimmune disease state.

In a further embodiment, the Exemplary Conjugates kill or inhibit the multiplication of cells that produce an autoimmune antibody associated with a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with the Exemplary Conjugates include, but are not limited to, Th2 lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn’s syndrome, systemic sclerosis, and graft versus host disease); Thl lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren’s syndrome, Hashimoto’s thyroiditis, Grave’s disease, primary biliary cirrhosis, Wegener’s granulomatosis, and tuberculosis); activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture’s syndrome, rheumatoid arthritis, and type I diabetes); and those disclosed in Table

5.

TABLE 5

Active Chronic Hepatitis

Addison’s Disease

Allergic Alveolitis

253

Allergic Reaction Allergic Rhinitis Alport’s Syndrome Anaphlaxis Ankylosing Spondylitis Anti-phosholipid Syndrome Arthritis

Ascariasis Aspergillosis Atopic Allergy

Atropic Dermatitis Atropic Rhinitis Behcet’s Disease Bird-Fancier’s Lung Bronchial Asthma Caplan’s Syndrome Cardiomyopathy Celiac Disease

Chagas’ Disease Chronic Glomerulonephritis Cogan’s Syndrome Cold Agglutinin Disease Congenital Rubella Infection CREST Syndrome

Crohn’s Disease Cryoglobulinemia Cushing’s Syndrome Dermatomyositis Discoid Lupus Dressier’s Syndrome Eaton-Lambert Syndrome Echovirus Infection Encephalomyelitis Endocrine opthalmopathy Epstein-Barr Virus Infection Equine Heaves Erythematosis Evan’s Syndrome Felty’s Syndrome Fibromyalgia Fuch’s Cyclitis Gastric Atrophy Gastrointestinal Allergy Giant Cell Arteritis Glomerulonephritis Goodpasture’s Syndrome

254

Graft v. Host Disease Graves’ Disease Guillain-Barre Disease Hashimoto’s Thyroiditis Hemolytic Anemia Henoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases Insulin-dependent Diabetes Mellitus Juvenile Arthritis

Juvenile Diabetes Mellitus (Type I) Lambert-Eaton Syndrome Laminitis

Lichen Planus

Lupoid Hepatitis

Lupus Lymphopenia Meniere’s Disease

Mixed Connective Tissue Disease

Multiple Sclerosis

Myasthenia Gravis Pernicious Anemia

Polyglandular Syndromes Presenile Dementia

Primary Agammaglobulinemia Primary Biliary Cirrhosis Psoriasis

Psoriatic Arthritis

Raynauds Phenomenon Recurrent Abortion

Reiter’s Syndrome Rheumatic Fever

Rheumatoid Arthritis Sampter’s Syndrome Schistosomiasis

Schmidt’s Syndrome Scleroderma

Shulman’s Syndrome Sjorgen’s Syndrome Stiff-Man Syndrome Sympathetic Ophthalmia

Systemic Lupus Erythematosis Takayasu’s Arteritis Temporal Arteritis

255

Thyroiditis

Thrombocytopenia Thyrotoxicosis Toxic Epidermal Necrolysis Type B Insulin Resistance Type I Diabetes Mellitus Ulcerative Colitis

Uveitis

Vitiligo Waldenstrom’s Macroglobulemia Wegener’s Granulomatosis

MULTI-DRUG THERAPY OF AUTOIMMUNE DISEASES

Methods for treating an autoimmune disease are also disclosed including administering to a patient in need thereof an effective amount of an

Exemplary Conjugate and another therapeutic agent known for the treatment of an autoimmune disease. In one embodiment, the anti-autoimmune disease agent includes, but is not limited to, agents listed in Table 6.

Table 6 cyclosporine cyclosporine A mycophenylate mofetil sirolimus tacrolimus enanercept prednisone azathioprine methotrexate cyclophosphamide prednisone aminocaproic acid chloroquine hydroxychloroquine hydrocortisone dexamethasone chlorambucil

DHEA danazol bromocriptine meloxicam infliximab

256

TREATMENT OF INFECTIOUS DISEASES

The Exemplary Conjugates are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of an infectious disease in a patient. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. In one embodiment, the Ligand unit binds to the infectious disease cell.

In one embodiment, the Conjugates kill or inhibit the multiplication of cells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with the Exemplary Conjugates include, but are not limited to, those disclosed in Table 7.

TABLE 7

Bacterial Diseases:

Diphtheria

Pertussis

Occult Bacteremia

Urinary Tract Infection

Gastroenteritis

Cellulitis

Epiglottitis

Tracheitis

Adenoid Hypertrophy

Retropharyngeal Abcess

Impetigo

Ecthyma

Pneumonia

Endocarditis

Septic Arthritis

Pneumococcal

Peritonitis

Bactermia

Meningitis

Acute Purulent Meningitis

Urethritis

Cervicitis

Proctitis

Pharyngitis

Salpingitis

257

Epididymitis Gonorrhea Syphilis Listeriosis Anthrax Nocardiosis Salmonella Typhoid Fever Dysentery Conjunctivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic Plague Tetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic Infections Syphilis Relapsing Fever Leptospirosis Lyme Disease Rat Bite Fever Tuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial Pneumonia Trachoma Inclusion Conjunctivitis

Systemic Fungal Diseases:

Histoplamosis Coccidiodomycosis Blastomycosis Sporotrichosis Cryptococcsis Systemic Candidiasis Aspergillosis Mucormycosis Mycetoma Chromomycosis

Rickettsial Diseases:

258

Typhus

Rocky Mountain Spotted Fever Ehrlichiosis

Eastern Tick-Borne Rickettsioses Rickettsialpox

Q Fever

Bartonellosis

Parasitic Diseases:

Malaria

Babesiosis

African Sleeping Sickness Chagas’ Disease Leishmaniasis

Dum-Dum Fever

Toxoplasmosis Meningoencephalitis Keratitis

Entamebiasis

Giardiasis

Cryptosporidiasis Isosporiasis Cyclosporiasis Microsporidiosis Ascariasis

Whipworm Infection

Hookworm Infection Threadworm Infection Ocular Larva Migrans Trichinosis

Guinea Worm Disease

Lymphatic Filariasis

Loiasis

River Blindness

Canine Heartworm Infection Schistosomiasis

Swimmer’s Itch

Oriental Lung Fluke Oriental Liver Fluke Fascioliasis

Fasciolopsiasis Opisthorchiasis Tapeworm Infections Hydatid Disease Alveolar Hydatid Disease

259

Viral Diseases:

Measles

Subacute sclerosing panencephalitis

Common Cold

Mumps Rubella

Roseola

Fifth Disease

Chickenpox

Respiratory syncytial virus infection

Croup

Bronchiolitis

Infectious Mononucleosis

Poliomyelitis

Herpangina Hand-Foot-and-Mouth Disease Bornholm Disease Genital Herpes Genital Warts Aseptic Meningitis Myocarditis

Pericarditis

Gastroenteritis

Acquired Immunodeficiency Syndrome (AIDS) Human Immunodeficiency Virus (HIV) Reye’s Syndrome

Kawasaki Syndrome

Influenza

Bronchitis

Viral “Walking” Pneumonia

Acute Febrile Respiratory Disease

Acute pharyngoconjunctival fever Epidemic keratoconjunctivitis Herpes Simplex Virus 1 (HSV-1) Herpes Simplex Virus 2 (HSV-2) Shingles

Cytomegalic Inclusion Disease Rabies

Progressive Multifocal Leukoencephalopathy Kuru

Fatal Familial Insomnia

Creutzfeldt-Jakob Disease Gerstmann-Straussler-Scheinker Disease Tropical Spastic Paraparesis Western Equine Encephalitis

260

California Encephalitis

St. Louis Encephalitis

Yellow Fever

Dengue

Lymphocytic choriomeningitis

Lassa Fever

Hemorrhagic Fever

Hantvirus Pulmonary Syndrome

Marburg Virus Infections

Ebola Virus Infections

Smallpox

MULTI-DRUG THERAPY OF INFECTIOUS DISEASES

Methods for treating an infectious disease are disclosed including administering to a patient in need thereof an Exemplary Conjugate and another therapeutic agent that is an anti-infectious disease agent. In one embodiment, the anti-infectious disease agent is, but not limited to, agents listed in Table 8.

TABLE 8 β-Lactam Antibiotics:

Penicillin G Penicillin V Cloxacilliin Dicloxacillin Methicillin Nafcillin Oxacillin Ampicillin Amoxicillin Bacampicillin Azlocillin Carbenicillin Mezlocillin Piperacillin Ticarcillin

Aminoglycosides:

Amikacin

261

Gentamicin Kanamycin Neomycin Netilmicin Streptomycin Tobramycin

Macrolides:

Azithromycin Clarithromycin Erythromycin Lincomycin Clindamycin

Tetracyclines:

Demeclocycline Doxycycline Minocycline Oxytetracycline Tetracycline

Quinolones:

Cinoxacin Nalidixic Acid

Fluoroquinolones:

Ciprofloxacin Enoxacin Grepafloxacin Levofloxacin Lomefloxacin Norfloxacin Ofloxacin Sparfloxacin Trovafloxicin

Polypeptides:

Bacitracin Colistin Polymyxin B

Sulfonamides:

262

Sulfisoxazole Sulfamethoxazole Sulfadiazine Sulfamethizole Sulfacetamide

Miscellaneous Antibacterial Agents:

Trimethoprim Sulfamethazole Chloramphenicol Vancomycin Metronidazole Quinupristin Dalfopristin Rifampin Spectinomycin Nitrofurantoin Antiviral Agents:

General Antiviral Agents:

Idoxuradine Vidarabine Trifluridine Acyclovir Famcicyclovir Pencicyclovir Valacyclovir Gancicyclovir Foscamet Ribavirin Amantadine Rimantadine Cidofovir Antisense Oligonucleotides Immunoglobulins Inteferons

Drugs for HIV infection:

Tenofovir Emtricitabine Zidovudine Didanosine Zalcitabine

263

Stavudine Lamivudine Nevirapine Delavirdine Saquinavir Ritonavir Indinavir Nelfinavir

EXAMPLES

Example 1 - Preparation of compound AB

<img file="IN520DEN2012A_D0133.tif" />

Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Patent No. 6214345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification.

264

Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al., (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M-H]

Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Patent No. 6214345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al., (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M-H].

The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66 mL, 21.0

265 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that was precipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). The solute was further precipitated with ether (ca. 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound AB which was 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%).

Example 2 - Preparation of compound 1

<img file="IN520DEN2012A_D0134.tif" />

Phenylalanine ί-butyl ester HC1 salt (868 mg, 3 mmol), /V-BocDolaproine (668 mg, 1 eq.), DEPC (820 pL, 1.5 eq.), and DIEA (1.2 mL) were diluted with dichloromethane (3 mL). After 2 hours (h) at room temperature (about 28 degrees Celsius), the reaction mixture was diluted with dichloromethane (20 mL), washed successively with saturated aqueous (aq.) NaHCO₃ (2x10 mL), saturated aq. NaCl (2x10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography in ethyl acetate. The relevant fractions were combined and concentrated to provide the dipeptide as a white solid: 684 mg (46 %). ES-MS m/z 491.3 [M+H]⁺.

For selective Boc cleavage in the presence of ί-butyl ester, the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 mL). 4M HCl/dioxane (960 pL, 3 eq.) was added, and the reaction mixture was stirred overnight at room temperature. Almost complete Boc deprotection was observed by RP-HPLC with minimal amount of /-butyl ester cleavage. The mixture was

266 cooled down on an ice bath, and triethylamine (500 pL) was added. After 10 min., the mixture was removed from the cooling bath, diluted with dichloromethane (20 mL), washed successively with saturated aq. NaHCO₃ (2x10 mL), saturated aq. NaCl (2 x 10 mL). The organic layer was concentrated to give a yellow foam: 287 mg (57 %). The intermediate was used without further purification.

The tripeptide Fmoc-Meval-val-dil-O-i-Bu (prepared as described in WO 02/088172, entitled “Pentapeptide Compounds and Uses Related Thereto”·, 0.73 mmol) was treated with TFA (3 mL), dichloromethane (3 mL) for 2 h at room temperature. The mixture was concentrated to dryness, the residue was co-evaporated with toluene (3 x 20 mL),and dried in vacuum overnight. The residue was diluted with dichloromethane (5 mL) and added to the deprotected dipeptide (287 mg, 0.73 mmol), followed by DIEA (550 pL, 4 eq.), DEPC (201 pL, 1.1 eq.). After 2 h at room temperature the reaction mixture was diluted with ethyl acetate (50 mL), washed successively with 10% aq. citric acid (2 x 20 mL), saturated aq. NaHCO₃ (2 x 10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography in ethyl acetate. The relevant fractions were combined and concentrated to provide Fmoc-Meval-val-dil-dapphe-O-i-Bu as a white solid: 533 mg (71 %). Rf 0.4 (EtOAc). ES-MS m/z 1010.6 [M+H]⁺.

The product (200 mg, 0.2 mmol) was diluted with dichloromethane (3 mL), diethylamine (1 mL). The reaction mixture was stirred overnight at room temperature. Solvents were removed to provide an oil that was purified by flash silica gel chromatography in a step gradient 0-10 % MeOH in dichloromethane to provide Compound 1 as a white solid: 137 mg (87 %). Rf 0.3 (10 % MeOH/CH₂Cl₂). ES-MS m/z. 788.6 [M+H]⁺.

Example 3 - Preparation of compound 2

267

<img file="IN520DEN2012A_D0135.tif" />

Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol) by treatment with 4M HCl/dioxane (4 ml) for 7 h at room temperature. The solvent was removed, and the residue was dried in a vacuum overnight to give provide Compound 2 as a hydroscopic white solid: 35 mg (120 % calculated for HC1 salt). ES-MS m/z 732.56 [M+H]⁺.

Example 4 - Preparation of compound 3

<img file="IN520DEN2012A_D0136.tif" />

Fmoc-Meval-val-dil-dap-phe-O-r-Bu (Example 2, 50 mg) was treated with 4M HCl/dioxane (4 ml) for 16 h at room temperature. The solvent was removed, and the residue was dried in vacuum overnight to give 50 mg of a hydroscopic white solid intermediate

The white solid intermediate (20 mg, 0.02 mmol) was diluted with dichloromethane (1 mL); DEPC (5 pL, 0.03 mmol, 1.5 eq.) was added followed by DIEA (11 pL, 0.06 mmol, 3 eq.), and r-butylamine (3.2 pL, 0.03 mmol, 1.5 eq.). After 2 h at room temperature, the reaction was found to be uncompleted by RP-HPLC. More DEPC (10 pL) and ί-butylamine (5 pL) were added and the reaction was stirred for additional 4 h. Reaction mixture was diluted with dichloromethane (15 mL), washed successively with water (5 mL), 0.1 M aq. HC1

268 (10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was diluted with dichloromethane and purified via flash chromatography in a step gradient 0-5 % MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide the Fmoc protected intermediate as a white solid: 7.3 mg (36 %). R_f 0.75 (10 % MeOH/CH₂Cl₂).

Fmoc protected intermediate was diluted with dichloromethane (0.5 mL) and treated with diethylamine (0.5 mL) for 3 h at room temperature. The reaction mixture was concentrated to dryness. The product was isolated by flash silica gel chromatography in a step gradient 0-10 % MeOH in dichloromethane to provide Compound 3 as a white solid: 4 mg (70 %). Rf 0.2 (10 % MeOH/CH₂Cl₂). ES-MS m/z 787 [M+H]⁺, 809 [M+Na]⁺.

Example 5 - Preparation of compound 4

<img file="IN520DEN2012A_D0137.tif" />

Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and triethyleneglycol monomethyl ether (164 pL, 1 mmol, 1 eq.) were diluted with dichloromethane (5 mL). Then, DCC (412 mg, 2 mmol, 2 eq.) was added, followed by DMAP (10 mg). The reaction mixture was stirred overnight at room temperature. The precipitate was filtered off. The solvent was removed in a vacuum, the residue was diluted with ethyl acetate, and purified by silica gel flash chromatography in ethyl acetate. The product containing fractions were pulled, concentrated, and dried in vacuum to give a white solid: 377 mg (91 %). Rf 0.5 (EtOAc). ES-MS m/z 434 [M+Na]⁺.

Removal of Boc protecting group was performed by treatment of the above material in dioxane (10 mL) with 4M HCl/dioxane (6 mL) for 6 h at

269 room temperature. The solvent was removed in a vacuum, the residue was dried in a vacuum to give a white solid.

The HC1 salt of Phenylalanine-triethyleneglycol monomethyl ether ester (236 mg, 0.458 mmol, leq.) and Ύ-Boc-Dolaproine (158 mg, 0.55 mmol, 1.2 eq.) were diluted with dichloromethane (3 mL). DEPC (125 pL, 1.5 eq.) and added to the mixture followed by DIEA (250 pL, 3 eq.). After 2 h at room temperature the reaction mixture was diluted with ethyl acetate (30 mL), washed successively with saturated aq. NaHCCh (2 x 10 mL), 10% aq. citric acid (2 x 10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography on silica gel in ethyl acetate. The relevant fractions were combined and concentrated to provide a white foam intermediate: 131 mg (50 %). R_f 0.25 (EtOAc). ES-MS mJz 581.3 [M+H]⁺.

Boc deprotection was done in dichloromethane (2 mL), TFA (0.5 mL) at room temperature for 2 h. Solvent was removed in vacuum, and the residue was co-evaporated with toluene (3 x 25 mL), then dried in vacuum to give 138 mg of dipeptide TFA salt.

Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.), and dipeptide TFA salt (138 mg) were diluted with dichloromethane (2 mL). To the mixture DEPC (63 pL, 1.5 eq.) was added, followed by DIEA (160 pL, 4 eq.). After 2 h at room temperature the reaction mixture was diluted with dichloromethane (30 mL), washed successively with 10% aq. citric acid (2 x 20 mL), saturated aq. NaCl (20 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in dichloromethane and was purified via flash chromatography on silica gel in a step gradient 0-5 % MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide white foam: 205 mg (81 %). R_f 0.4 (10 % MeOH/CH₂Cl₂). ES-MS mJz 1100.6 [M+H]⁺, 1122.4 [M+Na]⁺.

Fmoc protecting group was removed by treatment with diethylamine (2mL) in dichloromethane (6 mL). After 6 h at room temperature solvent was removed in vacuum, product was isolated by flash chromatography

270 on silica gel in a step gradient O-1O % MeOH in dichloromethane. The relevant fractions were combined and concentrated. After evaporation from dichloromethane/hexane, 1:1, Compound 4 was obtained as a white foam: 133 mg (80 %). R_f 0.15 (10% MeOH/CH₂Cl₂). ES-MS m/z 878.6 [M+H]⁺.

Example 6 - Preparation of compound 5

<img file="IN520DEN2012A_D0138.tif" />

Fmoc-Meval-val-dil-OH (Example 2,0.50 g, 0.78 mmol) and dapphe-OMe-HCl (0.3 g, 0.78 mmol, prepared according to Pettit, G.R., et al. AntiCancer Drug Design 1998,13, 243-277) were dissolved in CH₂C1₂ (10 mL) followed by the addition of diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (0.20 mL, 1.17, 1.5 eq.) was added and the contents stood over Ar. Reaction was complete according to HPLC in 1 h. The mixture was concentrated to an oil and purified by SiO₂ chromatography (300 x 25 mm column) and eluting with 100 % EtOAc. The product was isolated as a white foamy solid. Yield: 0.65 g (87 %). ES-MS m/z 968.35 [M+H]⁺, 991.34 [M+Na]⁺; UV X_max 215, 265 nm.

The Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene chloride (5 mL) was treated with diethylamine (2 mL) and the contents stood at room temperature for 2 h. The reaction, complete by HPLC, was concentrated to an oil, taken up in 2 mL of DMSO and injected into a preparative-HPLC (Ci₂-RP column, 5 μ, 100 A, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100 % in 40 min followed by 20 min at 100 %, at a flow rate of 25 mL/min). Fractions containing the product were evaporated to afford a white powder for the trifluoroacetate salt. Yield: 0.Γ26 g (98 %). R_f 0.28 (100 % EtOAc); ES-MS m/z 746.59 [M+H]⁺, 768.51 [M+Na]⁺; UV λ™» 215 nm.

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Example 7 - Preparation of compound 6

<img file="IN520DEN2012A_D0139.tif" />

nh₂

The trifluoroacetate salt of Compound 5 (0.11 g, 0.13 mmol), Compound AB (0.103 g, 0.14 mmol, 1.1 eq.) and HOBt (3.4 mg, 26 gmol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL, respectively). Diisopropylethylamine (22.5 gL, 0.13 mmol, 1.0 eq.) was added and the yellow solution stirred while under argon. After 3 h, an additional 1.0 eq. of DIEA was added. 24 hours later, 0.5 eq. of the activated linker was included in the reaction mixture. After 40 h total, the reaction was complete. The contents were evaporated, taken up in DMSO and injected into a prep-HPLC (C12-RP column, 5 g, 100 A, linear gradient of MeCN in water (containing 0.1 % TFA) 10 to 100 % in 40 min followed by 20 min at 100 %, at a flow rate of 50 mL/min). The desired fractions were evaporated to give the product as a yellow oil. Methylene chloride (ca. 2 mL) and excess ether were added to provide Compound 6 as a white precipitate that was filtered and dried. Yield: 90 mg (52 %). ES-MS m/z 1344.32 [M+H]⁺, 1366.29 [M+Na]⁺; UV λπ™ 215, 248 nm.

Example 8 - Preparation of compound 7

<img file="IN520DEN2012A_D0140.tif" />

<img file="IN520DEN2012A_D0141.tif" />

272

Compound 4 (133 mg, 0.15 mmol, 1 eq.), Compound AB, (123 mg, 0.167 mmol, 1.1 eq.), and HOBt (4 mg, 0.2 eq.) were diluted with DMF (1.5 mL). After 2 min, pyridine (5 mL) was added and the reaction was monitored using RP-HPLC. The reaction was shown to be complete within 18 h. The reaction mixture was diluted with dichloromethane (20 mL), washed successively with 10 % aq. citric acid (2 x 10 mL), water (10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was resuspended in dichloromethane and was purified via flash chromatography on silica gel in a step gradient 0-10% MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide Compound 7 as a white foam: 46 mg (21 %). R_f 0.15 (10 % MeOH/CH₂Cl₂). ES-MS m/z 1476.94 [M+H]⁺.

Example 9 - Preparation of MC-Val-Cit-PAB-MMAF t-butvl ester 8

<img file="IN520DEN2012A_D0142.tif" />

Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12 mmol, 1.1 eq.), and HOBt (2.8 mg, 21 gmol, 0.2 eq.) were taken up in dry DMF (1.5 mL) and pyridine (0.3 mL) while under argon. After 30 h, the reaction was found to be essentially complete by HPLC. The mixture was evaporated, taken up in a minimal amount of DMSO and purified by prep-HPLC (Ci₂-RP column, 5 μ, 100 A, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100 % in 40 min followed by 20 min at 100 %, at a flow rate of 25 mL/min) to provide Compound 8 as a white solid. Yield: 103 mg (71%). ES-MS m/z 1387.06 [M+H]⁺, 1409.04 [M+Na]⁺; UY 205, 248 nm.

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Example 10 - Preparation of MC-val-cit-PAB-MMAF 9

<img file="IN520DEN2012A_D0143.tif" />

nh₂

Compound 8 (45 mg, 32 gmol) was suspended in methylene chloride (6 mL) followed by the addition of TFA (3 mL). The resulting solution stood for 2 h. The reaction mixture was concentrated in vacuo and purified by prep-HPLC (C12-RP column, 5 μ, 100 A, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100 % in 40 min followed by 20 min at 100 %, at a flow rate of 25 mL/min). The desired fractions were concentrated to provide maleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF (MCval-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MS m/z 1330.29 [M+H]⁺, 1352.24 [M+Na]⁺; UV 205, 248 nm.

Example 11 - Preparation of MC-val-cit-PAB-MMAF tert-butyl amide 10

<img file="IN520DEN2012A_D0144.tif" />

Compound 3 (217 mg, 0.276 mmol, 1.0 eq.), Compound AB (204 mg, 0.276 mmol, 1.0 eq.), and HOBt (11 mg, 0.0828 mmol, 0.3 eq.) were diluted with pyridine/DMF (6 mL). To this mixture was added DIEA (0.048 mL), and the

274 mixture was stirred ca. 16 hr. Volatile organics were evaporated in vacuo. The crude residue was purified by Chromatotron® (radial thin-layer chromatography) with a step gradient (0-5-10% methanol in DCM) to provide MC-val-cit-PABMMAF tert-butyl amide 10. Yield: 172 mg (45 %); ES-MS m/z 1386.33 [M+H]⁺, 1408.36 [M+Na]⁺; UV ^ax 215, 248 nm.

Example 12 - Preparation of AC10-MC-MMAE by conjugation of AC10 and MC-MMAE

AC 10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 °C for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with ImM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-monomethyl auristatin E, i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to the chilled reduced antibody AC 10 in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and AC10-MC-MMAE is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 μιη filters under sterile conditions, and frozen for storage.

Example 13 - Preparation of AC10-MC-MMAF by conjugation of AC 10 and MC-MMAF

AC10-MC-MMAF was prepared by conjugation of AC 10 and MCMMAF following the procedure of Example 12.

Example 14 - Preparation of AC10-MC- val-cit-PAB-MMAE by conjugation of AC 10 and MC-val-cit-PAB-MMAE

275

AClO-MC-val-cit-PAB-MMAE was prepared by conjugation of AC 10 and MC-val-cit-PAB-MMAE following the procedure of Example 12.

Example 15 - Preparation of AC10-MC- val-cit-PAB-MMAF by conjugation of AC10 and MC-val-cit-PAB-MMAF (9)

AClO-MC-val-cit-PAB-MMAF was prepared by conjugation of AC 10 and MC-val-cit-PAB-MMAF (9) following the procedure of Example 12.

Example 16 - Determination of cytotoxicity of selected compounds

Cytotoxic activity of MMAF and Compounds 1-5 was evaluated on the Lewis Y positive cell lines OVCAR-3, H3396 breast carcinoma, L2987 lung carcinoma and LS174t colon carcinoma Lewis Y positive cell lines can be assayed for cytotoxicity. To evaluate the cytotoxicity of Compounds 1-5, cells can be seeded at approximately 5 - 10,000 per well in 150 μΐ of culture medium then treated with graded doses of Compounds 1-5 in quadruplicates at the initiation of assay. Cytotoxicity assays are usually carried out for 96 hours after addition of test compounds. Fifty μΐ of resazurin dye may be added to each well during the last 4 to 6 hours of the incubation to assess viable cells at the end of culture. Dye reduction can be determined by fluorescence spectrometry using the excitation and emission wavelengths of 535nm and 590nm, respectively. For analysis, the extent of resazurin reduction by the treated cells can be compared to that of the untreated control cells.

For 1 h exposure assays cells can be pulsed with the drug for 1 h and then washed; the cytotoxic effect can be determined after 96 h of incubation. EXAMPLE 17 - in vitro cytotoxicity cata for selected compounds

Table 10 shows cytotoxic effect of cAClO Conjugates of Compounds 7-10, assayed as described in General Procedure I on a CD30+ cell line Karpas 299. Data of two separate experiments are presented. The cAClO conjugates of Compounds 7 and 9 were found to be slightly more active than cAC10-val-cit-MMAE. ' ·

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TABLE 10

<td> Conjugate</td><td> IC50 (ng/mL)</td>

<td> c AC 10-val-cit-MMAE</td><td> 6</td>

<td> cAC10-7</td><td> 1.0</td>

<td> cAClO-8</td><td> 15</td>

<td> cAClO-9</td><td> 0.5</td>

<td> cAClO-10</td><td> 20</td>

In other experiments, BR96-val-cit-MMAF was at least 250 fold more potent than the free MMAF.

General Procedure I - Cytotoxicity determination. To evaluate the cytotoxicity of Exemplary Conjugates 7-10, cells were seeded at approximately 5 - 10,000 per well in 150 μΐ of culture medium then treated with graded doses of Exemplary Conjugates 7-10 in quadruplicates at the initiation of assay. Cytotoxicity assays were carried out for 96 hours after addition of test compounds. Fifty μΐ of the resazurin dye was added to each well during the last 4 to 6 hours of the incubation to assess viable cells at the end of culture. Dye reduction was determined by fluorescence spectrometry using the excitation and emission wavelengths of 535nm and 590nm, respectively. For analysis, the extent of resazurin reduction by the treated cells was compared to that of the untreated control cells.

Example 18 - Zn vitro cell proliferation assay

Efficacy of ADC can be measured by a cell proliferation assay employing the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488):

1. An aliquot of 100 μΐ of cell culture containing about 10⁴ cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each well of a 96well, opaque-walled plate.

2. Control wells were prepared containing medium and without cells.

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

4. The plates were equilibrated to room temperature for approximately 30 minutes.

277

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culture medium present in each well was added.

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

7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU = relative luminescence units.

Example 19 - Plasma clearance in rat

Plasma clearance pharmacokinetics of antibody drug conjugates and total antibody was studied in Sprague-Dawley rats (Charles River Laboratories, 250-275 gms each). Animals were dosed by bolus tail vein injection (IV Push). Approximately 300 μΐ whole blood was collected through jugular cannula, or by tail stick, into lithium/heparin anticoagulant vessels at each timepoint: 0 (predose), 10, and 30 minutes; 1, 2,4, 8, 24 and 36 hours; and 2, 3,4, 7, 14, 21, 28 days post dose. Total antibody was measured by ELISA ECD/GxhuFc-HRP. Antibody drug conjugate was measured by ELISA MMAE/MMAF/ECD-Bio/SA-HRP.

Example 20 - Plasma clearance in monkey

Plasma clearance pharmacokinetics of antibody drug conjugates and total antibody can be studied in cynomolgus monkeys. Figure 12 shows a two-stage plasma concentration clearance study after administration of H-MC-vcMMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg, administered at day 1 and day 21. Concentrations of total antibody and ADC were measured over time. (H = Trastuzumab).

Example 21 - Tumor volume in vivo efficacy in transgenic explant mice

Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many strains are suitable, but FVB female mice are preferred because of their higher

278 susceptibility to tumor formation. FVB males can be used for mating and vasectomized CD.l studs can be used to stimulate pseudopregnancy. Vasectomized mice can be obtained from any commercial supplier. Founders can be bred with either FVB mice or with 129/BL6 x FVB p53 heterozygous mice. The mice with heterozygosity at p53 allele can be used to potentially increase tumor formation. Some Fl tumors are of mixed strain. Founder tumors can be FVB only.

Animals having tumors (allograft propagated from Fo5 mmtv transgenic mice) can be treated with a single or multiple dose by IV injection of ADC. Tumor volume can be assessed at various time points after injection.

Example 22 - Synthesis of MC-MMAF via t-butyl ester

Synthesis 1:

<img file="IN520DEN2012A_D0145.tif" />

<img file="IN520DEN2012A_D0146.tif" />

<img file="IN520DEN2012A_D0147.tif" />

<img file="IN520DEN2012A_D0148.tif" />

MC-MMAF

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163 mmol) was suspended in CH2CI2 (0.500 mL). 6-Maleimidocaproic acid (68.9 mg, 0.326 mmol) and 1,3-diisopropylcarbodiimide (0.0505 mL, 0.326 mmol) were added followed by pyridine (0.500 mL). Reaction mixture was allowed to stir for

279

1.0 hr. HPLC analysis indicated complete consumption of starting compound 1. Volatile organics were evaporated under reduced pressure. Product was isolated via flash column chromatography, using a step gradient from 0 to 5% Methanol in CH₂C1₂. A total of 96 mg of pure MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (60% yield) was recovered. ES-MS m/z 981.26 [M+H]⁺; 1003.47 [M+Na]⁺; 979.65 [M-H]'.

MC-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 12, 74 mg, 0.0754 mmol) was suspended in CH₂C1₂ (2.0 mL) and TFA (1 mL) at room temperature. After 2.5 hr, HPLC analysis indicated complete consumption of starting material. Volatile organics were evaporated under reduced pressure, and the product was isolated via preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80A Column (250 x 21.20 mm). Eluent: linear gradient 10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05% TFA (aq) for an additional 20 minutes. ES-MS m/z 925.33 [M+H]⁺; 947.30 [M+Na]⁺; 923.45 [M-H]’.

Example 23a - Synthesis of MC-MMAF (11) via dimethoxybenzyl ester

Synthesis 2:

280

<img file="IN520DEN2012A_D0149.tif" />

Ή

<img file="IN520DEN2012A_D0150.tif" />

<img file="IN520DEN2012A_D0151.tif" />

Fmoc-Phe

<img file="IN520DEN2012A_D0152.tif" />

Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB

<img file="IN520DEN2012A_D0153.tif" />

<img file="IN520DEN2012A_D0154.tif" />

<img file="IN520DEN2012A_D0155.tif" />

<img file="IN520DEN2012A_D0156.tif" />

MC-MMAF

Preparation of Fmoc-L-Phenylalanine-2,4-dimethoxybenzyl ester (Fmoc-Phe-ODMB)

281

A 3-neck, 5-L round-bottom flask was charged with Fmoc-LPhenylalanine (200 g, 516 mmol Bachem), 2,4-dimethoxybenzyl alcohol (95.4 g, 567 mmol, Aldrich), and CH2C12 (2.0 L). Ν,Ν-dimethylformamide t-butyl acetal (155 mL, 586 mmol, Fluka) was added to the resulting suspension over 20 min under N2, which resulted in a clear solution. The reaction was then stirred at room temperature overnight, after which time TLC analysis (0.42, Heptane/EtOAc = 2:1) indicated that the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a light yellow oil, which was redissolved in CH2C12 (200 mL) and purified through a short plug of silica gel (25 cm x 25 cm, CH2C12) to give a colorless foam (250 g). MeCN (1 L) was added into the resulting foam, which totally dissolved the batch. It was then concentrated to dryness and redissolved in MeCN (1 L) and the resulting suspension was stirred for 1 h, filtered and the filter cake was rinsed with MeCN (2 x 200 mL) to give Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid (113.58 g, 41%, 95.5% AUC by HPLC analysis). Data: HPLC.

Preparation L-Phenylalanine-2,4-dimethoxybenzyl ester (PheODMB)

A 500-mL round-bottom flask was charged with Fmoc-Lphenylalanine-2,4-dimethoxybenzyl ester (26.00g, 48.3 mmol), CH2C12 (150 mL) and diethylamine (75 mL, Acros). Mixture was stirred at room temperature and the completion monitored by HPLC. After 4h, the mixture was concentrated (bath temp &lt;30 °C). The residue was resuspended in CH2C12 (200 mL) and concentrated. This was repeated once. To the residue was added MeOH (20 mL), which caused the formation of a gel. This residue was diluted with CH2C12 (200 mL), concentrated and the cloudy oil left under vacuum overnight. The residue was suspended in CH2C12 (100 mL), then toluene (120mL) was added. The mixture was concentrated and the residue left under vacuum overnight.

Data: HPLC, 1H NMR.

Preparation of Fmoc-Dolaproine (Fmoc-Dap)

Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HC1 in

1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC

282 analysis indicated the reaction was complete (10% MeOH/CH2C12) and the mixture was concentrated to near-dryness. Additional 1,4-dioxane was charged (50 mL) and the mixture was concentrated to dryness and dried under vacuum overnight. The resulting white solid was dissolved in H2O (400 mL) and . transferred to a 3-L, three-neck, round-bottom flask with a mechanical stirrer and temperature probe. Ν,Ν-diisopropylethylamine (214.3 mL, 1.23 mol, Acros) was added over one minute, causing an exotherm from 20.5 to 28.2 °C (internal). The mixture was cooled in an ice bath and 1,4-dioxane was added (400 mL). A solution of Fmoc-OSu (89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 mL) was added from an addition funnel over 15 minutes, maintaining the reaction temperature below 9 °C. The mixture was allowed to warm to room temperature and stir for 19 hours, after which the mixture was concentrated by rotary evaporation to an aqueous slurry (390 g). The suspension was diluted with H2O (750 mL) and Et20 (750 mL), causing a copious white precipitate to form. The layers were separated, keeping the solids with the organic layer. The aqueous layer was acidified using cone. HC1 (30 mL) and extracted with EtOAc (3 x 500 mL). The combined extracts were dried over MgSO4, filtered and concentrated to give 59.25 g of a yellow oil A. The Et2O extract was extracted once with sat. NaHCO3 (200 mL), keeping the solids with the aqueous layer. The aqueous suspension was acidified using cone. HC1 (50 mL) and extracted with Et20 (50 mL) keeping the solids with the organic layer. The organic layer was filtered and concentrated to give 32.33 g of a yellow oil B. The two oils (A and B) were combined and purified by flash chromatography on silica gel eluting with CH2C12 (3.5 L), then 3% MeOH/ CH2C12 (9 L) to give 68.23 g of Fmoc-dolaproine as a white foam (81%, 97.5% purity by HPLC (AUC)).

Preparation of Fmoc-Dap-Phe-ODMB

Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 mL, Acros) for 5 minutes and Fmoc-Dap (19.80g, 48.3 mmol) was added. The mixture was cooled in an ice bath and TBTU (17.08 g, 53.20 mmol, Matrix Innovations) was added. Ν,Ν-diisopropylethylamine (25.3 mL, 145.0 mmol,

283

Acros) was added via syringe over 3 min. After lh, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (1 L) and extracted with ethyl acetate (300 mL). After separation, the aqueous layer was re-extracted with ethyl acetate (2 x 150 mL). The combined organic layers were washed with brine (150 mL), dried (MgSO4) and filtered (filter paper) to remove the insolubles (inorganics and some dibenzofulvene). After concentration, the residue (41 g) was adsorbed on silica (41 g) and purified by chromatography (22 cm x 8 cm column; 65% Heptane/EtOAc (2.5 L); 33% Heptane/EtOAc (3.8 L), to give 29.4 g of product as a white foam (86%, 92% purity by HPLC).

Data: HPLC, 1H NMR, TLC (1:1 EtOAc/Heptane Rf= 0.33, red in vanillin stain).

Preparation of Dap-Phe-ODMB

A 1-L round bottom flask was charged with Fmoc-Dap-PheODMB (27.66 g), CH2C12 (122 mL) and diethylamine (61 mL, Acros). The solution was stirred at room temperature and the completion monitored by HPLC. After 7h, the mixture was concentrated (bath temp. &lt;30 °C). The residue was suspended in CH2C12 (300 mL) and concentrated. This was repeated twice. To the residue was added MeOH (20 mL) and CH2C12 (300 mL), and the solution was concentrated. The residue was suspended in CH2C12 (100 mL) and toluene (400mL), concentrated, and the residue left under vacuum overnight to give a cream-like residue.

Data: HPLC, 1H NMR, MS.

Preparation of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB

Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135 mL, Acros) for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94g, 39.1 mmol, see Example 2 for preparation) was added. The mixture was cooled in an ice bath and TBTU (13.8lg, 43.Ό mmol, Matrix Innovations) was added. N,NDiisopropylethylamine (20.5 mL, 117.3 mmol, Acros) was added via syringe over

284 minutes. After 1 hour, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (1.5 L) and diluted with ethyl acetate (480 mL). After standing for 15 minutes, the layers were separated and the aqueous layer was extracted with ethyl acetate (300 mL). The combined organic layers were washed with brine (200 mL), dried (MgSO4) and filtered (filter paper) to remove insolubles (inorganics and some dibenzofulvene). After concentration, the residue (49 g) was scraped from the flask and adsorbed on silica (49 g) and purified by chromatography (15 cm x 10 cm dia column; 2:1 EtOAc/Heptane (3 L), EtOAc (5 L); 250 mL fractions) to give 31.84 g of FmocMeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%, 93% purity by HPLC (AUC)).

Data: HPLC, TLC (2:1 EtOAc/heptane, Rf = 0.21, red in vanillin stain).

Preparation of MeVal-Val-Dil-Dap-Phe-ODMB

A 1-L, round-bottom flask was charged with Fmoc-MeVal-ValDil-Dap-Phe-ODMB (28.50 g), CH2C12 (80 mL) and diethylamine (40 mL). Mixture was stirred at room temperature overnight and then was concentrated under reduced pressure. The residue was adsorbed on silica (30 g) and purifed by flash chromatography (15 cm x 8 cm dia column; 2% MeOH/DCM (2 L), 3% MeOH/DCM (1 L), 6% MeOH/DCM (4 L); 250 mL fractions) to give 15.88 g of MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (69%, 96% purity by HPLC (AUC)).

Data: HPLC, TLC (6% MeOH/DCM, Rf = 0.24, red in vanillin stain).

Preparation of MC-MeVal-Val-Dil-Dap-Phe-ODMB ^v A 50-mL, round-bottom flask was charged with MeVal-Val-DilDap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4 mL),

285 maleimidocaproic acid (180 mg, 0.85 mmol), and TBTU (300 mg, 0.93mmol, Matrix Innovations) at room temperature. N,N-Diisopropylethylamine (450 pL, 2.57 mmol) was added via syringe. After 1.5 hours, the mixture was poured in water (50 mL) and diluted with ethyl acetate (30 mL). NaCl was added to improve the separation. After separation of the layers, the aqueous layer was extracted with ethyl acetate (25 mL). The combined organic layers were dried (MgSO4), filtered and concentrated. The resulting oil (1 g) was purified by flash chromatography [100 mL silica; 25% Heptane/EtOAc (100 mL), 10% Heptane/EtOAc (200 mL), EtOAc (1.5 L)] to give MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg, 57%, 94% purity by HPLC(AUC)).

Data: 1H NMR, HPLC.

Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF) (ID

A 50-mL, round-bottom flask was charged with MC-MeVal-ValDil-Dap-Phe-ODMB (Compound 13,428 mg, 0.39 mmol) and dissolved in 2.5% TFA/CH2C12 (20 mL). The solution turned pink-purple over 2 min. The completion was monitored by HPLC and TLC (6% MeOH/DCM, KMnO4 stain). After 40 min, three drops of water were added and the cloudy pink-purple mixture was concentrated to give 521 mg of a pink residue. Purification by chromatography (15% IPA/DCM) gave 270 mg of MC-MMAF (73%, 92% purity by HPLC) as a white solid.

Example 23b - Synthesis of analog of mc-MMAF

286 p

<img file="IN520DEN2012A_D0157.tif" />

ο

<img file="IN520DEN2012A_D0158.tif" />

<img file="IN520DEN2012A_D0159.tif" />

<img file="IN520DEN2012A_D0160.tif" />

<img file="IN520DEN2012A_D0161.tif" />

MB-MeM-Val-Dil-Dap-Phe -OtBu

<img file="IN520DEN2012A_D0162.tif" />

MB-MMAF

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol) was suspended in DMF (0.250 mL). 4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)benzoic acid (11 mg, 0.049 mmol) and HATU (17 mg, 0.044 mmol) were added followed by DIEA (0.031 mL, 0.17 mmol). This reaction mixture was allowed to stir for 2.0 hr. HPLC analysis indicated complete consumption of starting compound 1.

Product was isolated via preparatory RP-HPLC, using a Phenomenex Ci₂ Synergi Max-RP 80A Column (250 x 21.20 mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes. A total of 20 mg of pure product (14) was isolated (0.02 mmol, 46% yield). ES-MS m/z 987.85 [M+H]⁺; 1019.41 [M+Na]⁺; 985.54 [M-H]'.

MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14, 38 mg, 0.0385 mmol) was suspended in CH₂C1₂ (1 mL) and TFA (1 mL). Mixture was stirred

287 for 2.0 hr, and then volatile organics were evaporated under reduced pressure. Product was purified by preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80A Column (250 x 21.20 mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes. A total of 14.4 mg of MB-MMAF product was isolated (0.015 mmol, 40% yield). ES-MS mJz 930.96 [M+H]⁺ 952.98 [M+Na]⁺; 929.37 [M-H]'.

Example 23c - Preparation of MC-MeVal-Cit-PAB-MMAF (16)

<img file="IN520DEN2012A_D0163.tif" />

To a room temperature suspension of Fmoc-MeVal-OH (3.03 g, 8.57 mmol) and Ν,Ν’-disuccimidyl carbonate (3.29 g, 12.86 mmol) in CH2CI2 (80 mL) was added DIEA (4.48 mL, 25.71 mmol). This reaction mixture was allowed to stir for 3.0 hr, and then poured into a separation funnel where the organic mixture was extracted with 0.1 M HC1 (aq). The crude organic residue was concentrated under reduced pressure, and the product was isolated by flash column chromatography on silica gel using a 20-100% ethyl acetate/hexanes linear gradient. A total of 2.18 g of pure Fmoc-MeVal-OSu (4.80 mmoles, 56% yield) was recovered.

To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g, 4.84 mmol) in DME (13 mL) and THF (6.5 mL) was added a solution of Lcitrulline (0.85 g, 4.84 mmol) and NaHCO₃ (0.41 g, 4.84 mmol) in H₂O (13 mL). The suspension was allowed to stir at room temperature for 16 hr, then it was extracted into ?eri-BuOH/CHCl₃/H₂O, acidified to pH=2-3 with 1 M HC1. The

288 organic phase was separated, dried and concentrated under reduced pressure. The residue was triturated with diethyl ether resulting in 2.01 g of Fmoc-MeVal-CitCOOH which was used without further purification.

The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1 CH₂Cl₂/MeOH (100 mL), and to it was added p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g, 7.9 mmol). This suspension was allowed to stir for 125 hr, then the volatile organics were removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel using a 10% MeOH/CH₂Cl₂. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol, 18.5 % yield) was recovered. ES-MS m/z 616.48 [M+H]⁺.

To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55g, 0.896 mmol) in CH₂C1₂ (40 mL) was added STRATOSPHERES^tm(piperizine-resinbound) (&gt;5 mmol/g, 150 mg). After being stirred at room temperature for 16 hr the mixture was filtered through celite (pre-washed with MeOH), and concentrated under reduced pressure. Residue was triturated with diethyl ether and hexanes. Resulting solid material, MeVal-Cit-PAB-OH, was suspended in CH₂C1₂ (20 mL), and to it was added MC-OSu (0.28 g, 0.896 mmol), DIEA (0.17 mL, 0.99 mmol), and DMF (15 mL). This suspension was stirred for 16 hr, but HPLC analysis of the reaction mixture indicated incomplete reaction, so the suspension was concentrated under reduced pressure to a volume of 6 mL, then a 10% NaHCCh (aq) solution was added and the suspension stirred for an additional 16 hr. Solvent was removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel using a 0-10% MeOH/CH₂Cl₂gradient, resulting in 42 mg (0.072 mmol, 8% yield) of MC-MeVal-Cit-PAB-OH.

To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) and bis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in CH₂C1₂ (10 mL) was added DIEA (1.06 mL, 6.06 mmol). This suspension was stirred for 5.5 hr, concentrated under reduced pressure and purified by trituration with diethyl ether. MC-MeValCit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a 1:5 pyridine/DMF solution (3 mL), and to it was added HOBt (5 mg, 0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAF (compound 2, 150 mg, 0.205 mmol). This reaction

289 mixture was stirred for 16 hr at room temperature, and then purified by preparatory RP-HPLC (x3), using a Phenomenex C12 Synergi Max-RP 80A Column (250 x 21.20 mm). Eluent: linear gradient 10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05% TFA (aq) for an additional 20 minutes. MC-MeVal-Cit-PAB-MMAF (16) was obtained as a yellowish solid (24.5 mg, 0.0182, 0.45 % yield). ES-MS m/z 1344.95 [M+H]⁺; 1366.94 [M+Na]⁺.

Example 23d - Preparation of succinimide ester of suberyl-Val-Cit-PAB

MMAF (17)

<img file="IN520DEN2012A_D0164.tif" />

Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol, 1.5 eq.) were suspended in anhydrous pyridine, 5 mL. HOBt (10 mg, 0.076 mmol, 0.2 eq.) was added followed by DIEA (199 pl, 1.14 mmol, 3 eq.). Reaction mixture was sonicated for 10 min, and then stirred overnight at room temperature. Pyridine was removed under reduced pressure, residue was resuspended in CH2CI2. Mixture was separated by silica gel flash chromatography in a step gradient of MeOH, from 0 to 10%, in CH₂Cl₂.Product containing fractions were pulled , concentrated, dried in vacuum overnight to give 317 mg ( 59% yield) of Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m/z 1415.8 [M+H]⁺.

Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20% TFA/CH2CI2 (10 mL), for 2 hrs. Mixture was diluted with CH₂C1₂ (50 mL). Organic layer was washed successively with water (2 x 30 mL) and brine (1 x 30 mL). Organic phase was concentrated, loaded onto pad of silica gel in 10%

290

MeOH/CHiCh. Product was eluted with 30% MeOH/CIUCL. After drying in vacuum overnight, Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid, 38 mg, 40% yield. ES-MS m/z 1357.7 [M-H]'.

Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH2CI2 (2 mL) diethylamine (2 mL) and DMF (2 mL). Mixture was stirred for 2 hrs at room temperature. Solvent was removed under reduced pressure. Residue was coevaporated with pyridine (2 mL), then with toluene (2x5 mL), dried in vacuum. Val-Cit-PAB-MMAF was obtained as brownish oil, and used without further purification.

All Val-Cit-PAB-MMAF prepared from 67 mg of Fmoc-Val-CitPAB-MMAF, was suspended in pyridine (2 mL), and added to a solution of disuccinimidyl suberate (74 mg, 0.2 mmol, 4 eq.), in pyridine (1 mL). Reaction mixture was stirred at room temperature. After 3 hrs ether (20 mL) was added. Precipitate was collected, washed with additional amount of ether. Reddish solid was suspended in 30% MeOH/CfLCL, filtered trough a pad of silica gel with 30% MeOH/CH₂C12 as an eluent. Compound 17 was obtained as white solid, 20 mg (29% yield). ES-MS m/z 1388.5 [M-H]'

Example 24 - In vivo Efficacy of mcMMAF Antibody-Drug Conjugates

Efficacy of cAClO-mcMMAF in Karpas-299 ALCL xenografts: To evaluate the in vivo efficacy of cAClO-mcMMAF with an average of 4 drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL cells were implanted subcutaneously into immunodeficient C.B-17 SCID mice (5xl0⁶ cells per mouse). Tumor volumes were calculated using the formula (0.5xLxW²) where L and W are the longer and shorter of two bidirectional measurements. When the average tumor volume in the study animals reached approximately 100 mm³ (range 48-162) the mice were divided into 3 groups (5 mice per group) and were either left untreated or were given a single intravenous injection through the tail vein of either 1 or 2 mg/kg cAC10-mcF4 (Figure 1). The tumors in the untreated mice grew rapidly to an average volume of &gt;1,000 mm³ within Ί days of the start of therapy. In contrast, all of the cAC10-mcF4 treated tumor showed

291 rapid regression with 3/5 in the 1 mg/kg group and 5/5 in the 2 mg/kg group obtaining complete tumor response. While the tumor in one of the complete responders in the 2 mg/kg group did recur approximately 4 weeks later, there were no detectable tumors in the remaining 4/5 responders in this group and in the 3 complete responders in the 1 mg/kg group at 10 weeks post therapy.

Efficacy of cBR96-mcMMAF in L2987 NSCLC xenografts·. cBR96 is a chimeric antibody that recognizes the Le^Y antigen. To evaluate the in vivo efficacy of cBR96-mcMMAF with 4 drugs per antibody (cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC) tumor fragments were implanted into athymic nude mice. When the tumors averaged approximately 100 mm³ the mice were divided into 3 groups: untreated and 2 therapy groups. For therapy, as shown in Figure 3a, mice were administered cBR96-mcF4 at either 3 or 10 mg/kg/injection every 4 days for a total of 4 injections (q4dx4). As shown in Figure 3b, mice were administered cBR96-mcF4 or a non-binding control conjugate, cAC10-mcF4, at 10 mg/kg/injection every 4 days for a total of 4 injections (q4dx4). As shown in Figures 3a and 3b, BR96-mcF4 produced pronounced tumor growth delay compared to the controls.

Figure 2 shows an in vivo, single dose, efficacy assay of cAClOmcMMAF in subcutaneous L540CY. For this study there were 4 mice in the untreated group and 10 in each of the treatment groups.

Example 25 - in vitro efficacy of MC-MMAF Antibody-Drug Conjugates

Activity of cAClO-antibody-drug conjugates against CD30⁺ cell lines. Figures 4a and 16b show dose-response curves from a representative experiment where cultures of Karpas 299 (anaplastic large cell lymphoma) and L428 (Hodgkin’s Lymphoma) were incubated with serially diluted cAClOmcMMAF (Figure 4a) or cAClO-vcMMAF (Figure 4b) for 96 hours. The cultures were labeled for 4 hours with 50 μΜ resazurin [7-hydroxy-3Hphenoxazin-3-one 10-oxide] and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC₅₀ values are defined as the concentration where growth is

292 reduced 50% compared with untreated control cultures. Each concentration was tested in quadruplicate.

Activity of cBR96-antibody-drug conjugates against Leⁱ⁺ cell lines. Figures 5a and 5b show dose-response curves from a representative experiment where cultures of H3396 (breast carcinoma) and L2987 (non small cell lung carcinoma) were incubated with serially diluted cBR96-mcMMAF (Figure 5a) or -vcMMAF (Figure 5b) for 96 hours. The cultures were labeled for 4 hours with 50 μΜ resazurin and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC50 values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration is tested in quadruplicate.

Activity ofclF6-antibody-drug conjugates against CD70⁺ renal cell carcinoma cell lines. Figures 6a and 6b show dose-response curves from a representative experiment where cultures of Caki-1 and 786-0 cells were incubated with serially diluted clF6-mcMMAF (Figure 6a) or-vcMMAF (Figure 6b) for 96 hours. The cultures were labeled for 4 hours with 50 μΜ resazurin and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC50 values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration is tested in quadruplicate.

Example 26 - Purification of trastuzumab

One vial containing 440 mg HERCEPTIN® (huMAb4D5-8, rhuMAb HER2, U.S. Patent No. 5821337) antibody) was dissolved in 50 mL MES buffer (25 mM MES, 50 mM NaCl, pH 5.6) and loaded on a cation exchange column (Sepharose S, 15 cm x 1.7 cm) that had been equilibrated in the same buffer. The column was then washed with the same buffer (5 column volumes). Trastuzumab was eluted by raising the NaCl concentration of the buffer to 200 mM. Fractions containing the antibody were pooled, diluted to 10

293 mg/mL, and dialyzed into a buffer containing 50 mm potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.

Example 27 - Preparation of trastuzumab-MC-MMAE by conjugation of trastuzumab and MC-MMAE

Trastuzumab, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 °C for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with ImM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-monomethyl auristatin E (MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to the chilled reduced antibody trastuzumab in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and trastuzumab-MC-MMAE is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 pm filters under sterile conditions, and frozen for storage.

Example 28 - Preparation of trastuzumab-MC-MMAF by conjugation of trastuzumab and MC-MMAF

Trastuzumab-MC-MMAF was prepared by conjugation of trastuzumab and MC-MMAF following the procedure of Example 27.

Example 29 - Preparation of trastuzumab-MC- val-cit-PAB-MMAE by conjugation of trastuzumab and MC-val-cit-PAB-MMAE

294

Trastuzumab-MC-val-cit-PAB-MMAE was prepared by conjugation of trastuzumab and MC-val-cit-PAB-MMAE following the procedure of Example 27.

Example 30 - Preparation of trastuzumab-MC- val-cit-PAB-MMAF by conjugation of trastuzumab and MC-val-cit-PAB-MMAF 9

Trastuzumab-MC-val-cit-PAB-MMAF was prepared by conjugation of trastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of Example 27.

Example 31 - Rat toxicity

The acute toxicity profile of free drugs and ADC was evaluated in adolescent Sprague-Dawley rats (75-125 gms each, Charles River Laboratories (Hollister, CA). Animals were injected on day 1, complete chemistry and hematology profiles were obtained at baseline, day 3 and day 5 and a complete necropsy was performed on day 5. Liver enzyme measurements was done on all animals and routine histology as performed on three random animals for each group for the following tissues: sternum, liver, kidney, thymus, spleen, large and small intestine. The experimental groups were as follows:

<td> Grou P</td><td> Administered</td><td> mg/kg</td><td> pg MMAF/ 2 m</td><td> MMAF/ MAb</td><td> N/Sex</td>

<td> 1</td><td> Vehicle</td><td> 0</td><td> 0</td><td> 0</td><td> 2/F</td>

<td> 2</td><td> trastuzumab-MC-val-citMMAF</td><td> 9.94</td><td> 840</td><td> 4.2</td><td> 6/F</td>

<td> 3</td><td> trastuzumab-MC-val-citMMAF</td><td> 24.90</td><td> 2105</td><td> 4.2</td><td> 6/F</td>

<td> 4</td><td> trastuzumab-MC(Me)-valcit-PAB-MMAF</td><td> 10.69</td><td> 840</td><td> 3.9</td><td> 6/F</td>

<td> 5</td><td> trastuzumab-MC(Me)-valcit-PAB-MMAF</td><td> 26.78</td><td> 2105</td><td> 3.9</td><td> 6/F</td>

<td> 6</td><td> trastuzumab-MC-MMAF</td><td> 10.17</td><td> 840</td><td> 4.1</td><td> 6/F</td>

<td> 7</td><td> trastuzumab-MC-MMAF</td><td> 25.50</td><td> 2105</td><td> 4.1</td><td> 6/F</td>

<td> 8</td><td> trastuzumab-MC-val-citPAB-MMAF</td><td> 21.85</td><td> 2105</td><td> 4.8</td><td> 6/F</td>

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For trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-valcit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PABMMAF, the pg MMAF/m² was calculated using 731.5 as the MW of MMAF and 145167 as the MW of Herceptin.

The body surface area was calculated as follows: [{(body weight in grams to 0.667 power) x 11.8)/10000]. (Guidance for Industry and Reviewers, 2002).

The dose solutions were administered by a single intravenous bolus tail-vein injection on Study Day 1 at a dose volume of 10 mL/kg. Body weights of the animals were measured pre-dose on Study Day 1 and daily thereafter. Whole blood was collected into EDTA containing tubes for hematology analysis. Whole blood was collected into serum separator tubes for clinical chemistry analysis. Blood samples were collected pre-dose on Study Day -4, Study Day 3 and Study Day 5. Whole blood was also collected into sodium heparin containing tubes at necropsy and the plasma was frozen at -70°C for possible later analysis. The following tissues were collected and placed in neutral buffered formalin at necropsy: liver, kidneys, heart, thymus, spleen, brain, sternum and sections of the GI tract, including stomach, large and small intestine. Sternum, small intestine, large intestine, liver, thymus, spleen and kidney were examined.

Liver associated serum enzyme levels at each timepoint were compared to a range (5th and 95th percentile) from normal female SpragueDawley rats. White blood cell and platelet counts at each timepoint were compared to a range (5th and 95th percentile) from normal female SpragueDawley rats.

High dose study in normal female Sprague-Dawley rats: Group 1: Vehicle

Group 2: trastuzumab-MC-MMAF, 52.24mg/kg, 4210pg/m²

Group 3: trastuzumab-MC-MMAF, 68.25mg/kg, 5500pg/m²

Group 4: trastuzumab-MC-MMAF, 86.00mg/kg, 6930pg/m²

Tissues from 11 animals were submitted for routine histology. These animals had been part of an acute dose-ranging toxicity study using a

296 trastuzumab-MC-MMAF immunoconjugate. Animals were followed for 12 days following dosing.

Example 32 - Cynomolgus Monkey Toxicity/Safety

Three groups of four (2 male, 2 female) naive Macacafascicularis (cynomolgus monkey) were studied for trastuzumab-MC-vc-PAB-MMAE and trastuzumab-MC-vc-PAB-MMAF. Intravenous administration was conducted at days 1 and 22 of the studies.

<td> Sample</td><td> Group</td><td> Dose</td>

<td> Vehicle</td><td> 1 1M/1F</td><td> day 1 day 22</td>

<td> H-MC-vc-PAB-MMAE</td><td> 2 2M/2F</td><td> 180 gg/m² (0.5 mg/kg) at day 1 1100 gg/m² (3.0 mg/kg) at day 22</td>

<td> H-MC-vc-PAB-MMAE</td><td> 3 2M/2F</td><td> 550 gg/m² (1.5 mg/kg) at day 8 550 gg/m² (1.5 mg/kg) at day 29</td>

<td> H-MC-vc-PAB-MMAE</td><td> 4 2M/2F</td><td> 880 gg/m² (2.5 mg/kg) at day 15 880 gg/m² (2.5 mg/kg) at day 36</td>

<td> Sample</td><td> Group</td><td> Dose</td>

<td> Vehicle</td><td> 1 1M/1F</td><td> day 1 day 22</td>

<td> H-MC-vc-PAB-MMAF</td><td> 2 2M/2F</td><td> 180 gg/m² (0.5 mg/kg) at day 1 1100 gg/m² (3.0 mg/kg) at day 22</td>

<td> H-MC-vc-PAB-MMAF</td><td> 3 2M/2F</td><td> 550 gg/m² (1.5 mg/kg) at day 1 550 gg/m² (1.5 mg/kg) at day 22</td>

<td> H-MC-vc-PAB-MMAF</td><td> 4 2M/2F</td><td> 880 gg/m² (2.5 mg/kg) at day 1 880 gg/m² (2.5 mg/kg) at day 22</td>

H = trastuzumab

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Dosing is expressed in surface area of an animal so as to be relevant to other species, i.e. dosage at gg/m² is independent of species and thus comparable between species. Formulations of ADC contained PBS, 5.4 mM sodium phosphate, 4.2 mM potassium phosphate, 140 mM sodium chloride, pH 6.5.

Blood was collected for hematology analysis predose, and at 5 min., 6 hr, 10 hr, and 1, 3, 5, 7,14, 21 days after each dose. Erythrocyte (RBC) and platelet (PLT) counts were measured by the light scattering method. Leukocyte (WBC) count was measured by the peroxidase/basophil method. Reticulocyte count was measured by the light scattering method with cationic dye. Cell counts were measured on an Advia 120 apparatus. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were measured in U/L by UV/NADH; IFCC methodology on an Olympus AU400 apparatus, and using Total Ab ELISA - ECD/GxhuFc-HRP. Conj. Ab ELISA - MMAE/MMAF//ECDBio/SA-HRP tests.

Example 33 - 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 in Fendly et al. (1990) Cancer Research 50:1550-1558. Briefly, NIH 3T3/HER2-34oo cells (expressing approximately 1 x 10⁵ ErbB2 molecules/cell) produced as described in Hudziak et al. Proc. Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were harvested with phosphate buffered saline (PBS) containing 25mM EDTA and used to immunize BALB/c mice. The mice were given injections i.p. of 10⁷ cells in 0.5ml PBS on weeks 0, 2,5 and 7. The mice with antisera that immunoprecipitated ³²P-labeled ErbB2 were given i.p. injections of a wheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract on weeks 9 and 13. This was followed by an i.v. injection of 0.1 ml of the ErbB2 preparation and the splenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for ErbB2-binding by ELISA and radioimmunoprecipitation.

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Epitope mapping and characterization

The ErbB2 epitope bound by monoclonal antibody 4D5 was determined by competitive binding analysis (Fendly et al. Cancer Research 50:1550 -1558 (1990)). Cross-blocking studies were done by direct fluorescence on intact cells using the PANDEX™ Screen Machine to quantitate fluorescence. The monoclonal antibody was conjugated with fluorescein isothiocyanate (FITC), using established procedures (Wofsy et al. Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi (eds.) San Francisco: W.J. Freeman Co. (1980)). Confluent monolayers of NIH 3T3/HER2-34oo cells were trypsinized, washed once, and resuspended at 1.75 x 10⁶ cell/ml in cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN₃. A final concentration of 1% latex particles (IDC, Portland, OR) was added to reduce clogging of the PANDEX™ plate membranes. Cells in suspension, 20 μΐ, and 20 μΐ of purified monoclonal antibodies (100pg/ml to 0.1 pg/ml) were added to the PANDEX™ plate wells and incubated on ice for 30 minutes. A predetermined dilution of the FITC-labeled monoclonal antibody in 20 μΐ was added to each well, incubated for 30 minutes, washed, and the fluorescence, was quantitated by the PANDEX™. Monoclonal antibodies were considered to share an epitope if each blocked binding of the other by 50% or greater in comparison to an irrelevant monoclonal antibody control. In this experiment, monoclonal antibody 4D5 was assigned epitope I (amino acid residues from about 529 to about 625, inclusive within the ErbB2 extracellular domain.

The growth inhibitory characteristics of monoclonal antibody 4D5 were evaluated using the breast tumor cell line, SK-BR-3 (see Hudziak et al. (1989) Molec. Cell. Biol. 9(3):1165-1172). Briefly, SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsin and suspended in complete medium at a density of 4 x 10⁵ cells per ml. Aliquots of 100 μΐ (4 x 10⁴ cells) were plated into 96-well microdilution plates, the cells were allowed to adhere, and 100 μΐ of media alone or media containing monoclonal antibody (final concentration 5 pg/ml) was then added. After 72 hours, plates were washed twice with PBS (pH 7.5), stained with crystal violet (0.5% in methanol), and analyzed for relative cell proliferation as

299 described in Sugarman et al. (1985) Science 230:943-945. Monoclonal antibody 4D5 inhibited SK-BR-3 relative cell proliferation by about 56%.

Monoclonal antibody 4D5 was also evaluated for its ability to inhibit HRG-stimulated tyrosine phosphorylation of proteins in the M_r 180,000 range from whole-cell lysates of MCF7 cells (Lewis et al. (1996) Cancer Research 56:1457-1465). MCF7 cells are reported to express all known ErbB receptors, but at relatively low levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical molecular sizes, it is not possible to discern which protein is becoming tyrosine phosphorylated when whole-cell lysates are evaluated by Western blot analysis. However, these cells are ideal for HRG tyrosine phosphorylation assays because under the assay conditions used, in the absence of exogenously added HRG, they exhibit low to undetectable levels of tyrosine phosphorylation proteins in the M_T 180,000 range.

MCF7 cells were plated in 24-well plates and monoclonal antibodies to ErbB2 were added to each well and incubated for 30 minutes at room temperature; then rHRGpl 177.244 was added to each well to a final concentration of 0.2 nM, and the incubation was continued for 8 minutes. Media was carefully aspirated from each well, and reactions were stopped by the addition of 100 μΐ of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μΐ) was electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane. Antiphosphotyrosine (4G 10, from UBI, used at 1 pg/ml) immunoblots were developed, and the intensity of the predominant reactive band at Af_rD 180,000 was quantified by reflectance densitometry, as described previously (Holmes et al. (1992) Science 256:1205-1210; Sliwkowski et al. J. Biol. Chem. 269:1466114665 (1994)).

Monoclonal antibody 4D5 significantly inhibited the generation of a HRG-induced tyrosine phosphorylation signal atM_r 180,000. In the absence of HRG, but was unable to stimulate tyrosine phosphorylation of proteins in the M_r180,000 range. Also, this antibody does not cross-react with EGFR (Fendly et al.

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Cancer Research 50:1550-1558 (1990)), ErbB3, or ErbB4. Monoclonal antibody 4D5 was able to block HRG stimulation of tyrosine phosphorylation by D50%.

The growth inhibitory effect of monoclonal antibody 4D5 on MDA-MB-175 and SK-BR-3 cells in the presence or absence of exogenous rHRGfil was assessed (Schaefer et al. Oncogene 15:1385-1394(1997)). ErbB2 levels ip MDA-MB-175 cells are 4-6 times higher than the level found in normal breast epithelial cells and the ErbB2-ErbB4 receptor is constitutively tyrosine phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 was able to inhibit cell proliferation of MDA-MB-175 cells, both in the presence and absence of exogenous HRG. Inhibition of cell proliferation by 4D5 is dependent on the ErbB2 expression level (Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). A maximum inhibition of 66% in SK-BR-3 cells could be detected. However this effect could be overcome by exogenous HRG.

The murine monoclonal antibody 4D5 was humanized, using a gene conversion mutagenesis strategy, as described in U.S. Patent No. 5821337, the entire disclosure of which is hereby expressly incorporated by reference. The humanized monoclonal antibody 4D5 used in the following experiments is designated huMAb4D5-8. This antibody is of IgGl isotype.

REFERENCES CITED

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

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