Monomethylvaline compounds capable of conjugation to ligands

MONOMETHYLVALINE COMPOUNDS CAPABLE OF CONJUGATION TO LIGANDS

CONTINUITY

1. FIELD OF THE INVENTION

The present invention is directed to a Drug Compound and more particularly to Drag-Linker-Ligand Conjugates, Drag-Linker Compounds, and DrugLigand 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 antibodÿ-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.

2. 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 fat 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

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WO 2005/081711 PCTAJS2004/038392 often limits the dose of a drag (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitor agents and antiviral or antimicrobial drugs) that can be administered. By comparison, although oral administration of drags 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 drag has been absorbed into the systemic circulation. Further complications involve problems with oral bioavailability and residence of drag in the gut leading to additional exposure of gut to the drug and hence risk of gut toxidties. 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-drag conjugates for the local delivery of cytotoxic or cytostatic agents, e.g., drags to kill ch* inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticanccr Research 19:605-614; 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 drag 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 fa Cancer Therapy: A Review, in Monoclonal Antibodies *84: Biological And Clinical Applications, A. Piochera et al. (ects), pp. 475506). 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, fajmnnother. 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 fast 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.

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Letters 10:1025-1028; Mandlerei aL (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu etaL, (1996) Proc. Natl. Acad. Sd. USA 93:86188623), and calicbeamicin (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, DJL 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.

ZEVAUN® (ibritumomab tiuxetan, Biogen/Idcc) is an antibodyradioisotope conjugate composed of a murine IjgGl kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and ^,nIn or 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):2453-63; 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 cytopcnias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicbeamicin, 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 Π trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm, BZL Biologies, Immunogen Inc.), ah 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.1 (Chari el aL (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 “nonclcavable.”

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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 Drag Design 13:243-277). 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 monomethylanristatin (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 (Ktussman, et aL (2004), Bioconjugate Chemistry 15(4):765-773: Doronina et aL (2003) Nature Biotechnology 21(7):778-784; “Monomethyl valine Compounds Capable of Conjugation to Ligands*’; Francisco et al. (2003) Blood 102(4):1458-1465: U.S. Publication 2004/0018194; (Hi) anti-CD20 antibodies such as RTTUXAN® (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) Eselectin antibody (Bhaskar et aL (2003) Cancer Res. 63:6387-6394); and (vi) other antiCD30 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.

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The ErbB family of receptor tyrosine kinases are important mediators of cel! growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, EibBl, HER1), HER2 (ErbB2 or pl85*“), 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-EfbB2 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 etaL (1994) J. Biol. Chem. 269(20): 14661-14665; Scott etaL (1991) J. Biol. Chem. 266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994)91:7202-7206; Lewis et aL (1996) Cancer Research 56:1457-1465; and Schaefer er 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 4:543548 (1989); Maier et aL Cancer Res. 51:5361-5369 (1991); Bacus etaL Molecular Carcinogenesis 3:350-362(1990); Stancovski etaL Proc. NatL Acad. Set USA 88:86918695 (1991); Bacus et aL Cancer Research 52:2580-2589 (1992); Xu et aL Int. J. Cancer 53:401-408 (1993); W094/00136; Kasprayk 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 etaL (1994) Cancer Res. 54:3758-3765; Harwerth et aL (1992) J. Biol. Chem. 267:15160-15167; U.S. Patent No. 5783186; and Klapper et aL (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.8. etaL (1989) Proc. NatL Acad. Sci USA 86:9193-9197) andErbB4 (EP 599274; Plowman et aL(\993) Proc. NatL Acad. ScL USA 90:1746-1750; and Plowman et aL

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WO 2005/081711 PCÏ7US2004/038392 (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 cell-based assay (Kd « 5 nM) to the extracellular domain of the human epidermal growth factor receptor2 protein, HER2 (ΕΛΒ2) (U.S. Pateart 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; Slamon DJ, et aL (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 HER2. 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-efbB2) 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 antibody-dependent cellular cytotoxicity, ADCC (Hotaling TE, etaL (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471 ; Pegram MD, etaL (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

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WO 2005/081711 PCT/US2004/038392 patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselgaetal,(1996) J. Qin· 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 at, (1993) Cancer Immunol. Immunother. 37:255-263). Based on this observation, antibody 4D5 was humanized (hnMAb4D5-8, rhuMAb HER2, U.S. Patent No. 5821337; Carter et aL, (1992) Proc. Natl. Acad. Sci. USA 89:42854289) and tested in breast cancer patients whose tumors overexpeess HBR2 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 ErbB2overexpressing breast cancers that have received extensive prior anti-cancer 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 HER2directed 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.

3. SUMMARY OF THE INVENTION

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

<img file="CA2841741C_D0001.tif" />

or a pharmaceutically acceptable salt or solvate thereof wherein,

L~ is a Ligand unit;

-Aa-W&lt;-Y^ is a Linker unit (LU), wherein the Linker unit includes: -A-is a Stretcher unit aisOorl,

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WO 2005/081711

PCT/US2004/038392 each -W- is independently an Amino Acid unit, w is an integer ranging from Oto 12, -Y- is a Spacer unit, and yisO, 1 or 2;

p ranges from 1 to about 20; and

-D is a Drag unit having the Formulas Dg and Df:

R² O R⁴ R⁵ R⁶ R⁸ O R⁸ O

<img file="CA2841741C_D0002.tif" />

R² o R R R⁸ R⁸ 0 R⁸ O

R10 wherein, independently at each location:

R² is selected from H and Cj-Cg alkyl;

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

R* is selected from H, Cj-Cg alkyl, Cj-Cg carbocycle, aryl, Cj-Cg alkylaryl, Ci-Cg alkyl-fCs-Ce carbocycle), CyCt heterocycle and Ci-C₈ alkyl-fCs-Ce 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* 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 C₍-Cg alkyl;

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R⁷ is selected from H, Cj-Cg alkyl, Cj-Cg carbocycle, aryl, C|-Cg alkylaryl, Cj-Cg alkyl-(Cj-Cg carbocycle), Cj-Cg heterocycle and Cj-Cg alkyHCj-Cg heterocycle);

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

R⁹ is selected from H and Cj-Cg alkyl;

R¹⁰ is selected from aiyl or Cj-Cg heterocycle;

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

R^u is selected from H, Ci-C» alkyl, aryl, CyCg heterocycle, -&lt;R^,3OVR^M» or-(R¹³O)_m-CH(R^,5)2;

m is an integer ranging from 1-1000;

R¹³ is Cj-Cg alkyl;

R¹⁴isHorCi-Cg alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH^-NiR¹⁶)* -(CH^-SChH, or -(CH^-SQrCj-Cg alkyl;

each occurrence of R¹⁶ is independently H, C|-C₈ alkyl, or -(CHaKCOOH; where; n is an integer ranging from 0 to 6; and

R¹⁸ is selected from -C(R⁸)r-C(RVaryl, -C(R*)2-C(RV(C3-Cj heterocycle), and-C(R⁸)r-C(R⁸)r-(C3-Cecafbocycle).

•

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

<img file="CA2841741C_D0003.tif" />

Hj or pharmaceutically acceptable salts or solvates thereof, wherein:

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

R³ is selected from hydrogen, -Cj-Cg alkyl, -Cj-Cg carbocycle, aryl, -Ci-Cg alkyl-aryl, -Cj-Cg alkyi/Cg-Cg carbocycle), -Cj-Cg heterocycle and -Cj-Cg a!kyl-(Cj-Cg heterocycle);

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R⁴ is selected from hydrogen, -Cj-Cg alkyl, -Cj-Cg cafbocycle, -aryl, -CjCg alkyl-aryl, -Ci-Cg alkyl-fCj-Cj carbocycle), -CyCe heterocycle and -Ci-Cg alkyi-(C_rCg heterocycle) wherein R⁵ is selected from -H and -methyl; or R⁴ and R⁵ jointly, have the formula -fCR^¹*),- wherein R* and R^b are independently selected from -H, -Ci-Cg alkyl and -CyCg 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-Cg alkyl;

R⁷ is selected from H, -Cj-Cg alkyl, -Cs-Cg carbocycle, aryl, -Cj-Cg alkylaryl, -Cj-Cg alkyl-fCyCg carbocycle), -Cj-Cg heterocycle and -Ci-Cg alkyl-fCyCg heterocycle);

each R⁸ is independently selected from H, -OH, -Cj-Cg alkyl, -CyCg carbocycle and -O-fCj-Cg alkyl);

R⁹is selected from H and -Cj-Cg alkyl;

R¹⁰ is selected from aryl group or -CyCg heterocycle;

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

R^u is selected from H, Cj-C» alkyl, aryl, -CyCg heterocycle, -(R¹³O)_mR¹⁴, or-iR^OVCHCR¹⁵^;

m is an integer ranging from 1-1000;

R” is -CrCg alkyl;

R^,4isHor-C|-C₈alkyl;

each occurrence of R¹⁵ is independently H, -COOH, -(CH2)a-N(R¹⁶)2, (CH^-SOjH, or -(CH^-SOj-Ci-Cg alkyl;

each occurrence of R¹⁶ is independently H, -Cj-C₈ alkyl, or COOHiand 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.

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In still another aspect, the invention provides pharmaceutical compositions comprising an effective amount of a Drug-Linker Compound and a pharmaceutically acceptable carder 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 Drag-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 Drag-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-LinkerLigand 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 Drug-Linker 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 Drug-Linker-Ligand Conjugate.

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Id 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 Drug-Ligand Conjugate having a cleavable Drag 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 Drag-Linkcr-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 cleavablc 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 Drag-Linker-Ligand Conjugate.

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

Tn yet another aspecU 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

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WO 2005/081711 PC17US2004/038392 thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drag unit from the Drag-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 Drug5 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 Drag-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 Drag-Ligand 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 Drug-Linker Compound.

hi 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-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to 20 a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drag-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 Drag-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 Drag unit from the Drag-Ligand Conjugate.

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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 Drag-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 Drag-Ligand Conjugate having a cleavable Drag 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 Drag 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 Drag-Linker-Ligand Conjugate.

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

Ab-&lt;A_a-W_w-Y_y-D)p 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 ωιΐζ w is an integer ranging from 0 to 12,

Y is a Spacer uniLand yisO, 1 or 2, p ranges from 1 to about 20, and

D is a Drug unit selected foom Formulas De and Dpt

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PCTAJS2004/038392

R² 0 ^R R® R⁸ O R® O

<img file="CA2841741C_D0004.tif" />

&lt;5 wherein, independently at each location:

R² is selected from H and Cj-Cg alkyl;

R⁵ is selected from H, C_t-Cg alkyl, C₃-Cg carbocycle, aryl, Cj-Cg alkylaryl, Ci-Cj alkyl-(C3-Cg carbocycle), C₃-C₈ heterocycle and Cj-Cg alkyHC₃-Ce heterocycle);

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

R^s is selected from H and methyl;

or R⁴ and R^$ jointly form a carbocyclic ring and have the formula

-(CR^R⁶)*- wherein R^e and R^b are independently selected from H, Cj-Cg alkyl and CyCg carbocycle and n is selected from 2,3,4,5 and 6;

R⁶ is selected from H and Cj-Cg alkyl;

R⁷ is selected from H, Cj-Cg alkyl, C₃-Cg carbocycle, aryl, C|-Cg alkylaryl, Ci-Cg alkyl-(C₃-Cg carbocycle), Cj-Cg heterocycle and C_t-C_s alkyl-fCrCg heterocycle);

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

R⁹ is selected from H and Cj-Cg alkyl;

R¹⁰ is selected from aryl or Q-Cg heterocycle;

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

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R^u is selected from H, Ci-Ca alkyl, aryl, CrCg heterocycle, -(R^O^-R¹⁴, or-(R¹³OVCH(R^I3)2;

m is an integer ranging from 1-1000;

R¹³ is CrCg alkyl;

R^I4isH or Ci-Cg alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)n-N(R¹⁵)2, -(CHzX-SCbH, or -{CH^QrCi-Q alkyl;

each occurrence of R¹⁶ is independently H, C|-Cg alkyl, or -(CHzjnCOOH;

R¹⁸ is selected from -OiR’jj-CiR^aryl, -C(R⁸)r-C(R⁸)HC3-C8 heterocycle), and -C(R⁸)j~C(R’)z-(Q-Cs 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, mesothdin, Genbank accession no. NML005823);

(6) Napi3b (NAPI-3B, NPTUb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type Π sodium-dependent phosphate transporter 3b, Genbank accession no. NMJJ06424);

(7) Sema 5b (FLH0372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hog, sema domain, seven thrombospondin repeats (type 1 and type 1like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);

CA 02841741 2014-02-03

WO 2005/081711 PCT/DS2004/038392 (8) PSCAhlg (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. PCAN API, STAMP1, STEAP2, SIMP, 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. NML.017636);

(13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGF1, teratocarcinomaderived growth factor, Genbank accession no. NPJ003203 or NM_003212);

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

(15) CD79b (1Gb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);

(16) FcRU2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NWL030764);

(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. NMJXJ4442);

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WO 2005/081711 PCT/US2004/038392 (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, îmmnnogîobulin-associaîed alpha, a B cellspecific 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, Genbank accession No. NPJX)1774.1);

(29) CXCR5 (Burkitts lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, fonctions 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 ΓΓΑΜ domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); or

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WO 2005/081711 PCT/US2004/038392 (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).

hi 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-LinkerAntibody 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 DrugAntibody Conjugate having a cleavable Drag unit from the Drug-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 Drug-Linker-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 Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

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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 Drag-Antibody Conjugate having a cleavable Drag unit from the DrugAntibody 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 Drag-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 aspecti 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 Drag-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.

hi 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-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to

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WO 2005/081711

PCT/US2004/038392 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 DrugAntibody 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 Drag-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 Drag unit from the Drag-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 Drag unit from the Drag-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 Drag-Unker-Antibody Conjugates (also referred to as antibody-drug conjugates) having Formula Ic

Ab (’A_a‘W_wYy”D)p 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. NMj001203);

(2) El6 (LAT1, SLC7A5, Genbank accession no. NM_003486);

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

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WO 2005/081711 PCT/US2004/038392 (4) 0772P (CA125, MUC16, Genbank accession no. AF3614B6);

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

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

(7) Sema 5b (FLI10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. ABO4O878);

(8) PSCA big (2700050C12Rik, C530008016Rik, RISEN cDNA 27OOO5OC12.RIKEN cDNA2700050C12 gene. Genbank accession no. AY358628);

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

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. ΝΜ..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, Geobank accession no. NM_017636);

(13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGF1, teratocarcinomaderived growth factor, Genbank accession no. NP_003203 or NM_003212);

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

(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accession no. NNL000626);

(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);

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WO 2005/081711 PCTÆJS2004/038392 (19) MDP (Genbank accession no. BCO17O23);

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

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

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

(23) ASLG659 (Genbank accession no. AXO92328);

(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, CD79O, immunoglobulin-associated alpha, a B cellspecific 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, 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, Geubank 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 ΓΓΑΜ domains, may have a role in B-lymphocyte differentiation. Genbank accession No. NP_443170.1); or

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PCT/US2004/038392 (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoieceptor 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, aisOor 1, each W is independently an Amino Add unit, w is an integer ranging from 0 to 12,

Y is a Spacer unit, and yisO, lor2, p ranges from 1 to about 20, and

D is a Drug moiety selected from Formulas Dg and Dp:

<img file="CA2841741C_D0005.tif" />

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

R² is selected from H and C₍.Cg alkyl;

R³ is selected from H, Cj-Cg alkyl, C₃-Cg carbocycle, ary], Cj-Cg alkylaryl, Cj-Cg alkyl-fCj-Cg carbocycle), Cj-Cg heterocycle and Cj-Cg alkyl-iCyCg heterocycle);

R⁴ is selected from H, Cj-Cg alkyl, CrC₈ caibocycle, aryl, Cj-Cg alkylaryl, Cj-Cg alkyl-(C3-Cg carbocycle), C₃-C₈ heterocycle and Ci-C₈ aIkyl-(C₃-C₈ heterocycle);

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R^s is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula -(ΟΙό^η- wherein R* and R^b are independently selected from H, Ct-C₈ alkyl and CyCg 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, C|-C₈ alkyl, C₃-C₈ carbocycle, aryl, Ci-C₈ alkylaryl, C|-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C_rC₈ alkyI-(C₃*C₈heterocycle);

each R⁸ is independently selected from H, OH, Cj-C₈ alkyl, CyC₈carbocycle and O-fC|-C₈ alkyl);

R⁹ is selected from H and Cj-Cg alkyl;

R_w is selected from aryl or CyCe heterocycle;

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

R^u is selected from H, Ci-Cæ alkyl, aryl, C₃-C₈ heterocycle, -(R¹³0)mR¹⁴, or-(R^l3O)m-CH(R¹⁵)2;

m is an integer ranging from 1-1000;

R” is Q-Cg alkyl;

R^,4isHorC^,-C⁸alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CEh)n-N(R¹⁶)2, -(CH^n-SOjH, or^CH^n-SQj-Ct-Cg alkyl;

each occurrence of R¹⁶ is independently H, C|-C₈ alkyl, or -(CHî),COOH;

R¹⁸ is selected from -C(R⁸)2-C(R⁸)z-aryl, -C(R⁸brC(R⁸)z-(C^îGe heterocycle), and -C(R⁸)2-C(R⁸)r-(C3-C8 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).

Tn another aspect the invention includes an article of manufacture comprising an antibody-drug conjugate compound of the invention; a container; and a

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WO 2005/081711 PCT7US2004/038392 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 thé antibody until the antibody-drug conjugate compound enters a cell with a cellsurface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug 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.

hi another aspect, the drag 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-fonning 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 drag 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.

Various embodiments of this invention relate to an antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof, wherein the conjugate comprises an antibody covalently attached to one or more drug moieties, the antibody-drug conjugate having Formula Ic:

Ab —(-A_a-W_w-Y_y-D ) p _k wherein:

Ab is an antibody which binds to CD79b (IGb (immunoglobulin-associated beta), B29);

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;

y is 0,1 or 2;

p ranges from 1 to 20;

D has Formula De :

<img file="CA2841741C_D0006.tif" />

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

CA 2841741 2017-06-14

R² is H or C|-Cs alkyl;

R³ is H, C|-Cg alkyl, Cj-Cg carbocycle, aryl, C|-Cg alkyl-aryl, C|-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle, or C|-Cg alkyl-(C3-Cg heterocycle);

R⁴ is H, Ci-Cg alkyl, C3-C8 carbocycle, aryl, Ci-Cg alkyl-aryl, C|-Cg alkyl-(C3-Cg carbocycle), C3-C8 heterocycle, or C|-Cs alkyl-(C3-C8 heterocycle);

R⁵ is H or methyl;

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

R⁶ is H or C|-Cs alkyl;

R⁷ is H, C|-Cg alkyl, C3-C8 carbocycle, aryl, C|-Cg alkyl-aryl, C|-Cg alkyl-(C3-Ce carbocycle), C3-C8 heterocycle, or Ci-Cs alkyl-(C3-Cs heterocycle);

each R⁸ is independently H, OH, CpCe alkyl, C3-C8 carbocycle, or O-(Ci-Cs alkyl);

R⁹is H or C1-Cs alkyl; and

R¹⁸ is -C(R⁸)2-C(R⁸)2-aryl, -C(R⁸)₂-C(R⁸)2-(C3-C8 heterocycle), or

-C(R⁸)2-C(R⁸)2-(C₃-C8 carbocycle).

The invention will best be understood by reference to the following detailed description of the exemplary embodiments, taken in conjunction with the accompanying

27a

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CA 02841741 2016-05-16 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 cACl 0-mcMMAF in subcutaneous Karpas-299 ALCL xenografts.

Figure 2 shows an in vivo, single dose, efficacy assay of cACl0-mcMMAF 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 3a and arrows in Figure 3b indicate the days of therapy.

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

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

Figures 6a and 6b show in vitro activity of ClF6-antibody-drug conjugates against CD70⁺ renal cell carcinoma cell lines.

27b

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Figure 7 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with antibody drug conjugates (ADC): -·- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -O— Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and —Δ- TrastuzumabMC-MMAF, 4.8 MMAF/Ab, measured in Relative Fluorescence Units (RLU) versus pg/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, -oTrastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 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, -oTrastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab, and -A- Trastuzumab-MCMMAF, 4.1 MMAF/Ab.

Figure 10 shows an in vitro, cell proliferation assay with MDA-MB-468 cells treated with ADC: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab, -oTrastuzumab-MC-vc-PAB-MMAB, 3.3 MMAE/Ab, and -Δ- Trastuzumab-MC-vc-PABMMAF, 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 SpragueDawley 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: 0.5,13, 23, 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 athymie nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 pg/m²) and Trastuzumab-MC-vcPAB-MMAF (555 pg/m²). (H=Trastuzumab).

Figure 14 shows the mean tumor volume change over time in athymie nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10

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WO 2005/081711 PCT/ÜS2004/038392 mg/kg (660 gg/m²) of Trastnzumab-MC-MMAE and 1250 gg/m² Trastuzumab-MC-vcPAB-MMAE.

Figure 15 shows the mean tumor volume change over time in athymie 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 athymie 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 foe 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 (Mean± SD) after administration of Vehicle, trastuzumab-MC-val-citMMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF.

Figure 18 shows the Group mean change in animal (rat) body weights (Mean± SD) after administration of 9.94 mg/kg H-MC-vc-MMAF, 24.90 mg/kg H-MCvc-MMAF, 10.69 mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg H-MC(Me)-vc-PABMMAF, 10.17 mg/kg H-MC-MMAF, 25.50 mg/kg H-MC-MMAF, and 21.85 mg/kg HMC-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 (Mean ± SD) after administration of trastuzumab (H)-MCMMAF at doses of 2105,3158, and 4210 gg/m². The MC linker is attached via a cysteine of trastuzumab for each conjugate.

4. DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

4.1 DEFINITIONS AND ABBREVIATIONS

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

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

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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, Le., a molecule that contains an antigen binding site that

I 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 (&amp;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, Ffab’h fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-M) antibodies, CDR‘s, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.

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The term “monoclonal antibody’* as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, Le, 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 bybridoma method first described by Kohler el cd. (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 Maries 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 snch antibodies, so long as they exhibit the desired biological activity (U.S. Patent mp/ 4816567; and Morrison etaL (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 MAbs 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

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WO 2005/081711 PCT/US2004/038392 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 die 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’h, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment^).

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 and CH3. The constant domains may be native sequence constant domains (&amp;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.

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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 threedimensional 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, 82:6497-650! (1985) and Yamamoto et aL, (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 pl85oeu. 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 ΕΛΒ 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 223:10791082); amphiregulin also known as schwanoma or kératinocyte autocrine growth factor (Shoyab er aL (1989) Science 243:1074-1076; Kimura et aL, Nature, 348:257-260 (1990); and Cook et aL, MoL Cell. BioL, 11:2547-2557 (1991)); betacellulin (Shing et aL, Science, 259:1604-1607 (1993); and SasadaetaL,Biochem. Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal growth factor (ΗΒ-EGF) (Higashiyama et aL, Science, 251:936-939 (1991)); epiregulin (Toyoda et aL, J. BioL Chem., 270:74957500 (1995); and Komurasaki etaL, Oncogene, 15:2841-2848 (1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway er aL, Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et aL, Proc. NatL Acad. ScL, 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et aL, Oncogene, 18:2681-89 (1999)) or cripto (CR-1) (Kannan etaL, J. BioL Chem., 272(6):3330-3335 (1997)). ErbB ligands which bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, ΗΒ-EGF and epiregulin. ErbB ligands which bind ΕΛΒ3 include heregulins. ErbB ligands capable of binding ΕΛΒ4 include betacellulin, epiregulin, ΗΒ-EGF, NRG-2, NRG-3, NRG-4 and heregulins. The

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ErbB ligand may also be a synthetic BrbB 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/BGF chimera biregulin (see, for example, Jones et aL, (1999) FEBS Letters, 447:227-231 )·

...............“HeTCguIin” (HRG) refers to a'polypeptide encoded by the beregulin gene product as disclosed in US. Patent Na 5641869 or Marchionni er nt, Nature, 362:312318 ¢1993). Examples of hcregulins include heregulin-a, heregulin-βΐ, heregulin~P2 and beregulin-p3 (Holmes et aL, Science, 256:1205-1210 (1992); and U.S. Patent Nno. 5641869); neu differentiation factor (NDF) (Peles etaL, Cell 69:205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (Falls er aL (1993) Cell 72:801-815); glial growth factors (GGFs) (Marchionni etaL, Nature, 362:312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et aL, J. BioL Chan., 270:14523-14532 (1995)); γ-heregulin (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-PAGB as described in Sliwkowski et aL, J. BioL Chem., 269(20):14661-14665 (1994), for example. Examples of such ErbB heterooligomers include EGFR-ErbB2 (also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ΕΛΒ3-ΕΛΒ4 (HER3/HER4) complexes. Moreover, the ErbB hetero-oligomer may comprise two or more ErbB2 receptors combined with a different ErbB receptor, such as ErbB3, EibB4 or EGFR (ErbBl). 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 add 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

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PCT/ÜS2004/038392 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 tumor-associated 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 Genentcch, 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 FcyRHI only, whereas monocytes express FcyRI, FcyRII and FcyRIH. FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravctch and Kinct, (1991) Anna. 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 etaL, Prco. NatL Acad. Sci. USA, 95:652-656 (1998).

The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to tile 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)

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WO 2005/081711 PCI7US2004/038392 and includes receptors of the FcyRI, FcyRII, and Fey Rm subclasses, including allelic variants and alternatively spiked forms of these receptors. FcyRH receptors include FcyRUA (an “activating receptor”) and FcyRHB (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 (ΤΓΑΜ) in its cytoplasmic domain. Inhibiting receptor FcyRHB contains an immunoreceptor tyrosine-based inhibition motif (ΠΊΜ) in its cytoplasmic domain. (See review M. in Daëron, 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 et aL, J. Lab. Clin. Med., 126:330-41 (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 etaL,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, die 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 β-sheet 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

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Institutes of Health, Bethesda, MD). The constant domains are not involved directly in binding an antibody tn an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellule* 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” (eg., 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), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 2632 (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 antigen-binding 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’)j fragment that has two antigen-binding sites and is still capable of crosslinking antigen.

Fv” is the minimum antibody fragment which contains a complete antigenrecognition 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, ft is in tins 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

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WO 2005/081711 PCT/US2004/038392 bear at least one free thiol group. F(ab’)z 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 (k) 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 Plticktbun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Veriag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigenbinding 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 arc 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.

“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

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'.· Ί 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) Cuit. Op. Struct Biot, 2:593-596.

Humanized anti-Eri>B2 antibodies include buMAb4D5-l, huMAb4D5-2, buMAb4D5-3,huMAb4D5-4,huMAb4D5-5,huMAb4D5-6,huMAb4D5-7and ‘ * huMAb4D5-8 (HERCEPTIN®) as described iu Table 3 of U.S. Patent No. 5821337;

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 wife diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonprotemaceous 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 N-termin al or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nomeducing 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 inducts 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

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WO 2005/081711 PCT/US2004/038392 nuclear/chnxnatin 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. Nonlimiting 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 (Le., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (Le., 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, Le. >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-LinkerLigand Conjugate, an an antibody drug conjugate (ADC) or the like whereby the covalent

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WO 2005/081711 PCT/US2004/038392 attachment, e.g._t 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 moictics of the Drug-Ligand Conjugate, a Drag-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 antiproliferation effect of an antibody drug conjugate compound or an intracellular metabolite of an antibody drug conjugate compound. Cytotoxic activity may be expressed as the ICso 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, nonsmall 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 EibB2 receptor and/or

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WO 2005/0817Π PCT/US2004/038392 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 ΕΛΒ2 receptor overexpression by measuring shed antigen (e.g., ΕΛΒ2 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.

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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 = 0-10,000 copies/cel], 14- — at least about 200,000 copies/cell, 24=at least about 500,000 copies/cell, 34 = about 1-2 x 10⁶ copies/cell. Overexpression of HER2 at the 34 level, which leads to ligand-independent activation of the tyrosine kinase (Hudziak et aL, (1987) Proc. NatL Acai ScL 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; Slamon et aL, (1987) Science, 735-ΛΊΊ4&amp;).

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,²¹¹ At, ^,3,1.¹²⁵1, “Y, ¹⁸⁶Re, ^t88Re, ^,sSm. ^2,2Bi, ^ΠΡ, “C, 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; ethylcnimines and methylamdamines including altretamine, triethyicncmclamine, trietylenephospboramide, triethiylenethiophosphoramide and trimethyiolomelamine; TLK 286 (TELCYTA™); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); betalapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotccan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); doiastatin; duocarmydn (including the synthetic

CA 02841741 2014-02-03 analogues, KW-2189 and CB1-TM1); elentherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramnstine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterinc, prednimnstine, trofbsfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustinc, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calichearnicin gammall and calicheamicin omegall (see, e.g.jAngew. Chem. Int. Ed. Engl.·. . 33:183-186 (1994)) and anthracyclines such as annamycin, AD 32, aicarubitin, daunorabicin, dexrazoxane, DX52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detonrbicin, 6-diazo-5-oxo-L-norlencine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorabicin), esorubicin, marccllomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromyem, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuberoidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as ftudarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azanridine, carmoftir, 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 add (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as AUMTA®, LY231514 pemetrexed, dihydrofolaie reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouraciJ (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^¹¹, TDX); inhibitors of dihydropyrimidine dehydrogenase such as enitaracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrcne; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;

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WO 2005/081711 PCT/US2004/038392 gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubidn; losoxautrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic add; triaziquone; 2,2',2-trichlorotriethylaminc; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vtndesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustme; 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 (Rhône-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 5FU and leucovorin.

Also included in this definition are anti-honnonal 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, 4-hydroxytamoxifen, 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, fodrozole, RIVISOR® vorozole, FEMARA® letrozoie, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamidc, leuprolide, and goserelin; as well as troxacitabine (a

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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, PKC-alpha, Raf, Η-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECRN® 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 2D1839 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 EtbB receptor. Examples of such inhibitors include the EGFR-targeted drugs noted in the preceding paragraph as well as quinazolines such as PD153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyiimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines, curcumin (diferuloyi 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); PTK787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac

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WO 2005/081711 PCI7US2004/038392 (ISIS 3521; Isis/UUy); Imatimb mesylate (Gleevac; Novartis); PKI166 (Novartis); GW2016 (Glaxo SmithKline); 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 anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor of growth factor receptor involved in promoting angiogenesis. In one embodimenL the antiangiogenic 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-a 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 -Π; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytemacrophage-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, IL-9, ΙΙΛ0, IL·! 1,1H2; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including UF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources

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WO 2005/081711 PCT/ÜS2004/038392 or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

The terra “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_t “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et dL, (ed.), pp. 247-267, Humana Press ¢1985). The prodrugs of this invention include, but are not limited to, phosphatecontaining prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptido-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, βlactam-containing prodrags, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrags, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drags that can be derivatized into a prodrag 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 drag (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 tiie 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

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PCT/US2004/038392 acid molecule contained in cells that ordinarily express the antibody where, for example, foe nucleic acid molecule is in a chromosomal location different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary for foe 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 add 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 Che 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 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 foe originally transformed cell are included. Where distinct designations are intended, it will be 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

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WO 2005/081711 PCT/ÜS2004/038392 scleroderma and sclerosis, responses associated with inflammatory bowd 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, IgE-mediated 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 (SUE) such as cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis (MS) such as spmoeptieal 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 Rheumatics and Giant Cell (Takayasu’s) Arteritis), Medium Vessel vasculitis (including Kawasaki's Disease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis, such as ChurgStrauss vasculitis or syndrome (CSS)), aplastic uremia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (ΑΠΪΑ), pernicious anemia, pure red cell aplasia (PRCA), Factor Vm 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-glomeralar 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, Stevens-Johnson 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 polyeudocrinopathies, Reiter’s

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WO 2005/081711 PCT/US2004Æ38392 disease, stiff-mao syndrome, immune complex nephritis, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ΓΓΡ), thrombotic throbocytopenic purpura (TTP), thrombocytopenia (as developed by myocardial infarction patients, for example), including autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto’s Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison’s disease, 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-Barrë 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 disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (QMS), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cel! 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 Cj-Cig 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, -CHfCHs)^, 1-butyl (n-Bu, nbutyl, -CH2CH2CH2CH3), 2-methyl-l-propyl (i-Bu, i-butyl, -CH2CH(CH3)₂), 2-butyl (s-Bu, s-butyi, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1pcntyl (n-pentyl, -CH2CH2CIfeCH2CH3)_t 2-penty] (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (^CHs^^CHa), 3-methyI-2-butyl (ŒfCIWCHiCH^), 3-metbyl-l-butyI (-Œ2Œ2CH(CH3)_a), 2-methyl-l-butyl (CH2CH(CH3)CH2CH3), Lhexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3XCH2CH2CH3)), 2-metbyl-2

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WO 2005/081711 PCT/ÜS2004/038392 pentyl (-C(CH3)2CH2CH2CH3), 3-mcthyl-2-pentyl (&lt;H(CH3)CH(CH3)CH2CH3), 4methyl-2-pentyl (-CH(CH3)CfÎ2CH(CH3)₂), 3-methyl-3-pentyl (-C(CH3XCH2CH₃)₂), 2-mefoyl-3-pentyl (-01(012013)01(013^), 23-dimethyl-2-butyl (C(CH3)₂CH(CH₃)₂), 3,3-dimethyl-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. Le. a carbon-carbon, sp²double bond. Examples include, but are not limited to: ethylene or vinyl (-CH=CH2), allyl (-CH₂CH=CH2), cyclopentenyl (-C₅H₇), and 5-hcxenyl (-CH₂CHjCHjCHjCHzzCHa).

“Alkynyl is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, Le. a carbon-carbon, sp triple bond. Examples include, but are not limited to: acetylenic (-001) and propargyl (-OI₂OOi).

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 foe removal of two hydrogen atoms from foe same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CHr) 1,2-ethyl (-CHjCHr), 1,3-propyl (-CHzO^CHr), 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 foe 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: 12ethylene(-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 arc not limited to: acetylene (-OC-), propargyl (-CH₂OC-), and 4-pentynyl (-CII2CH2CH2OCH-).

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

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WO 2005/081711 PCT/ÜS2OT4/0M392 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, 2phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. The arylalkyl group comprises 6 to 20 carton 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-benzimidazoly!methy!, 2-farylethyl, 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 carton 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 ac a bicycle having 7 to 10 ring members (4 to 9 carton 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, ary], 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, -0*. -OR, -SR, -S, -NR₂, -NR₃, =NR, *CX₃, -CN, -OCN, -SCN, . -N=C=0, -NCS, -NO, -NO* =N2» -N* NC(=O)R, -C(=O)R, -C(=O)NR₂, -SOf, -SQsH, -S(=OhR, OSfsOhOR, -S(=O)2NR, -S(=O)R, -OP(=OXOR)2, -P(=OXOR)2, -PO ₃, -POsH₂, -C(=0)R, -C(=O)X, -C(=S)R, -COzR, -CO/, -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-C)g alky], Ce-Cæ ary], CyCu heterocycle, protecting group or prodrng moiety. Alkylene, alkeaylenc, and alkynylene groups as described above may also be similarly substituted.

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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 to 7 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], [5,61, °^r [6,6] system.

Heterocycles are described in Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly Chapters 1,3, 4,6,7, and 9; The Chemistry of Heterocyclic Compounds, A series of Monographs’ (John Wiley &amp; Sons, New York, 1950 to present), in particular Volumes 13,14,16,19, and 28; and J. Am. Chan. 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, pynolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthaleny], indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyI, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-l,2,5-thiadiazinyl, 2H,6H-1,5»2dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofbranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyi, pyrazinyi, pyridazinyl, indolizinyl, isomdolyl, 3H-indolyl, IH-indazolyl, purinyl, 4H-quinolizinyI, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenantbridinyl, acridinyl, pyrimidinyl, pbenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyi, 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,

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WO 2005/081711 PCT/CS2004/038392 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, 3pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridaziny), 6pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3pyrazinyl, 5-pyiazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles are bonded at position I of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3pyrroline, imidazole, imidazolidine, 2-imidazoIine, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoIine, 3-pyrazoline, piperidine, piperazine, indole, indolinc, IH-indazoIe, position 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, 1azetedyl, 1-pynolyl, 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 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 [44], [54]. [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, l-cyclopent-l-enyl, l-cyclopent-2-enyl, 1cyclopent-3-enyl, cyclohexyl, 1-cyclobex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cyclobeptyl, and cyclooctyl. ¹

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 drag moiety, hi various embodiments, a linker is specified as LU. Linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: -(CRaJnOfCRaîhrepeating units of alkyloxy (e.g., polyethylenoxy, PEG, polymcthyleneoxy) and alkylamino (e.g., polyethylcncamino, Jeffamine™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.

The term chiral refers to molecules which have the property of nonsuperimposability of the mirror image partner, while die term achiral refers to molecules which are superimposable on their minor image partner.

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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 nonsuperimposable 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 Elid, 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 centers). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of planepolarized 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, raU 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

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WO 2005/081711 PCT/US2004/038392 more groups including, but not limited to, -Ci-Cg alkyl, -O-(Ci-Cg alkyl), -aryl, -C(O)R’, -OC(O)R\ -C(0)OR’, -€(O)NH₂, -C(O)NHR’, -C(O)N(R’)2 -NHC(O)R’, -SfO^R’, S(O)R’, -OH, -halogen, -N3, -NH₂, -NH(R’), -N(R’)j 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 “C₍-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 -n-decyl; while branched Ci-Cg alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, 10 tert-butyl, -isopentyl, 2-methylbutyl, unsaturated Ci-Cg alkyls include, but are not limited to, -vinyl, -allyl, -l-butenyl, -2-butenyl, -isobutylcnyl, -1-pentenyl, -2-pentenyI, 3-methyl-l-butenyl, -2-methyl-2-butenyl, -23-dimethyi-2-butenyl, l-hexyl.'2-hexyl, 3hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, 3-methyH butynyl. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert] 5 butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyi, 2~methylpcntyl, 3-methylpentyl, ·

2,2-dimethylbutyl, 23-dimethylbutyl, 2,2-dimethylpentyl, 23-dimethylpentyl, 33dimethylpentyl, 23,4-trimethylpcntyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4dimcthylhexyl, 23-dimethylhexyl, 33-dimethylhexyl, 2,4-dimethylpentyl, 2methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A Cj-Cg alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-C₈ alkyl, -O-(Ci-C₃ alkyl), -aryl, -C(0)R’, -OC(O)R’, -C(O)OR’, C(O)NH₂, -C(O)NHR‘, -QOjNiR’^ -NHC(O)R’, -SChR’, -SfOhR’, -S(O)R’, -OH, halogen, -Nj, -NH₂, -NH(R*), -NfR’^ and -CN; where each R’ is independently selected from H, -C|-C» alkyl and aryl.

A “Cj-Cg carbocyclc” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative Cj-Cg carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, cyclohexyl, -cyclobexenyl, -13-cydohexadienyl, -1,4-cyclobexadienyl, -cycloheptyl, 13-cycloheptadienyl, -1,3,5-cycloheptatrieny], -cyclooctyl, and -cyclooctadienyl. A C330 Cg carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -Cj-Cg alkyl, -O-(Ci-Cg alkyl), -aryl, -C(O)R’, -OC(O)R‘, C(0)OR‘, -C(0)NH₂, -C(0)NHR’, -C(O)N(R’)2 -NHC(O)R’, W, -S(0)R’, -OH,

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PCT/US2004/038392 halogen, -N3, -NHz, -NH(R*), -N(R’)z and -CN; where each R’ is independently selected from H, -Ci-Cg alkyl and aryl.

A “CyCg carbocyclo” refers to a Cj-Cs 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 -(CHzh-io** Examples of a Ci-Cjo 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="CA2841741C_D0007.tif" />

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

A “CyCg heterocycle” refers to an aromatic or non-aromatic Cs-Cj 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 C3Cg 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, isoxazolyi and tetrazolyl. A Cg-Cg heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, -Cj-Cg alkyl, -O-(Ci-C₈ alkyl), -aryl, -C(O)R', -OC(O)R’, -C(O)OR', -C(0)NH₂, -C(O)NHR’, C(O)N(R’)i -NHC(O)R’, -SfO^R’, -S(O)R’, -OH, -halogen, -N₃, -NH2, -NH(R’), N(R’)z and -CN; wherein each R* is independently selected from H, -C]-Cg alkyl and aryl.

“Cj-Cg heterocyclo” refers to a Cj-Cg heterocycle group defined above wherein one of the heterocycle group’s hydrogen atoms is replaced with a bond. A CyCe

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WO 2005/081711 PC17US2M4/038392 heterocyclo can be unsubstituted or substituted with up to six groups including, but not limited to, -Cj-Cg alkyl, -O-(C|-C8 alkyl), -aryl, -C(O)R’, -OC(O)R’, -C(O)OR’, C(O)NH₂, -C(O)NHR’, -C(0)N(R’)₂ -NHC(O)R’, -5(0^’, -S(O)R’, -OH, -halogen, N3, -NHz, -NH(R’), -N(R’)z and -CN; wherein each R’ is independently selected from H, -CrCg alkyl and aryl.

An “Exemplary Compound” is a Drug Compound or a Drug-Linker Compound.

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

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, triethyisilyl 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 (inflate), 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 arc 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,

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WO 2005/081711 PCT/ÜS2004/038392 succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e, 1,1 ’-methylene-bis -(2-hydioxy-3- naphthoate)) 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 die 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-(i-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicaiboxylate, DEPC is diethylphosphorylcyanidatc, DIAD is diisopropylazodicafboxylate, DEA is MV-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimetboxyethane), DMF is iV,N-dimethylfonnanitde, DMSO is dimethylsulfoxide, doe is dolaphenine, dov is W,R-dimethylvaline, DTNB is 5,5*-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetrianrinepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-l -ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is N-(9-fluorenylmetboxycarbonyl), gly is glycine, HATU is (?-(7-azabenzotriazol-l-yl)-lVJV,lV’^'-tetramethyluronium hexafluoropbosphate, HOBt is l-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, 2Λ>(+)norephedrine, PAB is p-aminobenzyl, PBS is phosphatc-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pop is p-nitrophenyl, MC is 6-maleimidocaproyl, phe

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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 inaleiinidocaproyl- polyethylene glycol; SPP is N-Succinimidyl 4-(2-pyridylthio) pentanoate; and SMCC is N-Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-1 carboxylate. ^x

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 (Le., 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.

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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-mcthyl auristatin E (MW 718); MMAF is Nmethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW 731.5); MMAFDMAEA 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 Cterminus 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-tenninal Phenylalanine of Auristatin F (MW 732).

1ΉΕ 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:

L-4A_a~W_w-Y_y-D) _pla or a pharmaceutically acceptable salt or solvate thereof wherein,

L-is a Ligand unit;

-A^Ww-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

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-D is a Drug unit having the Formulas De and Dp:

<img file="CA2841741C_D0008.tif" />

<img file="CA2841741C_D0009.tif" />

wherein, independently at each location:

R² is selected from H and C|-Cg alkyl;

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

R⁴ is selected from H, C_rC₈ alkyl, CyCg carbocycle, aryl, C_rCg alkylaryl, Cj-Cg alkyl-(C₃-Cg carbocycle), C₃-Cg heterocycle and Cj-Cg alkyl-(C₃-Cg heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R^s jointly form a carbocyclic ring and have the formula

-(CR*R*^>)_O- wherein R^a and R^b are independently selected from H, Cj-Cg alkyl and Cj-Cg 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-Cg carbocycle, aryl, Cj-Cg alkyl20 aryl, Cj-Cg alkyl-(C3-Cg carbocycle), C₃-Cg heterocycle and Ci-Cg alkyl-(C₃-Cg heterocycle);

each R⁸ is independently selected from H, OH, C_rC₈ alkyl, C₃-Cg carbocycle and O-(Cj-Cg alkyl);

R’is selected from H and Cj-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-Cg heterocycle;

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Z is O, S, ΝΗ, or NR¹², wherein R¹² is C,-C₈ alkyl;

R¹¹ is selected from H, Cj-C® alkyl, aryl, CyCs heterocycle, -(R^OVR¹⁴, or-(R^I3O)_ro-CH(R^,s)2;

m is an integer ranging from 1-1000;

R¹³ is Q-Qj alkyl;

R^wisH or C)-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, ^CHz)n-N(R¹⁶)2.

-(CHîin-SQjH, or -(CH^-SQj-Cj-Cg alkyl;

each occurrence of R*⁶ is independently H, Ci-Cg alkyl» or -(CH^COOH;

R*⁸ is selected from -C(R⁸)r-C(R⁸)2-aiyl, -CiR^r-CCR^Cj-Cg heterocycle), and -C(R^e)2-C(R⁸)2-(C3-Cg carbocycle); and n is an integer ranging from 0 to 6.

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

<img file="CA2841741C_D0010.tif" />

lb or pharmaceutically acceptable salts or solvates thereof, wherein:

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

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

R⁴ is selected from hydrogen, -CpCg alkyl. -CrC8 carbocycle, -aryl, -C|Cg alkyl-aryl, -C;-C8 alkyl-CCj-Cg carbocycle), -Cs-Cg heterocycle and -Ci-Cg alkyHCr C8 heterocycle) wherein R⁵ is selected from -H and -methyl; or R⁴ and R³ jointly, have the formula -(CR*R^b)e- wherein R* and R^b are independently selected from -H, -Cj-Cg alkyl and -CyCj carbocycleand n is selected from 2,3,4,5 and 6, and form a ring with the carbon atom to which they are attached;

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

R⁷ is selected from H, -Cj-Cg alkyl, -Cy-Cg caibocycle, aryl, -Cj-Cg alkylaryl, -Ci-Cg alkyl-(C3-Cg caibocycle), -C₃-C₈ heterocycle and -C|-Cg alkyHCs-Cg heterocycle);

each R⁸ is independently selected from H, -OH, -Cj-Cg alkyl, -Cs-Cg carbocycle and -O-(C|-Cg alkyl);

R⁹is selected from H and -C|-Cg alkyl;

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

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

R¹¹ is selected from H, Ci-C» alkyl, aryl, -Ca-Cg heterocycle, -(R¹³O)_mR’⁴, or-(R‘³O)m-CH(R^,5)2;

m is an integer ranging from 1-1000;

R¹³ is -CrCg alkyl;

R^,4isHor-Ci-Cgalkyl;

each occurrence of R^,s is independently H, -COOH, -(CHzVNfR¹⁶)?, (CH^-SOjH, or^CHîXrSOrCi-C» alkyl;

each occurrence of R¹⁶ is independently H, -C]-Cg alkyl, or-(CH2)_aCOOH; and n is an integer ranging from 0 to 6.

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

Ab-4A.-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, aisOorl, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

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

D is a Drug moiety selected from Formulas Dg and Dtf

<img file="CA2841741C_D0011.tif" />

De

<img file="CA2841741C_D0012.tif" />

wherein, independently at each location:

R² is selected from H and Ci-C₈ alkyl;

R³ is selected from H, Cj-C₈ alkyl, C3-C₈ carbocycle, aryl, C|-C₈ alkylaryl, Ci-C₈ alkyl-(C3-Ce carbocycle), C₃-Cg heterocycle and C]-C₈ alkyl-fCj-Cg heterocycle);

R⁴ is selected from H, Cj-Cs alkyl, Cs-Cb carbocycle, aryl, Ci-Cg alkylaryl, C)-C₈ alkyl-(C₃-C₈ carbocycle), C₃-Cg heterocycle and Cj-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^n- wherein R* and R^b are independently selected from H, Ci-Q alkyl and CyCg carbocycle and n is selected from 2,3,4,5 and 6;

R^s is selected from H and C|-Cs alkyl;

R⁷ is selected from H, Cj-Cg alkyl, Cj-Cg carbocycle, aryl, Cj-Cg alkylaryl, Cj-C₈ alkyl-(C3-C₈ carbocycle), CrC₈ heterocycle and Cj-C₈ alkyl-(C₃-Cg heterocycle);

each R* is independently selected from H, OH, Cj-C₈ alkyl, C₃-C₈carbocycle and O-(Ci-Ce alkyl);

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R⁹is selected from H and C|-C₈ alkyl;

R¹⁰ is selected from aryl or Cj-Ce heterocycle;

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

R^u is selected from H, Cj-C» alkyl, aryl, C3-C8 heterocycle, -(R¹³O)m-R¹⁴, or-(R'MrCH(R¹⁵)2;

m is an integer ranging from 1-1000;

R¹³ is Cz-Cg alkyl;

R¹⁴is H or Cj-Cg alkyl;

each occurrence of R¹⁵ is independently H, COOH, -(CH2)n-N(R^l6)2, -{CHîJn-SOîH, or-iCHjJa-SQs-Ci-Cs alkyl;

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

R^,g is selected from -Î(R*)r&lt;:(R⁸)r-aryl, -C(RVG(R⁸)r-(C3-C8 heterocycle), 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. NMJXB486);

(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, NPTUb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type Π sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);

(7) Serna 5b (ED10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Kog, sema domain, seven thrombospondin repeats (type 1 and type Ιό?

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(8) PSCA big (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 (HGNCJJ639, IPCA-1, PCANAP1, STAMP1, STEAP2, SIMP, 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, FLÏ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4,Genbank accession no. NM_017636);

(13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGF1, teratocarcinomaderived growth factor, Genbank accession no. NPJJ03203 or NMJJ03212);

(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);

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(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with ïg beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell 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. NPJDO17O7.1);

(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_002U1.1);

(31) P2X3 (Purinergjc 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 (Ft receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ΙΓΑΜ 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 lymphomagenesis; deregulation of the gene by translocation occurs in 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 secbutyl 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.

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

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH₃, and R⁹ is -H.

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

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R^t0 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 R¹⁵ is (CH^-NfR¹^, and R¹⁶ is -Ci-C_g alkyl or -(CH₂)_n-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R^,5)2, wherein R¹⁵ is (CH^-SOjH.

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

Exemplary embodiments of Formula la have the following structures:

L-MC-vc-PAB-MMAF

<img file="CA2841741C_D0013.tif" />

<img file="CA2841741C_D0014.tif" />

L-MC-vc-PAB-MMAE

<img file="CA2841741C_D0015.tif" />

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<img file="CA2841741C_D0016.tif" />

L-MC-MMAF wherein L is an antibody, Val 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, fa some instances, separation, purification, and characterization of homogeneous Ligand-Drug-conjugates where p is a certain value from Ligand-DnigConjugates 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="CA2841741C_D0017.tif" />

or a pharmaceutically acceptable salt or solvate thereof, wherein:

R² is selected from -hydrogen and -C|-C₈ alkyl;

R³ is selected from -hydrogen, -Cj-C₈ alkyl, -Cj-Cg carbocycle, aryl, -CjCg alkyl-aryl, -Cj-C₈ alkyI-(C₃-C₈ catbocycle), -C₃-C₈ heterocycle and -Ci-C₈ alkyl-&lt;C₃C₈ heterocycle);

R⁴ is selected from -hydrogen, -C|-C₈ alkyl, -C₃-C₈ carbocycle, -aryl, -C_rC₈ alkyl-aryl, -Cj-C₈ alkyl-fCrCs caibocycle), -C₃-C₈ heterocycle and -C]-C₈ a!kyl-(Cr

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Cg heterocycle) wherein R³ is selected from -H and -methyl; or R⁴ and R⁵ jointly, have the formula -(CR’R’V wherein R* and R^b are independently selected from -H, -C|-C₈alkyl 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;

R⁶ is selected from-Hand-C|-Cg alkyl;

R⁷ is selected from -H, -Cj-Cg alkyl, -C₃-Cg carbocycle, aryl, -C|-Cg alkylaryl, -Cj-Cg alkyl-(C₃-Cg carbocycle), C₃-Cg heterocycle and -Cj-Cg alkyi-(C₃-Cg heterocycle);

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

R⁹ is selected from -H and -Cj-Cg alkyl;

R¹⁰ is selected from aryl group or -Cj-Cg heterocycle; Z is-O-,-S-,-NH-, or-NR¹²-, wherein R¹² is Cj-Cg alkyl;

Rⁿ is selected from -H, C1-C20 alkyl, aryl, -C3-C8 heterocycle, -(RⁿO)ro15 R*⁴, or-iR¹³O)_m-CH(R^,5)2;

m is an integer ranging from 1-1000;

R¹³ is-CrCg alkyl;

R¹⁴ is-Hor-Cj-Cg alkyl;

each occurrence of R^1S is independently -H, -COOH, -(CH2)_n-N(R^l6)2, 20 (CH^-SOsH, or -(CH^-SQrCi-Cg alkyl;

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

In one embodiment, R³, R* and R⁷ are independently isopropyl or sec25 butyl and R^s is -H. In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is -H, and R⁷ is sec-butyl.

In another βιηΐχχΐίιηβηζ R² and R⁶ are each methyl, and R⁹ is -H. In still another embodiment, each occurrence of R is -OCH₃.

In an exemplary επΛοάΐιηβηζ R³ and R⁴ are each isopropyl, R² and R⁶ are 30 each methyl, R^s is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH₃, and R⁹ is -H.

In one embodiment, Z is -0- or -NH-.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

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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(R^1S)2, wherein R¹⁵ is (CH2)n-N(R¹⁶>2, and R¹⁶ is -CrC, alkyl or -{CH^-COOH.

In another embodiment, when Z is -NH, R¹¹ is -CH(R^,5)z, wherein R^1S is 5 (CHîJn-SOîH.

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

<img file="CA2841741C_D0018.tif" />

<img file="CA2841741C_D0019.tif" />

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<img file="CA2841741C_D0020.tif" />

<img file="CA2841741C_D0021.tif" />

/^N- 6,

<img file="CA2841741C_D0022.tif" />

<img file="CA2841741C_D0023.tif" />

SO3H g.

<img file="CA2841741C_D0024.tif" />

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<img file="CA2841741C_D0025.tif" />

<img file="CA2841741C_D0026.tif" />

NHz 10 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-f-A.-W_w-Y_y-D)_p comprising an antibody covalently attached to one or more drag units (moieites). The antibody-drag conjugate compounds include pharmaceutically acceptable saltsor solvates thereof.

Formulaic 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_OO12O3);

(2) E16 (LA.T1, 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 (MIT, MSLN, SMR, megakaryocyte potentiating factor, mesothelia. Genbank accession no. NM_005823);

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

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WO 2005/081711 PCT/US2004/038392 (7) Sema 5b (FU10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphonn 5b Hog, sema domain, seven thrombospondin repeats (type 1 and type 1likc), 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 (Endothélia type B receptor, Genbank accession no. AY275463);

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

(11) STEAP2 (HGNC_8639,1PCA-1, PCANAPI, 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, FLJ2OO41, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);

(13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGF1, teratocarcinomadcrivcd growth factor, Genbank accession no. NP_003203 or NMJJ03212);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3dÆpstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004);

(15) CD79b (CD79B, CD79p, 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);

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WO 2005/081711 PCI7US2004/038392 (25) GEDA (Genbank accession no. AY260763;

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

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

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cellspecific 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. 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 H 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 erythcmatosis, Genbank accession No. NPJDO5573.1);

(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ΓΓΑΜ 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,

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PCT/ÜS2004/038392 aisOorl, each W is independently an Amino Acid unit, w is an integer ranging from 0 to 12,

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

D is a Drug moiety selected from Formulas Da and Dp:

<img file="CA2841741C_D0027.tif" />

<img file="CA2841741C_D0028.tif" />

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

R² is selected from H and Ci-C_s alkyl;

R³ is selected from H, C|-Cg alkyl, C₃-Cg caibocycle, aryl, Q-Cj alkylaryl, Cr-Cj alkyl-fCs-Cs caibocycle), C₃-Ce heterocycle and Cj-Cg alkyl-fCyCs heterocycle);

R⁴ is selected from H, CrQ alkyl, C₃-Ct carbocycle, aryl, Ci-Cg alkylaryl, Ci-Cg alkyl-(C₃-Cg carbocycle), Cj-Cg heterocycle and Ci-Cg alkyl-(C₃-Cg heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R^s jointly form a carbocyclic ring and have the formula -(CR^- wherein R’ andR^b are independently selected from H, C|-Cg alkyl and C₃-Cg carbocycle and n is selected from 2,3,4,5 and 6;

R^fi is selected from H and Ci-Cg alkyl;

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<td></td><td> WO 2005/081711 PCT7ÜS2004/038392 R⁷ is selected from H, Cj-Cg alkyl, Cj-Cg carbocycle, aryl, C]-C₈ alkylaryl, Ci-Cg alkyl-(C3-C₈ carbocycle), C₃-Ce heterocycle and Ci-C₈ alkyl-(C3-C₈heterocycle); each R* is independently selected from H, OH, Ci-Cg alkyl, C3-C8</td>

<td> 5</td><td> carbocycle and O-(Ci-C₈ alkyl); R⁹ is selected from Hand Cj-Cg alkyl; R¹⁰ is selected from aryl or Cj-Cg heterocycle; Z is 0, S, NH, or NR¹², wherein R¹² is Cj-Cg alkyl; R^u is selected from H, Cj-Cjo alkyl, aryl, Cj-Cg heterocycle, -(R’^O^-R¹⁴,</td>

<td> 10</td><td> or-iR^-CHrt; m is an integer ranging from 1-1000; R” is CrC₈ alkyl; R^,4isHorC|-Csalkyl; each occurrence of R¹⁵ is independently H, COOH, -(CH^n-NiR¹⁶^,</td>

<td> 15</td><td> -(CH^-SOjH, or -(CH^-SCh-Ci-Q alkyl; each occurrence of R¹⁶ is independently H, C|-C₈ alkyl, or -(CH^nCOOH; R¹⁸ is selected from -C(R⁸)r-C(R⁸)2-aryl, -C(R⁸)z-C(R^B)r-(C3-Cg heterocycle), and -C(R⁸)r-C(R⁸)2-(C3-C₈ carbocycle); and</td>

<td> 20</td><td> 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 secbutyl and R³ is -H. In an exemplary embodimenL R³ and R⁴ are each isopropyl, R³ is -H, and R⁷ is sec-butyl.</td>

<td> 25</td><td> In yet another embodiment, R² and R^e are each methyl, and R⁹ is -H. la 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, R^s is -H, R⁷ is sec-butyl, each occurrence of R⁸ is -OCH3, and R⁹ is -Η. In one embodiment, Z is -0- or -NH-.</td>

<td> 30</td><td> In one embodiment, R*° is aryl. In an exemplary embodimenfr R¹⁰ is -phenyl. In an exemplary embodiment, when Z is -0-, Rⁿ is -H, methyl or t-butyl.</td>

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PCT/DS2004/038392 fa one embodiment, when Z is -NH, Rⁿ is -CH(R^1S)2, wherein R^1S is (CH2)_n-N(R¹⁶)2, and R¹⁶ is -Cj-Cg alkyl or -(CH^-COOH.

In another embodiment when Z is -NH, R¹¹ is -CH(R¹⁵)2, wherein R¹⁵ is (CH^-SOjH.

I

Exemplary embodiments of Formula Ic ADC have the following structures:

<img file="CA2841741C_D0029.tif" />

Ab-MC'VC-PAB-MMAF

<img file="CA2841741C_D0030.tif" />

Ab-MC-vc-PAB-MMAE

<img file="CA2841741C_D0031.tif" />

Ab-MC-MMAE

<img file="CA2841741C_D0032.tif" />

Ab-MC-MMAF 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 drag loading is represented by p, the average number of drugs per antibody in a molecule of Formula L Drug loading may range from 1 to 20 drugs (D) per

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PCT/US2004/038392 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 20 in the antibodies of the compounds of the invention exist as disulfide bridges and must be reduced with a reducing agent such as ditbiothreitol (DIT). 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 drag-linker intermediate, or linker reagent followed by drug moiety reagent, then 30 the resulting product is a mixture of ADC compounds with a distribution of one or more drag moieties attached to an antibody. The average number of drags 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

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PCT/US2WM/038392 spectroscopy, and separated by HPLC, e.g., hydrophobic interaction chromatography (Effect of drag loading on the pharmacology, pharmacokinetics, and toxicity of an antiCD30 antibody-drug conjugate”, Hamblett, KJ., 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 Drug-LinkerLigand Conjugate has the formula:

•~“A_a W_w Yy“ wherein:

-A- is a Stretcher unit;

aisOor 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.

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43.1 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 (Trant’s reagent) or another sulfhydryl generating reagent

In one embodiment the Stretcher unit forms a bond with a sulfur atom erf 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 EŒa and mb, 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-, Ci-Cjo alkylene-arylene-, -arylene-C|-C)o alkylene-, -Ci-Cio alkylene-fCyCs carbocyclo)-, -(C3-C8 carbocyclo)-Ci-Cio alkylene-, -Cj-Cg heterocyclo-, -C4-C10 alkylene-fCj-Cg heterocyclo)-, -iC₃-Ce heterocyclo)-Ci-C_!0 alkylene-, -(CH2CH₂O)_r, and -(CHiCHjOjr-CHr; and r is an integer ranging from 1-10. It is to be understood from all the exemplary embodiments of Formula la, such as HI-VI, that even where not denoted expressly, from 1 to 20 drug moieties are linked to a Ligand ( p = 1-20).

<img file="CA2841741C_D0033.tif" />

Hla

H₂-CONH—R¹⁷-C(0)—W_w—Y_y—D mb

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

-{CHzjr:

<img file="CA2841741C_D0034.tif" />

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

-fCH₂CH₂O)_rCH₂-;andris2:

<img file="CA2841741C_D0035.tif" />

Still another illustrative Stretcher unit is that of Formula IHb wherein R¹⁷ is -(CHjjs-:

<img file="CA2841741C_D0036.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.

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.

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Representative Stretcher units of (his 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;

<img file="CA2841741C_D0037.tif" />

Va

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 (-CH0) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, 10 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, VDb, and Vic, wherein -R¹⁷-, L·, -W-, -Y-, 4>, w and y are as defined above.

<img file="CA2841741C_D0038.tif" />

Via

<img file="CA2841741C_D0039.tif" />

VBb

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<img file="CA2841741C_D0040.tif" />

<img file="CA2841741C_D0041.tif" />

Vic

432 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 5 Spacer unit is absent and links the Ligand unit to the Drug unit if the Stretcher unit and Spacer unit are absent.

Ww- 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, 10 and w is an integer ranging from 0 to 12:

<img file="CA2841741C_D0042.tif" />

wherein R^wis hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CHzOH, -CH(OH)CH3, -CHzCHîSCHj, -OfeCONHj, -CHjCOOH, -CHzCHîCONHj. GH₂ŒI₂COOH,-(CH₂)₃NHC(=NH)NH₂, -(CH₂)3NH2.-(CH2)3NHCOCH3,15 (CHîJîNHCHO, (CHzJz^HC^NHJNHî, -(CHiW^, -(CHî^COCHs, (CHzWCHO, -ÎCH₂)3NHCONH₂, -(OL^NHCONHz, -CH₂CH₂OT(OH)CH2NH₂,2pyridylmcthyi-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyi,

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<img file="CA2841741C_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 à Drug (D).

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

<img file="CA2841741C_D0044.tif" />

wherein R²⁰ and R²’ are as follows:

benzyl methyl isopropyl isopropyl benzyl isobutyl jec-butyl

R* (CHjWî; (CH₂)4NH₂; (CH2)4NH₂;

(CH₂)3NHCONH₂;

(CH₂)3NHCONH₂;

(CH₂)3NHCONH₂;

(CH₂)3NHCONH₂;

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<img file="CA2841741C_D0045.tif" />

(CH2)3NHCONH₂;

methyl; and (CH₂)₃NHC(=NH)NH₂;

<img file="CA2841741C_D0046.tif" />

wherein R²⁰, R²¹ and R²² are as follows:

<td> R®</td><td> R^</td>

<td> benzyl</td><td> benzyl</td>

<td> isopropyl</td><td> benzyl</td>

<td> H</td><td> benzyl</td>

^22 (CH2)4NH₂; (CWft; and (CH2)4NH₂;

<img file="CA2841741C_D0047.tif" />

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

<td> R®</td><td> r21</td><td> R®</td><td></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 -(CHzjiNH^ R²⁰ isopropyl and R²¹ is (CHzjiNHz; R²⁰ isopropyl and R²¹ is -(CH₂)3NHCONH₂. Another exemplary Amino 10 Acid unit is a unit of formula (VID) wherein R²⁰ is benzyl, R²¹ is benzyl, and R²² is (CH,)».

Usefill -Ww- units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease.

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WO 2005/081711 PCT/ÜS2004/038392 fa 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, -Ww is a dipeptide, tripeptide, tetrapeptide or pentapeptide.

When R”, R²⁰, R²’, 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 R*⁹, R²⁰, R²’, 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 valinecitrulline. fa another aspect, the Amino Acid unit is pbenylalanine-lysine (i.e. fk). fa yet another aspect of the Amino Acid unit, the Amino Acid unit is N-methylvaline-citrulline. In yet another aspect, foe Amino Acid unit is 5-aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine saine 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 non-uatural amino acids.

433 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 foe Drag moiety when foe 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

Spacer units are of two genera] types: self-immolative and non selfimmolative. 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 Diug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immolative Spacer unit include, but are not limited to a (glycineglycine) 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-cel] associated-protease, a cancer-cellassociated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycine-Drag moiety is cleaved from L-ArWw-. fa one embodiment, an

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PC17ÜS2004/038392 independent hydrolysis reaction takes place within the target cell, cleaving the glycineDrug moiety bond and liberating the Drug.

In another embodiment, -Yy- is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) whose phenylene portion is substituted with Q> wherein Q is -C)-C₈ alkyl, -O(C|-C₈ alkyl), -halogen,- nitro or -cyano; and m is an integer ranging from 0-4. Scheme 1

Ab- -A_a-W*—Gly—D enzymatic cleavage

GlyO hydrolysis |

Drug

Ab-f-Ae-W_w-Gly-Gly]-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-.

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 self-immolative Spacer unit can release -D without the need for a separate hydrolysis step. In this 15 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 Tokief aL (2002)1 Org. Chem. 67:1866-1872.

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<img file="CA2841741C_D0048.tif" />

Qm

<img file="CA2841741C_D0049.tif" />

<img file="CA2841741C_D0050.tif" />

1,6-elimlnadon

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

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.

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<img file="CA2841741C_D0051.tif" />

wherein Q is -Ci-Cg alkyl, -O-fCj-Cg 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 2amînoîmidazol-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 410 aminobutyric acid amides (Rodrigues et al, Chemistry Biology, 1995,2,223), appropriately substituted bicyclo[2.2.1] and bicyclo[22.2] ring systems (Storm, etaL, J. Amer. Chem. Soc., 1972,94,5815) and 2-aminophenylpropionic acid amides (Amsberry, etaL,]. Org. Chem., 1990,55,5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, etaL, J. Med. Chem., 1984,27,1447) are also examples of self-immolative spacer useful in Exemplary Compounds.

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

Scheme 4

<img file="CA2841741C_D0052.tif" />

L—rA_a—W_w.

^0(0(0))^-0 ^CH^OfCtOB-Dl

P enzymatic cleavage

2 drugs wherein Q is -Ci-C₈ alkyl, -O-(C|-Ce 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 (-Yy) are represented by Formulas (X)-(X11):

<img file="CA2841741C_D0053.tif" />

wherein Q is -C|-Ce alkyl, -O-(C_rC8 alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4;

HHN-CH₂-CO-4 xi and

Embodiments of the Formula la* and Ic antibody-drag conjugate compounds include:

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<img file="CA2841741C_D0054.tif" />

<img file="CA2841741C_D0055.tif" />

P wherein w and y are each 0,

<img file="CA2841741C_D0056.tif" />

<img file="CA2841741C_D0057.tif" />

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<img file="CA2841741C_D0058.tif" />

4.4 THE DRUG UNIT (MOIETY)

The drag 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 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 Drag unit (moiety) having a nitrogen atom that can form a bond with the Spacer unit when y=I 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 Dy:

R³ OR⁷ CH3 R⁹

SY/àVfAY'-·· r2 o R R R⁶ R⁸ O R⁸ O De

<img file="CA2841741C_D0059.tif" />

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WO 2005/081711 PCT/US2004/038392 wherein, independently at each location:

R^z is selected from H and C;-C₈ alkyl;

R³ is selected from H, Ci-Cg alkyl, Cg-Cg carbocycle, aryl, Ci-Cg alkylaryl, Cj-Ce alkyl-(Cg-Cg carbocycle), CyCg heterocycle and Ci-Cg alkyl-iCj-Cg heterocycle);

R⁴ is selected from H, C|-C₈ alkyl, C₃-C₈ carbocycle, aryl, Ci-C₈ alkylaryl, Cj-C₈ alkyi-(C₃-Cg carbocycle), Cg-Cg heterocycle and Ci-C₈ alkyl-fCyCi heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R³ jointly form a carbocyclic ring and have the formula -(CR'R¹^- wherein R* and R^k are independently selected from H, Ci-Cg alkyl and C₃-Cg carbocycie 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, C|-C₈ alkyl, Cg-Cg carbocycle, aryl, C|-Cg alkylaryl, Ci-C₈ aIkyI-(C3-C₈ carbocycle), Cg-C» heterocycle and Ci-Ce alkyl-(C3-Cs heterocycle);

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

R⁹is selected from H and Ci-Cg alkyl;

R¹⁰ is selected from aryl or Cg-Cg heterocycle;

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

Rⁿ is selected from H, Ci-C» alkyl, aryl, C3-Cg heterocycle, -(R^I3O)m-R¹⁴, or-fR’^VCHiR*⁵)/.

m is an integer ranging from 1-1000;

R¹³ is C^Cg alkyl;

R^l4isH or C,-C₈ alkyl;

each occurrence of R^IS is independently H, COOH, --(CHzVNiR¹⁶)^ -(CH^-SOgH, or -(CH^-SOi-Ci-Q alkyl;

each occurrence of R¹⁶ is independently H, C|-C₈ alkyl, or-fCHzV COOH;

R¹⁸ is selected from ^(R’MXR’h-aryl. heterocycle), and -C(R⁸)2-C(R⁸)r-&lt;C3-Cg carbocycie); and n is an integer ranging from 0 to 6.

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In one embodiment, R³, R⁴ and R⁷ are independently isopropyl or secbntyi and R^s is -H. In an exemplary embodiment, R³ and R* 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 R^s 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(R^,5)2, wherein R¹⁵ is (CH2)n-N(R^,6)2, and R¹⁶ is-CrC8 alkyl or -(CH^-COOR

In another embodiment when Z is -NH, R^u is -CH(R¹⁵)a, wherein R¹⁵ is (Cft^-SOjEL

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

MMAE

MMAF

<img file="CA2841741C_D0060.tif" />

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<img file="CA2841741C_D0061.tif" />

<img file="CA2841741C_D0062.tif" />

<img file="CA2841741C_D0063.tif" />

<img file="CA2841741C_D0064.tif" />

<img file="CA2841741C_D0065.tif" />

<img file="CA2841741C_D0066.tif" />

<img file="CA2841741C_D0067.tif" />

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<img file="CA2841741C_D0068.tif" />

SO₃H

<img file="CA2841741C_D0069.tif" />

<img file="CA2841741C_D0070.tif" />

Ό00Η ,and

T

NHj and pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited to triethyleue glycol esters (TEG), as shown above, can be attached to the Drag Unit at R¹ *. Without 10 being bound by theory, the hydrophilic groups assist in the internalization and nonagglomeration of the Drag Unit.

4.5 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, hi one aspect, the ligand unit acts to deliver the Drug unit to the particular target cell population with which the ligand unit

CA 02841741 2014-02-03 reacts. Such Ligands include, but are not limited to, large molecular weight proteins such as, for example, fall-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrienttransport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substance.

A Ligand unit can form a bond to à Stretcher unit, an Amino Acid unit, a Spacer UniL 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 embodimenL 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 embodimenL 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 reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Trant’s Reagent).

In another embodimenL 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 eg., 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).

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Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to, transfcmn, 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 TGF-β, 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-ofinteresL 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 lysoiecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arc 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 (mAb) to an antigen-of-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 Kdhler 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, I985, 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

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PCT/US2004/038392 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 el aL, 1983, Proc. Nad. Acad. ScL USA. 80,7308-7312; Kozbor et aL, 1983, Immunology Today 4,72-79; and Olsson et aL, 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 full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein etaL, 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 tiie correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker etaL,EMB0 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, C«2, and Ch3 regions. It is preferred to have the first heavy-chain constant region (C_H1) containing the site necessary for light chain binding, present in at least one of the fusions. Nucleic adds 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 oiganism. 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

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CA2841741 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).

For further details for generating bispecific antibodies see, for example. Surest) a aL, Methods in Enzymology, 1986,121:210; Rodrigues et al, 1993, J. of Immunology 151:6954-6961; Carteret aL, 1992, BioTTechnology 10:163-167; Carter et aL, 1995, Z 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 bifanctional antibodies can be derived either biologically, Le, 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.

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 antianti-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 die 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 (eg., the BIA core assay) (See, for eg., 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 usefol antibodies include fragments of antibodies such as, but not limited to, F(ab*)z 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

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CA284174I \ ;· antibody molecule, and Fab fragments, which can be 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 UJS. Paient No. 4946778; Bird, 1988, Science 242:423-42; Huston et aL, 1988, fmc. Nad Acad. Set USA 85:5879-5883; and Ward er al, 1989, Nature 334:544-54), 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 pardons, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions arc derived from different animal species, such as those having a variable region derived from a murine monoclonal and human trnnmnoglobulin constant regions. (See, e.g., Cabilly et at, U.S. Patent No. 4816567; and Boss rf of, U.S. Patent No. 4,816397.

Humanized antibodies are antibody molecules from nonhuman 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, U3. Patent No. 5,585,089).

Such chimeric and hamanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described tn 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; Better et dL, 1988, Science 240:1041-1043; Un et aL, 1987, Proc. Natl Acad. Sci. USA 84:3439-3443; Liu etaL, 1987, J. Immunol. 1393521-3526; Sun et aL, 1987, Proc. Nad. Acad. Sci. USA 84:214-218; Nishimura et dL, 1987, Cancer. Res. 47:999-1005; Wood et aL, 1985, Nature 314:446-449; and Shaw et aL, 1988, J. Nad. Cancer Inst 80:1553-1559; Μοιώοη, 1985, Science 229:1202-1207; Oi etaL, 1986, BioTechniques 4:214; U.S. PatentNo. 5225539; Jones etaL, 1986, Nature 321:552-525;

Veifioeyan etai. (1988) Science 239:1534; and Beadier er aM 988, J. Immuno). 141:4053-4060,

Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous

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CA 02841741 2014-02-03 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 daring 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. 13:6593). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies. See, eg., US. Patent Nos. 5625126; 5633425; 5569825; 5661016; 5545806.

Other hummt 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, eg., 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 Bid, 222:581 (1991); Quan, Μ. P. and Carter,?. 2002. The rise of monoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease, Jardieu, P. M. and Hck Jr, R. B, eds, Marcel Dekker, New York, NY, Chapter 20, pp. 427-469).

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 (eg., a peptide bond), at either the N-tcrminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10,20 or 50 amino add 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.

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Antibodies include analogs and derivatives that are either modified, Le_t by the covalent attachment of any type of molecule as long as such covalent attachment pennits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, die derivatives and analogs of the antibodies include those that have been further modified, &amp;g., by glycosylation, acetylation, pegyiation, 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 tnnicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino adds.

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 add 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

). 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, eg., from the GenBank 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, g.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; RlTUXAN® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin’s lymphoma; OvaRex (AltaRex

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Corporation, MA) which is a murine antibody for the treatment of ovarian cancer, Panorex (Glaxo Wellcome, NC) which is a murine IgG^ antibody for the treatment of colorectal cancer, Cetuximab ΕΛΐ tux (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 PH (Leukosite, MA) which is a humanized IgG_tantibody 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 nonHodgkin’s lymphoma; ADomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or non-Hodgkin’s lymphoma; Avastin (Genentech, Inc., CA) which is an anti-VEGF humanized antibody for the treatment of lung and colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for toe treatment of non-Hodgkin’s lymphoma; and CEAcide (Immunomedics, NJ) winch 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), CA15-3 (carcinomas), CA19-9 (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), MUC1-KLH (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

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PC17US2004/038392 include, but aie not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. I, Henderson, A. J., Hofstead, S. J., Casazza, A M., Firestone, R. A., Hellstrom, L, HeUsttâm, K. B., “Cure of Xenografted Human Carcinomas by BR96-Doxorabicin Immunoconjugates” Science 1993,261,212-215), BR64 (Trail, PA, Winner, D, Knipe, J., Henderson, A. J., Lasch, S. L, Zoecklet, 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, L ‘Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64~Doxorubicin 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, G B., and Wahi, A. F. “Agonistic properties and in vivo 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 AC10.(Bowen, ML 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 ). M»ny 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 Radiopham, 2000,15,459-76; Murray, J. L, “Monoclonal antibody treatment of solid tumois: 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, die 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 antiHERT, from hybridoma), rhu4D5 (transiently expressed, full-length humanized antibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8 (full-length, 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 nonglycosylated)).

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 innnunospccific for an antigen of a cell that is

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WO 2005/081711 PCT/ÜS2004/038392 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, Anti-Cardiolipin Antibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SM Antibody; Anti-Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo; Anti-UiRNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital Cells Antibody; Anti-Histones; AntiRNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti-GBM Antibody.

hi 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 intcgrin, 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, CDS, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Noo-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB. TNF-R1, TNFR-2, RANK, TACL BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAHz-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CDlla, CDllb, CD1 le, 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.

hi 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,

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WO 2005/081711 PCT/US2004/038392 polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, eg., IPS 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, eg., 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 (Medlnunune, 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 HTV 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 IgGj 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 diphtberiae, 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 cercus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella

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WO 2005/081711 PCT/US2004/038392 abortus. Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsntsngumushi, Chlamydia spp.); pathogenic fangi (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 vennicularis, 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 tumorassociated polypeptides that are specifically expressed on the surface of one or more particular typc(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 foe non-cancerous cells. The identification of such tumor-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

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PC17US2004/038392 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 targeted by antibodies include all amino acid sequence variants and isofonns possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the corresponding sequences listed (SEQ Π) 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):

(I) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NMJ001203, ten Dijkeæ.» etaL Science 264 (5155):101-104 (1994), Oncogene 14 (II) :1377-1382 (1997)); W02004063362 (Claim 2); W02003042661 (Claim 12); US2003134790-A1 (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); WO20Û298358 (Claim 1; Page 183); W0200254940(Page 100-101); WO200259377(Page 349-350); W0200230268 (Claim 27; Page 376); W020D148204 (Example; Fig 4) NP-001194 bone morphogenetic protein receptor, type IB /pid=NP_001194.1 Cross-references: MEÆ603248; NPJJ01194.1 ; NMJX)1203J

502 aa

MT,T.R3AmrTJi vrfi'K κ RDnRnTAPTPRPKVLRCKCHHHCPEDSVHNICS'roGYCFTMIEED Ρδσηρνντ8οαΛηΕβ3θΡζΧ3ΐΐ)ΤΡΐΡΗ0ΐυΐ3ΤΕοσΓΕΐΐΝΒαΝκηι₁ΗΡΤϋ?ρΐΛΝηηΓνη GPIHHRALMSVTVCSLLLVLIILPCYPRYKRQSTRPRYSIGLBQDETYIPPGESLRDLI BQSQSSGSGSC&amp;PIiVQRTIAKQIQMVKQieXSRYræVWMGKWRGEKVAVXVFFTTEEAS WFRBTKITQfT5/IiMRHBinXGFIASDIKlTQSWTQI&lt;YLITDYHEMGSLYDyLKSTTIiIlAKS MLiaAYSSVSQIXmiJiTHIFSTQGKTAIAEimi^tani»ViaQiGTCCIADIiGIAVKFISD TNEVDIPPWrRVGTKRYMPPSVLDESLHRNHFQSYIMAIMYSFGLILWEVARRCVSGGIV EEYQLPYHDLVPSDPSYBDMRKrVCIKKLRPSFPURWSSDBCLRQMGKLMTECWAHNPAS RLTALRVKKTIAKMSBSQDIKL·

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PCT/US2004/038392 (SBQ ID NO: 1) (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);

Biocbem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), GaugitschJLW., etaL (1992)1 Biol· Cbem. 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 133136); US2003224454 (Fig 3); W02003025138 (Claim 12; Page 150);

NPJ0O3477 solute earner family 7 (cationic amino acid transporter, y+ system), member 5 /pid=NP_0034773 - Homo sapiens

Cross-references: MDÆ600182; NPJJ03477.3; NM_015923; NM_003486_l

507 aa

NAGAGPKRRAIAAPAAEKKEEAREKMIAAKSADGSAPAGEGEGVTIjQRNITIjLNGVAHV

GTIIGSGIPVTPTGVLKBAGSPGLALVVHAACGVPSIVGALCYARLGTTISKSGCïJYAYM ΏκνγοεηΡΑκίΏαΜίΕηιιικρβεοΥΓν^νΡΑΤΥΐΛΚΡηρρτσρνΡΕΕΑΆΚίνΆα^ονΣ ηΐιΊΆνΝΟΥδνΚΑΑΤΗνΟηΑΡΑΑΑΚΙιΙιΑΙιΑΙιΙ ILLGFVQIGKGWSNLDPNFSFEGTKLDV GNIVIAiYSGIiFAYGGWNYIiNFVTBBMINPinun»MAIIISLPIVTI*VYVI»TNLAYFTrL· 3ΤΒΟΜΙ>δ8ΕΑνΑνΠΓΟΝΥΗ1θνΐ^ΝΪΙΡνΡνθΙ£ΟΒ35νΝ68Ι.ΡΤ88ΚΙ.ΡΡν08ΚΕΟΗηρ SIIÆMIHPQU,TpVPSLVFTCVimÆiYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRH RKPÉLERPIKVNlALPVFFÏIiACLFriIAVSFWKrPVECGIGFTIILSGIiP'VYFFGVWWKlï

KPKWLLQGIFSTTVLCQKLMQWPQBT (SEQ ID NO: 2) (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM-012449

Cancer Res. 61 (15), 5857-5860 (2001), Hnbert^.S., et al (1999) Proc. Nati. Acad. Sci. USA. 96 (25):14523-14528); W02004065577 (Claim 6); W02004027049 (Fig IL); EPI394274(Example II); W02004016225 (Claim2); W02003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Fig 2); WO200289747 (Example 5; Page 618-619); W02003022995 (Example 9; Fig 13A, Example 53; Page 173, Example 2; Fig 2A);

NP_036581 six transmembrane epithelial antigen of the prostate

Cross-references: M1M:6O4415; NPJ036581.1; NNf_012449„l

339 aa

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MBSRKDITNQESLWKMKPRRNIiEEDDYLIIKDTGETSMLKRPVLLHLHQTAHADEFE>CPSE ΒΟΗΤΟΕΕΡΡα»ΙΠ.ΡΙΚΙΑΑΙΙΑ8ΕΤπ.ΥΤΙΛΚΕνΐΗΡΙΑΤ5ΗΟΟΥΡΥΚΙΡΙΙ.νΐΝΚνΐ.ΡΜ VSIIT,LALVYLPGVIAAIVQIJINGTKYKKFPHWL>DKWMLTRKQFGLLSFFFAVI>HAIYSL· 5ΥΡΜΚΚ5ΥΕΥΚΠΠΝΗΑΪ(»ν00ΝΚΕηΑΗΙΒΗΠνΗΚΜΕΙΥν5ηαΐναΐΛΙΙιΑηηΑνΤ3ΙΡ5 VED£mTWREFHYlQSKlÆIVSlJiL>GTlHAL>Ii?ÀWNKW.IDIKQFVWYTPPTFMIAVFLPIV VLIFiœiDPLPCWCKimRHGWEDVTKINKrEICSQL (SEQ ID NO 3) (4) 0772Ρ (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: G134501467; AAK74120.3; AF361486_1

6995 aa

PWSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTAVT NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSI. tsglrbtssserissategstvlsevpsgattevsrtevissrgtsmsgpdqftispdis TBAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLiDTSTTTFWSGTHSTASPGFSHS EMTTLiMSRTPGDVPWPSLPSVEEASSVSSSLSSP/lMTSTSFFSTLPESISSSPIiPVTALL TLGPVKTTDMLRTSSEPETSSPPNLSSTSAEIIATSEVTKDREKIHPSSNTPVVNVGTVI YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPBTMMTQPTSSLTSGLREIS TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTSTITRIS TPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSR GPEDVSWPSRPSVEKTSPPSSIiVSLSAVTSPSPIiYSTPSESSHSSPIiRVTSIiFTPVMMKT TOiHDTSLEPVTTSPPSMNITSDESIiATSKATMETEAIQLSENTAVTQMGTISARQRFYS sypglpepskvtspwtsstikdivsttipasseitriemeststi>tptpretstsqeih satkpstvpykaltsatiedsmtqvmsssrgpspdqstmsqdistevitrlstspiktes tbmtittqtgspgatsrgtltldtsttfmsgthstascjgpshsqmtalmsrtpgevpwls ΗΡ8νΒΕΑ88Α8Ρ8Β88ΡνΜΤ883Ρν88ΤΙ_ίΡ0ΒΙΗ333Ι,ΡνΤ8ϋΤ801ινΚΤΤΕΙΛ<3Τ83Ε PBTSSPPNLSSTSAEIIATTEVTTOTEKLEMTNWTSGYTHESPSSVIiADSVTTKATSSM GITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEBTSSATRKSTVLSgVPT gattevsrtraisssrtsipgpaqstmssdtsmetitristpltrkestdmaitpktgps gatsqgtftldssstaswpgthsattqrfprsvvttpmsrgpedvswpspi>sveknspps 3&ν3338νΤ3Ρ3ΡΙ.Υ3ΤΡ3σ35Η83ΡνΡνΤ3ηΡΤ3ΙΜΜΚΑΤϋΜΙ<Ι1Α8Ι_ίΕΡΕΤΤ5ΑΡΝΜΝΙ TSDESIiAASKATTETBAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSS irdnmvsttmpgssgitrieiesmssltpglrbtrtsqditsstetstvlykmpsgatpe vsrtevmpssrtsipgpaqstmsldisdewtrlstspimtesaeitittcjtgysiatsq Vn.PIÆTSMTFLSGTHSTMSQGIiSHSBMTNLMSRGPESIjSWTSPRFVETTRSSSSLTSI.P 1ΤΤ3Ιι3Ρν38ΤΙιΙιΟ33Ρ85ΡΙιΡνΤ8Ι₄ΙΙιΡΟΙινΚΤΤΕνΐ₁ΟΤ38ΒΡΚΓ888ΡΝΙ₁88ΤενΕΙΡ ATSEIMTDTEKIHPSSNTAVAKVRTSSSVHBSHSSVLADSETTITIPSMGITSAVEDTTV

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FTSNPAFSETRRIPTRPTPSLTPGFRETSTSERTTSITETSAVLFGVPTSATTBVSMTEI MSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSKSTQMTITTQKSSPGATAQSTLTLA ΤΤΤΑΡΙΛΚΤΗ3ΤνΡΡΕί1ίΙ8ΕΜΤΤΙΜ5Η8ΡΚΝΡ3»Κ33ΡΡνΕΚΤ38383ΙιΙ.8Ι.ΡνΤΤ3Ρ3 VSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEriAASEVTTDTE KIHPSSSMAVTNVGTTSSGHKLYSSVSIHSBPSKATYPVGTPSSMARTSISTSMPANFET TGFEAEPFSHIiTSGIJlKTNMSIJXrSSVTPTimPSSPGSTHUiQSSKTDFTSSMCrSSPDW PPASQYTBIPVDIITPFNASPSTrBSTGÏTSPPESRFTMSVTESTHHLSTDLLPSAETIS TGTVMPSIÆBAKrePATTOVPRAISGSGSPFSRTBSGPGDATIiSTIAESLPSSTPVPFSS STFTTTOSSTIPALHEITSSSATPYRVDTSIXSTESSTTBGRLVMVSTIiDTSSQPGRTSSS PIUJTRMTESVELGTVTSAYÎJVPSLSTRLTRTDGIMEHITKIPNEAAHRSTIRPVKSPQT STSPASPKGmnXSOTKRMETTTTMiKITrTMamSIUW^^ hastiptemmittpyvfpdvpbttssîatsiæaetstaijiotpsvfsrbsbtiaslvsr SGABRSPVIQTLDVSSSBPDTTASWVIHPAETIPTVSKTTPNPFHSELDT7SSTATSHGA DVSSAIPTKISPSBUUa₁TPLmSCrDTSTTFPTLTKSPHETETRTTWL₁THPAETSSTI PRTIPNFSHHBSDATPSZATSPGABTSSAIPIMTVSPGAEDIiVTSQVTSSCHnRNMTIPT ηΤΙ^Ρ6ΒΡΚΤΙΑ3ΐνΤΗΡΒΆ0Τ38ΑΙΡΤ3ΪΙ8ΡΑν3ΒΙ_ίνΤ3ΜνΤ3ΙΛΑΚΓβΓΠίΚΑηΤΝ3 PGEPATTVSLVTHSAQTSPTVPWTTSIFFHSXSDTTPSMTTSHGAESSSAVPTPTVSTEV PGWTPLVTSSRAVISTTIPIIjTLSPGBPETTPSMATSHGEEASSAIPTPTVSPGVPGVV TSLVTSSRAVTSTTIPILTPSLGEPETTPSMATSHGTEAGSAVPTVLPEVPQtVT’SLVAS SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTV3PEVPGWTSLVT83SGVN3 TSIPTLILSPGELBT?TPSMATSHGÆAS8AVPTPTVSPGVSGWrPIjVTSSRAVTSTTIP ILTLSSSEPETTPSMATSBGVHASSAVLTVSPEVPGMVTFLVTS3RAVTSmPTLTISS ΟΕΡΕΤΤΓ3ΐνΤΗ3ΒΑΚΜΙ8ΑΙΡΤΙισν3ΡΤνοβΙ>νΤ8ηνΓ88β8ΕΤ8ΑΡ81Π_ίτνΆ380ΡΕΤ IDSWVAHPGTRASSVVmTVBTGBPFnnSLVTHPAESSSTLPRTTSRPSHSBLDTMPS ΤνΤδΡΒΑΕδδΒΑΙδΤΤΙδΡβΙΡβνΐ,ΤδΙινΤδδθωΐΗΑΤΡΡΤνΡΕδΡΗΒδΕΑΪΑδΗνΤΗΡ avtsttvprttphyshsepdttpsiatspgwsatsdfptitvspdvpdnvtsqvtssgtd Τ8ΙΤΙΡΤηΤΚ38βΕΡΕΤΤΤ3ΡΙΤΥ8ΕΤΗΤ88λΙΡΤΒΡν8ΡηΑ3ΚΜΙ_ίΤ3Κνΐ330Τ03ΤΤΤ FPn»TBTPYBPETTAIQI»IHPÀET8WVPRTTPKFSHSKSDTTLPVAITSPGPEASSAV3 TTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPBTTAæWLTaPAETSnVSGnPN FSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGWTSQVTSSATDTSTAIPTLTPSPG BPETTASSATHPGTQTGPTVPIRTVPSSBPDÏMASWVTHPPQTSTPVSRTTSSPSHSSPD ΑΤΡνΜΑΤ8ΡΕΤΕΑ38ΑνηΤΤΙ8Ρ6»ΒΙΝΤ80ΙΤ83βΑΑΤ3ΤΓνΡΤΕΤΗ8Ρα<ΡΕΤΤΑΙιϊ. STHPRIKTSKTFPAS'rVFPiiVSETrASLTIRPGÀETSTALPTQrrSSLl'TLLVTGTSRVD η3ΡΙλ3Ρσν3ΑΚΓΑΡΙ₁3ΤΗΡΟΤΒΤ3ΤΜΙΡΤ3ΤΜ1ΟΜ.ΕΤταιΙΑΤ888ΑΒΤεϊ’6Τ1.Τΐ_ίΤ 1^ΡΑν3βΙώ8Α8ΙΤ^Κ50ΤνΤ51ΒΙΤΕΤ8Ρ8νΤ3ν(3ΡΡΕΡ8ΚΤνταΤΤΜ1ΊιΙΡ3ΕΜΡΤΡΡ KTSHGEGVemiUmiiVBATNLATTGSSPTVAlOTmPNTIAGSLFTPLTTPGMSTIA SESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMV ΡΡΤΙΛΕΤΙΤΪΠ:0ΥΕΕΤ)ΜΚΕΡΟ8ΗΚΡΝΑΤΒΕΕΙ4}αΐΙ₍ϊα>ηΡΙΙΝ38Ι>ΕΥΙ₎Υ3βΟΒΙΑ3ΙΛΡΕ lOJSSATàVDMCTHRPDPBDIÆIJaiERLYWEIÆNLTNGIQELaPYTUJRI^YVNGFTHR SSMPTTSTPGTSTVDVGTSGTPSSSPSPTXAGPLLMPFTIiKFTITlilXiQYEBDMRRTGSRK FITOŒSVWXÎLIiiœLFraTOVGPLYSGCRLTWlPKXDGRATGVDAICTHRLDPÏCSPGLN REQLWEIÆlŒ.TNDIEEWlPYTIJlRmYVHGPTHQSSVSTTSTPGTSTVDIiRTSGTPSS LSSPTIMAAGPIiLVPFTLNFTITHIjQYCKDNCmPGSRKFNTTBRVIXXniIiGPIFlQnSVG PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQL'niGIKELGPYT ΙΟΒΝδηΥνΝσΡΤΗΚΤβνΡΤΤβΤΡατΒΤνΟΙιαΤδβΤΡΡβηΡβΡΑΊΤΙΟΡΙΛνίΡΤΙιΝΡΤΙΤΝ ns

CA 02841741 2014-02-03

WO 2005/081711 PCT/ÜS2004/038392 ηΚΥΕΕΏΜ1ΠΤΡα5ΗΚΕΪΓ^ΗνΐΖ?ΤΐνσΡΜΧ·ΚΝΤ3ναΐΛΥ3α0ΚΒΤΣΒΕΕΕΚησΑΑΤσνθΑ

ΙΟΤΗΕΙΛΡΚεΡΟνηΗΕΟΕΥΐίΕΕβοηΤΒΚΪΙΚΕΙΧίΡΥΤηοΚΝδηΥνΝΒΡΤΗνίΡνΡΤδεΤΡβ TSTVDIÆSGTPSSLPSPTSAIAGPI^VPPTIIIFTITNLKYBEDMHCPGSRKFNTTERVLQ δίΛβΡΜΡΚΝΤ^ΡΙ,ΥδβΟΚΙιΤΙΛΕδΕΚηΟΑΑΤσνΟΑίσΓΗκηϋΡΚβΡθνΟΕΕΟΒΥΜΕΒε QL'rtraiKELGPYTLDRNSLYVNGPTHQTSAPNTSTPGTSrVDLGTSGTPSSI.PSPTSAGP IiVPFTLNFTIT^YEEDMHHPGSRKFl«TTERVIi<)GUiGPMFiansVGLLYSGCRLTIX RPEKNGAATGMDAICSHRLiDPKSPGLNRSQLYWELSQLTHGIKELGPYTLDRNSLYVNGF ΤΗΕδδνΑΡΤδΤΡσΤβΤνΟΙιβΚΟπΡδδηΡδΡΤΤΑνΡΙ,ΙινΡΡτωίΡΤΙΤΝΙιΟΥβΕηΜΚΗΡβ SRKMn^raRVLQGUiGPLFiaKSVGPLySGCRLlSLRSEiaxaATGVDAICTHHLNPQSP ΟΙΒΗΒΟΒΥΜΟΜαΜΤΝδΙΚΕΙβΡϊΤΙΟΚΝδΙΥνΝσΡΤΗΡδββΙΤΤδΤΡίΤΜΤνΟΙιαΤδσΤ PSPVPSPTTAGPbLVPyTIJïFTlTNLQYEEDMHRPGSRKmATERVI>QGlLSPlFKNSSV σΡΚΥ56ΟΚΙ₁Τ5ηΗΡΕΚηβΑΑΤ6ΜηΑνα₎ΥΗΡΝΡΚΕΡαΐιηΕΕ0Ι₁ΥΜΒΙ»3ΰΙ.ΤΗΝΙΤΕΙΧ3ΡΥ SLDRDSLYVNGPTHQNSVPTTSTPGTSTVYWATTGTPSSFPGMTEPGPLLIPFTFNFTIT Μ^ίΥΕΕΝΜΟΗΡΘ3ΚΚΡ1^^νΐΟθω^1ΡΚΝΤ8νϋΡΚΥ5σ<3η-ΤΕΙΛΡΕΚΟΕΑΑΤσνθ TICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTP ΟΤδΤνΗΧΑΤδΟΤΡδδΧ,ΡΟΙΤΑΡνΡΙίΕΙΡΡΤυϊΡΤΙΤΜΙιΗΥΕΕΝΜΟΗΡβδΚΚΡΝΤΤΕΕνίιΟ σΏΙ₁ΚΡηΡΚ5ΤενσΡΒΥ33Ώ<ΒΤωΛΡΕΚΗεΆΑΤσνθΑΙ(?ΤΙιΚΜΡΤαΡ3ΐηΚΕΚΐ4ΥΚΕΙ;5 QLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPÀSLPGHTAPGP I^VPFTUJFTITî^YEWMRHPGSIUCFm^RVLQGLLKPLFKSTSVGPLYSGCRLTLL RPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGP THRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPG 8ΕΚΡΝΤΤΕΚνΐ>00Ιι1ΕΡηΡϊαη'3ΐαΡηΥ830ΕΕΤΙιΙιΚΡΕΚΟΚΑΑΤΚνΐ1ΑΐσΓΗΗΡΟΡ05Ρ GLNREQLYWELSQLTHGITKIjGPYTWRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGI PPSLPETTATGPLLVPFTIiNFTI'INLQYEENMGHPGSRKFNXTBSVLQGIiLKPbFKSTSV 6Ρ^Υ5σ€ΗΒΤΜΛΡΕΚοσνΑτκνηΑϊστΗΗΡηρκΐρσι.οκ(χ}ΐ.γν»Εη5οητΗ8ΐτΕΐβργ TIORDSLYVNGPTQRSSVPTTSTPGTFTVQPETSETP8SLPGPTATGPVU.PFTLNFTI X NIXJYKEDMHRPGSRKFîm^RVLQGLLMPLFKNTSVSSLYSGCRLTLLRPSKDGAATRVD ΑνσίΗΕΡΟΡΚδΡσ^ΟΗΕΗηΥϊηαώδηΊΉσΓΓΕΙΛΡΥΤΧ.ΟΕΗΒΙ.ΥνΝΒΡΤΗΟδδΜΤΤΤΗΤΡ DTSTMHLATSRTPASLSGPTTASPbLVLFTINFTIÏ2n^YEENMHHPG3RICFNTTERVLQ ΟΙ^ΡνΡΚΝΤ8νσΡΚΥ8Ο0ΕΛΤΙΛΚΡίααΧ3ΑΑΤΚνΠΑΐαΤΥΡΡΟΡΚ8ΡσΕΟΗΕζ}ΒΥ1ΪΕΙ»8 QLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASP ΙΛνΐ»ΡΤω^ΙΏπΛΥΕΕΝΜ0ΗΡβ8ΕΚΏΠΤΕΗνΐΧ2ΟΙ₁Ι₁Κ8Ι₁ΡΚ8Τ8ν6Ρ1Υ8βΟΚΒΤΙΛ RPEIOX^ATGVDAICTHHPDPmMJlREQLY^^ ΊΉΗ88ν8ΤΤ3ΤΡβΤΡΤνΥΙΛΑ8ΧΤΡΆ3ΙΡΘΡ3ΑΆ3ΗΙΛΐηΡΤΙιΝΡΤΙΏΪΙΛΥΕΕΝΜ»Ρσ8 ΗΚΜ^^νΒοοχ.ΐΛΡΐιΡΚΝΤδνορηΥδσσΕΐ.τχ.ΐΛΡΕκιχίΕΑτσνηΑίετΗΗΡηρτσρσ ΙΛΕΒ0ΙΥηΒΒ30ηΐΉ3ΙΤΕΙΧ3ΡΥΤΙΛΗη8ΒΥνΝσΡΤΗΚ83νΡΤΤ8Τθν5Τ5ΕΕΡΕΤΙ₁ΝΡΤΙ ΝΙ^ΗΥΜΑΓ»«ΟΡβ8ηΐ^ΝΙΤΟΝνΜΟΗΙιϋ8ΡΕΡΟΕ88ΙΧ1ΑΡΎΤΟΟΚνΐΑηΗ8νΚ1«3ΑΕΤΕν οηζτΓΥΐ»ςρι»8αροι.ρΐΕονΡΗΒΐ3θ2ΤΗσιτκι<θΡΥ8ΐ₎ηκη8ΐπ>ικϊΥΝΕΡσρηΕρρττ ΡΙΦΑΤΤΡΙΡΡηΒΕΑΤΤΑΜσΥΗΙιΚΤΠΤΙιΝΡΠδΝίΧΪΥβΡΟΝσΚύβΑΤΕΝβΤΕσνίΟΗΙΛΚΡ IæQKSSMGPF^ŒιGCQLISLRPEïœGAATCVDTTCTΏÎPDPVGPGL·DIQQL·YWEI.SQL·THG νΤ0Ιί6ΡΥνϊ4)ΚΙ)81ΡΙΝαΥΑΡ0ΝΙ₁3ΙΕσΕΥ0ΙΝΡΗΓνΝΗΝΙ₁3ΝΡΟΡΤ88ΕΥΙΠ₁ΙΛηΐΟΠ KVTTLYKGSQIJfflrFRFCLVTiaTWSVLVTVKALPSSNWPSLVEQVFLDKThNASFHW LGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLN'FTITNLPYSQDKAQPGTrNYQRNKRN ΪΕηΑΐ>Ν0Ι4^881Κ3ΥΡ3ΪΧΧ}ν3ΤΡΕ3νΡΐηΰηΠ^>ανθ3Ι>ΟΝΡ8ΡΙΛηΗνθΚνΑΧΥΕΕΠ. ΡΜτκκ5τοχ<}ΝΡΤΜ)Κ38νινηθΥ3ΡΚΚΝΚΡητσ»3θΐ₁ρρΗΑνΐϋΐσΐΛσΐΛαΒΐτα·χσ

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GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ (SEQ ID NO:4) (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelia, Genbank accession no. NM_OO5823

Yamaguchi Ji., etaL Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. USA.

(20):11531-11536 (1999), Proc. Nat). 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: MDÆ601051; NP_005814.2; NM_005823_l

622 aa

MALPTARPLLGSCGTPMiGSIiFU»PSIX3WQPSRTUtoBTGQBAAPl4DGVLANPPNISS Ε3ΡΚΟΙιΙ>Ι3ΡΡΟΑΕν86Ι»§ΤΕΗνΚΕΙΑ'νΆΙΛΰΚΝνΚΙι£>ΤΒΟΙΛΟΙΛΗΜ>8ΕΡΡΙϊηΐιηΑ1ΡΙ« ϋΙ4^ΐΜ^ΠΑΡ2ΟΡ0ΛσΠ^Ρ3ΕΙΤΚΑΝνηύηΡΚΟΑΡΕΚ0ΚΙ^ΡΑΑΙΛαΐσνΐ<3δ1^3ΕΑ DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW SVSTMDAJÙRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRRKVKKT ACPSGKKftREIDBSLIFYIOTiELEACVDAAIiIATQMDRVNAlPFTyEQLDVLKHKljDELY PQGYPESVTQRLGYLFIJO<SPEDrRKKNVTSLETLK?\LLEVNKGHEMSPQVATLIDRFVK grgqiæ>kdtldtltafypgyi>cslspbei.ssvppssiwavrj?qdldtcdprqldvlypkà ΚΙίΑΡΟΝΜΝΘδΚΥΡνίαΟβΡΙιΟαΆΡΤΕηηΚΑΒΞΟζ^ονβΜηίΑΤΡΜΚΙΛΤηΑνί.ΡηΤνΆΕνΟ ΚΙΠ^ΡΗνΕ0ΜΜΒ»ΗΗΡνΗΙΜΙΙΛ0ΗΟΠϋω7ΓΙΛΙΧ3>06βΙΡΐ*3ΥΕνηθΏΒΜ3ΕΑΏ5«Τ PCIÆ-GPGPVLTVLAimiASTIA {SEQ ID NO:5) (6) Napi3b (NAPI-3B, NPTUb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type Π sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424,

J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), Feild, J .A., et aL (1999) Biocham. Biophys. Res. Commun. 258 (3):578-582); W02004022778 (Claim 2); EP1394274 (Example 11); W02002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19); WÛ200157188 (Claim 20; Page 329); W02004032842 (Example IV); W0200175177 (Claim 24; Page 139-140);

Cross-references: MDÆ604217; NP.006415.1; NM_006424_1

690 aa

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ΜΑΡ^ΕΙ^ΑΟΡΝΡΠΚΥΙ.ΕΟΑΑαθΰΡΤΑΡθΚ5ΚΞΤΝΚΤΠΝΤΕΑΡνΤΚΙΕΕΒΡ3Υ3ΤΑΊΊ_ίΙ DEPTEVDDPWNLPTLQDSGÏKWSERDTKGKILCFFQGIGRLIbLLGFLYFFVCSLDILSS AFQLVGGKMAGQPFSNSSIMSNPLLGLVIGVLVTVLVQSSSTSTSIWSMVSeSLLTVRA AIPIIMGANIGTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLIiPVEVATHY LEIIŒQLIVESFHFKNGffiAPDUUCVTTKPFTKLIVQIiDKKVISQIAMNDEKAKtlKSLiVK IWCKTPTmCIOINVTVPSTANCTSPSIKZWIIXSIQNWTMKNVTYKENIAKCQHIFVNFHLP

DIAVQTnj>ILSLLVLCGCLIMIVKZI_lGSVLKl3QVAlVIKKrnJTDFPFPFAWI.TGYLAI

ΒνσΑΟΜΤΡΐν0383νΡΤ8ΑΙ>ΤΡΙ.ΙΟΐσνΐΤΙΕΚΑΏ>Ι_ίΤΙ₁63ΝΙΟΤΤΠΜΙΛΑΙΛ3ΡβΝΑ Ι.Ρ85Β0ΙΑΙ.€ΗΡΡΡΝΙ5ΘΙ1>1ιΚΥΡΙΡΡΤί41ιΡΙΚΜΑΚβΙΖ5ΝΙ5ΑΚΥΚΚΡΑνΡΥΙ>ΙΙΕΕΙ?ΙιΙ PLTVFGLSLAGWRVLVGVGVPWFII ILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSL

KPWDAWSKFTGCFQMRCCÏCCRVCCRACCLLCGCPKCCRCSKCCEDLEEAQEGQDVPVK APETFDNITISREAQGEVPASDSKTECTAI» (SEQ ID NO:6) (7) Sema 5b (FLJ10372, ΏΑΑ1445, 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, (semapborin) 5B, Genbank accession no. AB040878,

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

W02003003984 (Claim 1); W0200206339 (Claim 1; Page 50); WÛ200188133 (Claim 1; Page 4143,48-58); W02003054152 (Claim 20); W02003101400 (Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737; 1093 aa

MVLAGPIJ\VSUXPSLTimVSHLSSSQDVSSEPSSSQQLC7U_JSKHP^,IVAtWI£PWVSNF TYPGARDFSQLAWPSGi^LIVGARNYLFRLSIANVSimQATEWASSEDTRRSCQSKGKT BEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQV'GNLSRTTEKINGVARCPYDPRHNST Ανΐ83&lt;Χ3ΕϋΥΑΑΤνΐΟΡ8σΚΟΡΑΙΥΚ8ΙΧ385ΡΡΙΛΤΑΟΥΚ8ΚΗΙιΝΕΡΝϊνΆΑΥΟΙ(3ηΕΑΥ PFIJŒNAVEHlXX^TVYSRVARVCKNDVGGRFIiEDTWrrFMKARLNCSRPGEVPFYYNE IXJSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIAS

PIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDL· ν0ΑΚΟΤΙ.ΥΗνηΥΙ0ΤΕ3βΤΙΙιΚΑΙ>3ΤΑ3Ε8ηΗβσΏ₁ΒΕΙ₁ΗνΐΡΡβΚ»ΕΡΙΛ8ΙΛΐηΗ3ΆΗ

ALFVGLRDGVLRVPLERCAAYRSQGACbGARDPYCGWDGKQQRCSTLEDssNMSLWTQNi TACPVRNVTRDGGFGPWSPWQPCBHItDGDNSGSCÏiCRARSCDSPRPRCGGLDCIÆPAÏHI ANCSRNGAKTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRÏCVGKSREERPGNENTP CPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTP WTPWLPVNVTQGGARQEQRFRFTCRAPIADPHGLQFGRRRTETRTCPADGSGSCDTDALV EDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGTAAE YQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPÀPSPGBDICLGLHTEEAL CATQACPEGHSPNSEWSKCTDDGAQSR8RHCEEI>LPGSSACAGNSS(2SRPCPYSEIPVn> PASSMBEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPN

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ΗΙιΗΥΚΟ&lt;3&lt;3ΤΡΚΝΕΚΪΤΡΜΕΓΚΓΙιΝΚΝΝΙιΙΡΟΟΚΑ1ίΡΥΡΪ4)ΟΤΝνΥΤΤΤΎΥΡ8ΡΙ«ΝΚΗ8ΡΕ PEASPGQRCPPNS (SEQ ID N0:7) (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);

US2003129192 (Claim 2); US2004044180 (Claim 12); US2OO4O44179 (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; AY358628J

141 aa ΜΗνΐΧ3ΙΑΑ*ΓΡ€0ΙιΡΙΧΡβΡΑΙ>0Ι0εΥ00ΒΒΡαΐ>ΐηΠΧ:&8ΡΕΕΐνΝΟΤνΝν00Μ00ΚΕνΜΒ QSAGIMYRKSCASSAACLIASAGYQSFCSPGKUJSVCISCCNTPLCNGPRPKKRGSSASA LRPGLRTTILFLKLALFSAHC (SEQ ID NO:8) (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);

Nakamuta M., et aL Biocbem. Biophys. Res. Commun. 177,34-39,1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178,248-255,1991; Arai H., et al. Jpn. Cire. J. 56, 1303-1307,1992; Arai H., etaL J. Biol. Chem. 268,3463-3470,1993; Sakamoto A., Yanagisawa M.,etaL Biochem. Biophys. Res. Commun. 178,656-663,1991; Elshourbagy NA, et aL J. Biol. Chem. 268,3873-3879,1993; Hændler B., et aL J. Cardiovasc. Pharmacol. 20, sl-S4,1992; Tsutsumi ML, et aL Gene 228,4349,1999;

Strausberg R.L., et aL Proc. Natl. Acad. Sei. USA 99,16899-16903,2002; Bourgeois C.» et al. J. din. Endocrinol. Metab. 82,3116-3123,1997; Okamoto Y., et al. Biol. Chem. 272,21589-21596,1997; Verheij J.B., etaL Am. J. Med. Genet 108,223-225,2002; HofstraR.M.W., etaL Eut. J.Hum. Genet 5,180-185,1997; Poffenberger E.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., et aL Hum. Mol. Genet 5,355-357,1996; HofstraRMW., etaL Nat Genet 12,445447,1996; Svensson P.J., et aL Hum. Genet 103,145-148,1998; Fuchs S., et aL Mol. Med. 7,115-124,2001; Pingault V., etaL (2002) Hum. GeneL 111, 198-206; W02004045516 (Claim 1); W02004048938 (Example 2); W02004040000 (Claim 151); W02003087768 (Claim 1);

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W02003016475 (Claim 1); W02003016475 (daim 1); W0200261087 (Fig 1);

WG2003016494 (Fig 6); W02003025138 (daim 12; Page 144); W02Û0198351 (daim 1; Page 124-125); EP522868 (daim 8; Fig 2); W0200177172 (Claim 1; Page 297-299); US2003109676; US6518404 (Fig 3); US5773223 (data la; Col 31-34); W02004001004;

442 aa

MQPPPSIXMRALVALVIÀCGIÆRIWGEKRGFTPIMlATPIÆiCZiaElNTPPTKmHPKSSKA gLARSLAPABVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTWSCLVFVLGIIGNS TLLRI lYKNKCMRNGPNILIASLMWLLHIVIDIPnrVYKlJaAEDWPFCy^CKliVPFI 0ΚΑ5νθΙΤνΐι3Ι_Ι0ΑΙ»3ΙθηΧ1ΛνΑεΚ8ΚΙ1ΏΙ6νΡΚΝΤΑνΕΐνΐΙ«νν5ννΐΛνΡΕΑΙΟΡ DIIJWYrraSYLRICIÆOTVQKTAFMQFÏXTAKDHWLFSFYFCLPIAITAJ'FYTIJtTCBM ΙΛΚΚ3(»!ΟΙΑΙΛΪΒΗΒΚΟΚΚΕνΑΚΓνΡΟΙινΐ<νΡΑΒαΗηΡΙ>ΗΒεΕΙΙ₁ΐα.^,Π.ΥΝΟΝΠΡΝΚθΕΏ Ι>3ΒΙΐνΐ₁ΠΥΐαηη4ΑΒΜ30ΙΝΡΙΑ&Υ&ν8ΚΕΕ10ϊ0Ρ1φά»0αΗ£Ώ8ΡΒΕΚββ&ΒΒΚ0ΒΟ LKFKANDHGYDNFRSSNXYSSS (8 EQ ID NO:9) (10) MSG783 (RNF124, hypothetical protein FLI20315, Genbank accession no.

NM_017763);

W02003104275 (Claim 1); W02004046342 (Example 2); W02003042661 (Claim 12);

W02003083074 (Claim 14; Page 61); W02003018621 (Claim 1); W02003024392 (daim 2; Kg 93); WO200166689 (Example 6);

Cross-references: LocusID:54894; NP_060233.2; NM_017763_l

783 aa

MSGGHQLQLAALWPWIJJiATLQAGFGRTGLVIiAAAVESERSAEQKAIIRVIPLKMDPTGK ΐΛΟίΤίΒσνρΑονΑΕίτΡΑΕοκηΜύδΗΡηγίίΜΑΒοηηΝηΒΡσρίδτνκηΕβΡΒηΑΡΚΡα. SLASKARMAGERGASAVLFDITEDRAAAEQIjQQPLGLTWPWLIWGNDAEiajMEFVYKNQ ΚΑΗνΠΙΚυ0ΙΡΡΑΜΡΟΥΓΐννίΙΕΜΤννσΠΡνΐΙΕΑ5νηΗΙΚΰΚΡΡΗ3ΚΡΟΡΙι00ΕΙΑΝΑΙ 80£ΑΤΚΚΥζ}Α30Κ0ΑΚ08ΝΡ05Ο8Βσ58ΑΡν0ΑΐαΛΒΡ3Ε0<}ΚΙ>Κνΐ3σΐ₁ΗΕΡΗΚΝσνο ΡΝηΗΟΗητορησνΡΝίτηαηδΡβαΒίιβΡβκβΥΟΕΡΟΚΚίΗίικοΗΡαΗΑΗΥΗΐιΡΑΑΥί.ΐιβ PSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPGEQQRLAGAQHPYAQGWCTiSH IXJSTSQHPAACPVPLRRARPPDSSGSGESYCTERSOTLADGPASDSSSGPCHGSSSDSW ΙϊΉ7)Ι3Ι»ΟθνΗ0333ΤΡΟ33Ι<33ϋΡηΡΙ»νΥΟ8ΡΚ6ΟΡΰΕνΒΜΟΡ3νΤ8ΕΡΒ3ΪιΟ3ννΡΤ6 KTQVSSHVHYHKHRHHHYKXRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTG gwigxapsni?T.gtn>QrTOar.PRPAPnpvnASSlCPSTSSrJNr_lOKSSIiSARHP0R][RRO8P SBPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLICGPPGLDKRLLPBTPGPCYS NSQPVWLCI.TPRQPI1EPHPPGEGPSEWSSDTAEGRPCPYPHCQVLSAQPGSBBELEELCB

QAV (SEQ ID NO:10)

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Lab. Invest 82 (11 ): 1573-1582 (2002)); W02003087306; US2003064397 (Claim 1 ; Fig

1); WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; Fig 4B); W02003104270 (Claim 11); W02003104270 (Claim 16); US2004005598 (Claim 22); W02003042661 (Claim 12); US20Û3060612 (Claim 12; Fig 10); WO200226822 (Claim 23; Fig 2); WO2002I6429 (Claim 12; Hg 10);

Cross-references: GL22655488; AAN04080.1; AF455138J

490 aa

ΜΤ8ΒΤΒΜΜΠ5ΡΚ8Ε5ΕΤνηΡΝΟΙΝαΐΚΡΑΚΚνΤν0νΐ080ΡΕΑΚ5ηΤΙΚΙ.ΙΗ€0ΥΗννΐ08

RNPKFASEFFPHWDVTHHEDALTKTNIIFVAIHRBHYTSLWDLRHLLVGKILIDVStQM

ΜΜ0ΥΡΒ3ΝΑΒΥΙΛ5ηΡΡη8Ι»ΐνΚ5ΡΝνν8ΑηΑΙι0ΙιβΡΚηΑ5ΚζΐνΥΙθεΝ1ϊϊ0ΑΚ0ανΐΒ IAIŒIOTIPIDIiGSIÆSAREXEI^UîIdm.WRGPWVAISIATFFFLYSFVRI>VIHPYA ΚΝΟβεΟΕΥΚΙΡΙΕίνΝΚΤηΡίνΆΓΠΛβηνΥΙιΑΟηΐιΑΑΑΥΟΙ,ΥΥΟΤΚΥΗΙΙΓΡΡΚΙιΕΤΜηΟ rpKiQmT.T^PffPAMVWVAYSTm.PMRRSgRYT^IJMAYOOVHAMIKNSWNEEEVWRIEMY ISFGIMSTXmiiSLLAWSIPSVSHALSNlŒFSFIQSTrÆYVAIiLISTFHVLIYGWKRAFB

EEYYRFYTPPNFVLALVLPSIVILGKIILFLPCISQKLKRIKKGWEKSQFLEEGIGGTIP HVSPERVTVM (SEQ ID MO;11) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636

XuXZ., et al Proc. Natl. Acad. Sci. USA. 98 (19):10692-10697 (2001), Cell 109 (3):397407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003)); US2003143557 (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);

Cross-references: MIM:606936; NP_0601062; NMJ)17636_1

1214 aa mwpekeqswipkifkkktcttfivdstdpggtlcqcgrprtahpavamedafgaawtv iroSDAHTTEKPTDAYGBLDPTGAGRKHSNFLRLSDRTDPAAVYSLVTRTWGFRAPNLWS VLGGSGGPVIiQfrWLQDLLRRGLVRAAQSTGAWIVTGGItHTGIGRHVGVAVRDHQMASTGG TKVVAMGVAPWSVVRNRDTL INPKGS FPARYRWRGDPEDGVQFPWYNYSAFFLVDDGTH

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6ΟΛ3ΕΪ»ΡΒΙΛ0Λ8ΥΙ3ζΧ1ΚΤσν0βΤ5ΐηΐΡνϊα^ΙΙ)60ΕΚΜΕΤΒΙΕΗΑΤ0Λ0ηΡαΛ

VAGSGGAAIXZAEI^EIJTIAPGSGGARQGEARDRIRRFFPKGDLEVLQAQVERIMTRKEL

LTVYSSBDGS2EFETrVLKALVKAaeSSEA8AÏLDELRLAVAWNRVDIAQSBI>FRGDIQW

Κδ?Η^ΕΑ5Ι><αΑ[.ηΝΠΗΡΒΚ\ηυΛα3Ηεΐ>3ΙΧ3ΤΡΤ,ΤΡΜΚΙΛ0ΏΥ5ΰυ^3Ν3ΏΙ^ίιΊΖ>0Α

SHSAflTKAPALKSGAAEIiRPPDVGHVLRMLLGKMCAPRYPSGGAWDPHPGQGFGBSMYIiL

ΚΟΚΆΤ8ΡΓΛΙΖ1Α3ΕΟ0ΑΡΗ3ηΓ₁ΙχΕΗΑΙιΙ₁ΒΝΚΑ0ΝΑΜΥΡνΠίΜ08ΝΆν35ΑΙΧΐΑΰΙΛΛΕνΜΑ

ΗΙΛΡΟΑΕΕΑΑΚΚΐωΐΛΡΚΡΕεΜσνθΙ»ΡΟΕσϊΚδ8Βνΐυ«ϋαηΒΚΗσΡΙ,«(Π)ΑΤ(ΧΟΙΛΜΟ

ADARAFPAQDGVQSLLTQKWWGMiASTTPIWALVLAFFCPPLIYTRLITFRKSEEBPTRE

EI_JEFIXto9V12XnSGPVGTMPAI3CrPIXJVPRQSGRP&lt;XXGGRCGGRRCI.RRWi’HFWGIAPV

ΤΙΡΜ(11Γνν8ΥΙ₎1ΡϋΙΛΡ8!^1ϋνΡΡζ)ΡΑΡΡ(^ΙίΒθωΛΥΡ«ΆΡΤΙ₁ΙΛΈΕΙ_ίΚθαΠ30ααθ3η

Α50&lt;3Ρ0Ρ0ΗΑ5Ι^ΰΕΒΜ,ΥΙΑ03^ζ2€Ώΐν7)ΒΤσΡτΐΧ3νααΚ1.ΤΡΟΕΥΗΐαΚΤνΐΧ2ΙΟΓΜ

VFTVRUUilFTVNKQIXSPKIVIVSKWiKDVFFFIiPFLGVWLVAYGVATEGLLRPRDSDFP

SIUWVFYRPYIiQIFGQIPQEDMDVMWHSKCSSEPGFWAHPPGAqASTCVSQYANWLV νίΠΛνίΡΙΟΜΠΧΠνΝΙιΙιΙΑΜΡβΥΤΡΟΚνΟΟΝΒϋΙ.ΥΗΚΑΟΗΥΗΙιΙΗΒΡΗβΚΡΑΙΑΡΡΡΙ νΐ8ΗΙιΐαΛΛΪ®ΙΛΪ»ΡΚ8Ρ0Ρ88ΡΑ1ΕΗΡΡνΤΜΚΕΆΒΚΐαΛΤΚ85νΗΚΕΗΡΪιΕΑΚΑΙΙΙΪΚ RESDSEIU^TSQKAmiALKQLGEIREYEQRLKVLEREVQQCSRVLGWVAEALSRSALI.P PGGPPPPDLPGSKD (SEQ ID NO:12) (13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NMJW3212, dccodicola,A., et al. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555565 (1991)); ÜS2003224411 (Claim 1); W02003083041 (Example 1); W02003034984¹(Claim 12); W0200288170 (Claim 2; Page 52-53); W02003024392 (daim 2; Hg 58); W0200216413 (Claim 1; Page 94-95,105); W0200222808 (daim 2; Fig 1); US5854399 (Example 2; Col 17-18); US5792616 (Hg 2);

Cross-references: MIM:187395;NP_003203.1;NML.003212_1

188 aa

MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYIAFRDDSIWPQEBPAIR

PRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPSyYGRNCEHDVRKKNCGSVPH ΐηνΠιΡΚΚ28ΙιαθΗΗΘΟΙ«ΟΡΡΟΛΡηΡΟ&lt;2ΧΪΙ.νΜΟΒΗηνΑ8βΤΡΕΕΡΡ8ΑΒΤΤΓΡΜΙ,νθΙ 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,

Fujisaku et aL (1989) J. Biol. Chetn. 264 (4):2118-2125); Weis JUL, etaL I

Exp. Med. 167,1047-1066,1988; Moore M., et aL Proc. Natl. Acai Sci. USA.

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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; QI 3866; Q14212; EMBL; M26004; AAA35786.1.

1033 aa MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGE KSLIZLITIOTKVIXrrWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFAaKT NFSMNGHKSVWCQAIQMWQPTRIiPTCVSVFPI_lECpAIJWHNŒMraEliVQ3IAPGI»SVT ySCESGÏLX.VGEiailKl.SSGKMSAVPPTCBBARtSCSU^PMGKVKSPPIIAVGVTAMF FGDEGYRLQGPPSSRCVTAGQGVAWTKMPVCKEIFCPSPPPILMJRHIGNSLANVSYGSI VTYTCDPDPEBGVNFILIGESTI>RCTVDSQKTGIWSGPAPRCEIiSTSAVQCPHPQn»RGR ΜνΒ6ςΐΦΕΥΤΪΝΟΤνΐΡΑ£Μ1ΌΡΤ1ΚβΒΚ0ΐΚσΝΑ0βΤΜΕΡΒ21ΡνθΕΚΕ00ΑΡΡΝΙΙ₁Να0 IOEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACKATGRQDÏiT KPQHQFVRPDVNSSCGBGYKLSGSVYQECQGTIPWFMEIRLCKBITCPPPPVIWGAHTG SSLEDFPYGTTVTYTCNPGPERGVBFSLIGBBTIRCTSNDQERGTWSGPAPIjCiaSUAV QCSKVHXANGYKISGKKAPYFYNDTVTFKCYSGFTIiKGSSQIRCKADNTWDPEIPVCEKE Τα0ΗνΚ08ΒαΕηΡΑα3κνΕηνΝΤ8€0ηθΥ0ΕΤΟΗΑΥ0Μα2ηΑΕΝβΙΚΡΚΚΙΡηθΚνΐΗ€Η PPPVTVNGKHTGMMAENFLYGNEVSYBCDQGFYLLGEKKrQCRSnSKGHGSWSGPSPQCL . RSPPVTRCPNPBVKHGYKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCSrrENTWVPGV ptcikkafigctpppktpngnhtggniarfspgmsxlyscdqgyllvgealllcthegtw SQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAWTEECEDGYMLEGSPQSCJCQSDHQ ΝΚΡΡΙΑναΚΒΚΒΙΑΡνΣασίΑΑβηΐΙΛΤΡΣΓνίΤΣΥνίΒΚΗΗΕΕΝΥΎΤηΤΒΟΚΕΑΡΗηΒΑ REVÏSVDPYNPAS (SBQ ID NO:14) (15) CD79b (CD79B, CD79P, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NMJXJ0626 or 11038674, Proc. Nad. Acad. Sci. USA (2003) 100 (7):4126-4131, Blood (2002) 100 (9)^068-3076, Muller et aL (1992) Eur. J. Immunol 22 (6):1621-1625)-, W02004016225 (claim 2, Kg 140); W02003087768, US2004101874 (claim 1, page 102); W02003062401 (claim 9); WO2Û0278524 (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: NUM: 147245; NP_000617.1; NMJXXJ626J

229 aa

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ΜΑΚΙΛΓ3ΡνΡ5ΗΗΜνΑηηΐΑΙ₁8ΑΒΡνΡΑΆΚ5ΕυΗΥΚΝΡΚ05ΑΩ5ΕΓΗ05ΡΚΡΙΆΚΚΚεΚΤ VmiCYMNSASGNVSWLWKÇEMDEMWnKLKKGRMSESQl'reSLATLTIQGIRFEDNGIY FCQQKann^,6EVYQGŒTEt>RWFSTIJ«IiKQmrn«DGIIMIQTI₁I.IIlFIIVPIFLL· LDKDDSKACSMgBDHTÏEGLDIDQIATYBDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO:15) (16) FcRH2 ÇCFGP4, 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), XuMJ., etaL (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775; 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 .030764.1

508 aa

MI^SimVIFDAVTEQADSLTLVAPSSVFEGDSIVI₁KCQGBQJWKIQKMAYHKDNKBI»SV PKKFSDFLlQSAVLSDSGNYrcSTKQQLFLHDKTSNIVKTKVQBLFQRPVLTASSFQPIE GGPVSLKCETRLSPQRLDVQLQFCFFRENt^VLGSGNSSSPELQlSAVWSEDTGSYWCKAE τντί®ΙίΟζ03Ι»050ΙΗν0ΚΙΡΙ8ΝνΕ1ΕΙ1«ΡβαθνΤΕβ0ΚΙΙΙ>ησ5νΑΘ(3Τβ»νΤΡ8ΜΤ REATCTSMGKKTORSLSAELEIPAVÏŒSDAGltYYCRADNGHVPIQSKVVNIPVRIPVSRP vltiæspgaqaavgdu.klhcbalrgspptlyqfyhedvtlgnssapsggGasfnlslta EHSGNYSCBANNGIiGAQCSEAVPVSISGPDGYRRDIiMTAGVÎJ»GI>FGVIiGFTGVMLI»ÏA LFHKXSGBSSATNKPRGASRPNPQEFTYSSPTPTWEELQPVYVNVGSVDVDWYStJVWSM QQPESSANIRTLLBXrKDSÇfVTYSSVKKS (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., etaL Proc. Natl. Acad. Sci. USA. 82,6497-6501,1985; Swiercz JJÆ, et aL J. Cell Biol. 165, 869-880,2004; Kuhns JJ.,etaL J. BioL Chem. 274,36422-36427,1999; ChoH.-S., et aL Nature 421,756-760,2003; Ehsani A., etaL(1993) Genomics 15,426-429; W02004048938 (Example 2); W02004027049 (Fig 11); W02004009622; W02003081210; W02003089904 (Claim 9); W02003016475 (Claim 1);

US2003118592; W02003008537 (Claim 1); W02003055439 (Claim 29; Fig 1A-B);

W02003025228 (Claim 37; Hg 5C); WO200222636 (Example 13; Page 95-107);

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W0200212341 (Claim 68; Hg 7); WO200213847 (Page 71-74); W0200214503 (Page

114-117); WO200153463 (Claim 2; Page 41^6); W0200141787 (Page 15);

W0200044899 (Claim 52; Hg 7); WÛ200020579 (Claim 3; Hg 2); US5869445 (Claim 3; Col 31-38); WO9630514 (daim 2; Page 56-61); EP1439393 (Claim 7); W02004043361 (Claim 7); W02004022709; W0200100244 (Example 3; Hg 4); Accession: P04626; EMBL; Ml 1767; AAA358O8.1. EMBL; Ml 1761;

AAA35808.1.

1255 aa

ΜΒΙΛ«υύΟΚΜ^ΜΑΙΑ*ΡΡΟΑΑ£ΤανσΤαΤΧ>ΜΚω^ΡΑ3ΡΕΜ_<Ι)ΜϊιΚΗΙΥ0(Κ^νν0(2α_ιBLTYLPTNASLSFL^lIQEVQGYVLIAHNQVRQVPLQRhRIVRGTQLFKDNYATAVLDNG DPLNNTTPVTGASPGGLRELQLtRSLTEILKGGVLIQRNPQLCVQDTILWKDiraKNNQLA ltlidtnrsrachpcspmckgsrcwgessedcqsltrtvcaggcarckgplptdccheqc AAGCTGPKHSDCLACLHFNHSGICELHCPALVTINTDTFESMPNPBGRYTPGASCVTACP ΥΝΥΙ,ΒΤΟναΒΙΙΊΈνϋΡΙΛΝΟΒνΤΑΒηβΤΟΚΟΒΚΟβΚΡαΑΚνσΥΟΙΧΧΒΗΙίΒΒνΚΑνΤδΑΙϊ Ι0ΒΡΑθαααΡβ8ΙΑΡΒΡΒδΡΙΧϊ0ΡΑ8ΚΤΑΡΙι0ΡΕ0Ιι0ν?ΕΤΕΕΕΙΤσΥΠΥΙ8ΑΗΡη8ηΡ ΟΙι8νΡ0ΝΙ₁0νΐΚΟΡΙΙιΗΜ»Υ3ηΤΙ4ΧΠΧ518ΙίΜΙιΗ5ίΛΕΙΛ30ΙΑ^ΠΪΗΝΤΗΙΛΓνΗΤν PKDQI>FRlII«QAIJ^AiraPBDECVGEGI>ACHQLCARGHCWGPGPTQCVNCSQFLRGQKC VBBCRVIiQGLPRBYVNARHCIjPCHPBCQPQNGSVTCFGPBADQCVACAHYKDPPFCVARC PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPABQRASPLTSIISAWG ΙΙ^νννίισννΡΟΙΙΙΚΚΚΟΟΚΙΙΰζΧΤΜΚΚΙ^βΕΤΕΙινΒΡΕΤΡδαΑΜΡΝύΑζ^ΠίΙΙΚΕΤΕΙι RKVKVLGSGAFGTVYKGIWlPDGENVKIPVAIKVLRENTSPKANKEIIDEAYVMAGVGgP YVSRLI«GICÏ₎TSTVQI»VT5IiMPYGCI>LDHVRENRGRIK3SQDIJJMaMQIAKGMSYIJa>VR LVHRDIAARNVLVKSPNHVKITDFGLARLLDIDETKYHADGGKVPIKWMALESrLRRRFT HQgDVWSYGVTVWEIlMTFGAKPYDGIPARBIPDIiIiBKGERLPQPPÏCTIDVYMIMVKCWM IDSBCRPRFREIiVSEFSRMARDPQRFWIQNEDIXIPASPIiDSTFÏRSLLEDDDMGDLVDA BBYLVPQQGFPCPDPAPGAGGNVHHRHRSS8TR8GGGDLTLGLEPSEBBAPRSPIAP8EG ΑβδηνΡΟβοίΛΜβΑΑΚβίΏδΐ.ρτΗηρεΡΏακγβΒηρτνΡΕΡδΕτησγνΆΡΐ.τσδΡΟΡΒΥν NQPDVRPQPPSPREGPI»PAARPAGMI»BRPKTIiSPG10ïGVVKDVFAFGGAVENPBYLTPQ GGAAPQPHPPPAP8PAPDMI.YYTOQDPPBBGAPPSTFKGTPTAEliPEÏIÆIiDVPV (SEQ ID NO:17) (18) NCA (CEACAM6, Genbank accession no. M18728);

Barnett T., étal Genomics 3,59-66,1988; Tawaragi Y., et aL Biochcm. Biophys. Res.

Commun. 150,89-96,1988; Strausberg R.L., et aL Proc. Natl. Acad. Sci. USA.

99:16899-16903,2002; W02004063709; EP1439393 (Claim 7); W02004044178 (Example 4); W02004031238; W02003042661 (daim 12); WO200278524 (Example

2); WO20Q286443 (daim 27; Page 427); W0200260317 (daim 2);

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Accession: P40199; Q14920; EMBL; M29541; AAA599I5.1. EMBL; M18728; 344 aa

MQPPSAPPCRLHVPWKEVLLTASIiLTFîOiPPTTAKLTlESTPFNVAEGKBVI^LAHNLPQ 102IOTSWYKrWVDœSLTVGYVIGTQQATPGPAYSGaŒTIYPNAShMQNVTQNDTGFY TLQVIKSDIjVNEEATGQPHVYPKLPKPSISSNNSNPVEDKDAVAFTCEPEVQjn'TYLWWV NGQSLPVSPRIiQLSNGNMTIjTLIjSVKlUJDAGSYECEIQNPASANRSDPVTIJiVLYGPDVP TISPSKAJTYRPGKKLNLSCHAASKPPAQYSWyllJGTFQQSTQEIjFIPNTTVNNSGSYMCQ AHNSATGLNR'TIVI’MITVSGSAPVLSAVATVGTTIGVLARVALI (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: MDÆ179780; AAH17023.1; BC017023J

411 aa

ΜΝΒσΗν^^^νΑλΖΟΤΑΕΡΕΡΟΚΑΕΚΙΜΚΟδΡνίΟΟΗΝΟΏΡΗΟΏΕΟΜΡΝΝΡΕΰΟΕΚΑΝΤίΤΤ IAeiHOTIPKIiRAfiFVGGQFWSVÏTPCDTC»nŒlAVRRTLEQMDVVHRMCRMYPETFLYVT ΕδΆβΙΕΟΑΕΜσΚνΑβηίΟνΒΟσΗβΐηδηΐιβνΐ,ΗΑηΥαίιΟΜΗΥΕΤΙΤΗβαϊΤΡΚΑηΝΗΠν DT9DSEPQSQGl^PFGQRVVKEUmiX3VIiIDI^nZSV%TMK^TLQLSRAPVIFSHSSAYS να^δΗΐηΐνΡηπνίΛηνΚΟΊΒδΠνΜνΝΕίαΟΙΠβΏΙΠΏΝΙιάΟνΜΗηηΗΙΚΕνί^ΑηΑνβ FGGDFIXTVP^VPEGIÆDVSKYPDLnÆIJJiRNWrEAEVKI^U₁ADNI₁LRVFEAVEQASNLT ΟΑΡΕΒΕΡΠΊ4ΧΪΙιΟ(33ΟΗΤΚΥΟΥ83αΑ98ΜΒΗ»6ΙΛΙιΑ3ΙΑΡΕνΜΜ1ιΙ.

(SEQ ID »0:19) (20) IL20Ra (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);

Clark H.F., et al. Genome Res. 13,2265-2270,2003; Mungall AJ., et aL

Nature 425,805-811,2003; Blumberg H., et aL Cell 104,9-19,2001;

Dumontier L·, etaL J. Immunol 167,3545-3549,2001; Panish-Novak J., et al.

J. BîoL Chem. 277,47517-47523,2002; Pletnev S., et aL (2003) Biochemistry

42:12617-12624; Sheikh F., et aL (2004) J. Immunol 172,2006-2010;

EP1394274 (Example 11); US2004005320 (Example 5); W02003029262 (Page 74-75); W02003002717 (Claim 2; Page 63); WO200222153 (Page 4547); US2002042366 (Page 20-21); W0200146261 (Page 57-59);

W0200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59);

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

553 aa

ΜΚΑΡ®^ΑΙΛΡΙ^1ΡΡΙ.ΙΛΙ>ΐα*ΑΑΡΝΟΚΑνΜ^3σσΐΡΚΡΑΝΙΤΡη8ΙΝΜΚΝνϊ_ι0νΠ’ΡΡΒ GLQGVKVTYTVQYyiYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKC 8ΚΗΑΒ3ΟΒΡΥΡΡηΒΤ0Ι6ΡΡΒνΆΙ>ΤΤΟΕΚεΐΒννΐ>τΑΡΒΚΗΚΝ1ΠΦΙ>Ρν9Ν00ΙΥ8ΐπ>Κ YNVSVIjmSimVSQCVTNim>VLTWWPNTLYCVHVESPVPGPPRRAQPSEKQCARTL KDQSSEFKAKHFWYVLPISITVFLFSVMGYSIYRYZHVGKEiaiPANLII.IYGNEFDKRF ρνΡΑΕΚίνίΝΡΙΊΊιΝΙδηΟβΚΙΗΗΟηΜδΙίΙΛΚΒΒΟνΒΚωιηΡΟΡδβΝηΚΡΡΟΕΕΕΒνΚΗΐ GYASHLMBIFCDSEBNTEGTSFTQQESLSRTIPPDKTVIBYEYDVRTTDÏCAGPBBQKLS Ι43ΒΕν8ΤΟ6ΤΜ£30ΑΑΙΑνηθΡ0ΊΊιΟΥ3ΥΤΡΟΙ4}πωΡΕΑ0ΚΗΤη8Ε8σΡΕΕΕΡΒΤΤΙ₎ν DWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGREGLLSRLYREPAPDRPPGENETYLMQ FMEEWGLYVQMEN (SEQ ID NO :20) (21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)

Gary S.C., étal. Gene 256,139-147,2000; Clark H.F., etaL GenomeRcs. 13, 2265-2270,2003; Strausberg RJ-, et aL Proc. Natl. Acad. Sci. USA. 99, 16899-16903,2002; US2003186372 (Claim 11); US2003186373 (daim 11); · US2003119131 (Claim 1 ; Fig 52); US2003119122 (daim 1; Rg 52); US2003119126 (daim 1); US2003119121 (daim 1; Rg 52); US2003119129 (daim 1);US2003U9130(daim 1);US2003119128 (daim l;Rg52); US2003119125 (Claim I); W02003016475 (daim 1); W0200202634 (daim 1);

911 aa

MAQLFLPLLAALVLAïQAPAAIADVLEGDSSEDRAFRVRIAGDAPLQGVLGGAL'nPCHVH YUlPPPSRI»VIXKPRVlCirrM^»3lKABVI₍VARGVRVKVNEAYRFRVM.PAYPASIiTDV SIALS ELRPNDSGIYRCKVQHGIDDSSDAVEVKVKGWFLYREGSARYAPSPSGAQSACA ΒίαΑΗίΑΤΡΕοηΥΑΑΥίΛΟΥΕοαΛβϊίηβηοτνχγΡΣΟΤΡηΕΑΟΥαηΜησρρσνκΝΥσνν dpddlyotyctaedimjelfwdpp^'ILKEARAycqkrgaeiattgqlyaawdggldh CSPGWLADGSVRYPrVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPS AIPBASNPASNPASDGLEAIVTVTETLEEIiQLPQEATKSESRGAIYSIPIMEDGGGGSST PBDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYBDESEKEEESEEEEVEDEÀLW AWPSBLSSPGPRASLPTRPAAQBKSLSQÀPÀRAVIjQPGASPUPDGESEASRPPKVHGPPT ΒΤίΡΤΡΒΒΚΝηΑ8Ρ8Ρ8ΤηνΒΑΚΕνβΒλΤΟΟΡΕΙ>86νΡΒ£Β8ΕΕΏ388ΕαΑΡ8ΙιΙ>ΡΑΤΕΑ PEGTRKIÆAPSKnKSGRTAPAGTSAnSAQPVLPTDSASRGGVAWPASGDCVPSPCHNGGT CI^EEEGVRCXCLPaYGGDLŒVGI^ŒPGWDAPCXACYKHFSTRRSWEKAETQCRMYG AHIASISTPE3QDFINNRYREYQWIGLNDRTIEGDFLNSDGVPLLYEWNPGQPDSYFLS GENCWMVWKDQGQW3DVPCNYHLSYT?CKMG1.VSCGPPPELPIAQVPGRPRLRYEVDTVL RYHCREGUV2RNl4PLIRCQENGRWBAPQISCVPRRPARALHPEEDPEGRl2GRLLGRWKAL

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LIPPSSPMPGP (SEQ ID NO:21) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NMJXJ4442) ChanJ. and Watt,VJ4„ Oncogene 6 (6), 1057-106] (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosd. 21:309-345 (1998), Int Rev. Cytol. 196:177-244 (2000)); W02003042661 (Claim 12); WO2Û0Û53216 (daim 1; Page 41); W02004065576 (□aim 1); W02004020583 (Claim 9); W02003004529 (Page 128-132); W0200053216 (Claim 1; Page 42);

Cross-references: MIM:600997·; NP_004433.2; NML004442J

987 aa

ΜΑηΚΚΙβΑΜ.ΙιΙΛΡΒΙΛΑνΚΕΤΙ^8ΤΤΑΤΑΒΙΛ»ηίνΗΡΡ8σΝΕΕν5αΥηΕΝΜΝΤΙ1ΚΓΪΰ VCNVFESSQNNWLRTKFIRRRGAHRIEVEIIKFSVRDCSSIPSVPGSCKETFNLYYYEADF DSATKTFPNWMENPWVKVDTIAADESFBQVDIÆGRVMKINTEVRSFGPVSRSGFYIiAFQD YGGCMSLIAVRVmKCPRIIQNGAIFQETISGAESTSLVAARGSCIANAEEVDVPIKI.Y CNGDGEWLVPIGRCMCKAGFEAVENGTVC3fcGCPSGTFiCANQGDEACTHCPINSRTTSEGA TNCVa»GYYRADIDPIDMPCmPSAPQAVISSVNETSLMLBWrPPRDSGGREDI.VYNI ICKSCGSGRGACTRCGDNVQYAPRQL8LTEPRIYISDLIAHTQYTFEIQAVNGVTDQSPF SPQFASVNITlNQAAPSAVSIMHQVSRTVDSITIiSWSQPDQPNGVII>DYEIiQYYEKEI«SR YNATAJKSPTOTVWQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTSAEYQTSIQEK ηΡΒΙΐα83ΑΑ5ΕνΡΙ.ΐΑνννΐΑΐν0ΝΚΚΚεΡΕΗΆη3ΕΥΤΌΚΐΧΪΗΥΤ53ΗΜΤΡ®ίΚΙΥΐηΡ FTYBDPNEAVREFAlŒIDIS™<IEQVlGAGEFGEVCSQimŒP(3<REIFVAIKTLKSGY TBKQRRDFLSBASIMGQFDHPNVIRIiEGVVTKSTPVMI ITKFMENGSLDSFLRQNDGQFT VIQLVGMLRGIAAGMKYLADMNYVHRDIAARNTLVNSNLVCKVSDFGLSRFLEDDTSDPT YTSALGGKIPXRWTAPEAIQYRKFTSASDWSYGrVMWHVMSYGERPYWDM’INQDVINAI Ε0ΙΠΉΙ^ΡΡΜθεΡδΑΧ1φΙ>ΜΕϋαΝζ^ΡΝΗΚΡΚΪϋ0ΐνΝΤΙ*ΠΚΜΙΝηΡΗ31ΚΑΜΑΡΙ>Βάα nnùPLWRTiPDYTSFNTVDinniI^KMGQYKBSFANAGFTSFDVVSQMMMEDinRVGVT IAGHQKKILNSIQVMRAQMNQIQSVEV (SEQ ID NO:22) (23) ASLG659 (B7h, Genbank accession no. AX092328)

US20040101899 (Claim 2); W02003104399 (Claim 11); W02004000221 (Eg 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Eg 60);

W02002102235 (Claim 13; Page 299); US2003091580 (Example 2); W0200210187 (Claim 6; Eg 10); W0200194641 (daim 12; Eg 7b); W0200202624 (daim 13; Eg IA-IB); US2002034749 (daim 54; Page 45-46); W0200206317 (Example 2; Page 320321, Claim 34; Page 321-322); W0200271928 (Page 468-469); W0200202587

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WO200271928 (Page 233-234,452-453); WO0116318;

282 aa

MASI^IIaFWSIISIIIIIAGAIALIIGPGISGRHSITVTTVASAGNIGBDGILSCTFEP

ΟΙΚ18ΰΐνΐΟ«ΙΛζ8σνΐΧ^νΗΒ?^(^£ΜΕΟΟΕΜΡΚ^ΤΑνΡΆΙΧ2νΐνθΝΑ5ΙΛΐΐ2ΚΝν QLTDAGTYKCYI ITSKGKKNANLKYKTGAFSMPBVNVDYNASSETLRCEAPEWFPQPTW’ HASQVDQGANFSEVSNTSFBUtSENVTMKWSVDÎNVTINNTÏ'SCMXENDIAKATGDIKV 'lESEIKRRSHLQLWÎSKASLCVSSFFAISWALLPLSPyLMLK (SBQ ID NO:23) (24) PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436) Reiter R.E., et al. Proc. Natl. Acad. Sci. USA. 95,1735-1740,1998; Gu Z., et aL Oncogene 19,1288-1296,2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; W02004022709; EP1394274 (Example 11); US2004018553 (daim 17); W02003008537 (daim 1); WO200281646 (daim 1; Page 164); W02003003906 (Claim 10; Page 288); W0200140309 (Example 1; Fig 17); US2001055751 (Example 1; Fig lb); W0200032752 (daim 18; Fig 1); WO9851805 (daim 17; Page 97); WO9851824 (daim 10; Page 94); W09840403 (daim 2; Fig IB);

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

123 aa

ΜΚΑνιίΑϊώΜΑβυα.οροτΑϊΛαγδατΑανδ^οαχϊνΕΝσΓοι.ΟΕοαίίΤΑηιηΑνοίΛτ VISKGCSLNCVDDSQDYYVGKKNITCCDTDLaïASGAHÀLQPAAAIIALLPALGLLLWGP 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)

WÛ2003054152 (daim 20); W02003000842 (daim 1); W02003023013 (Example 3,

Claim 20); US2003194704 (daim 45);

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Cross-references: GL30102449; AAP14954.1; AY260763J

236 aa

MPGAAAAAAAAAAAMLPAQEAAKLYHn9YVRNSRAIGVI>WAIFTXCFATVItWCFIQPYW IGDGVDTPQAGYFGLFHYCIGNGFSRRLTCRGSPTDFSTLPSGAFKAASFFIGLSMMLII ACXICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKRMCGEKTDKYT IXJACSVRWAYILMIGIWALILSFLAFVLGNRQDSIJ'ÎAEELKAEïrKVLLSQYSLH (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 JS., et aL Science 293 (5537), 2108-2111 (2001); W02004058309; W02004011611; W02003045422 (Example; Page 32-33); W02003014294 (Claim 35; Eg 6B); W02003035846 (Claim 70; Page 615-616); WO200294852.(Col 136-137); WO200238766 (Claim 3; Page 133); W0200224909 (Example 3; Eg 3); Cross-references: MIM:606269; NP.443177.1; NM_052945_l

184 aa

MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ Ε8ν<3ΑΟΑΟΒΑΑΙ_>ΡΙ^σΐ₁ηΡΟΑΡΑΙΛ3ΙΛΙ.νϊΑΙ.5η.νθΙ₁ν3ΜΚΚΕΟΕΗΙ«ΟΑ83ΑΕΑΡησθ KDAPEPLDKVin>SPGISDATAPASiPPPGRDPGTTPPGHSVPVPATEI_lGSTBLVTrKTAG PEQQ (SEQ ID NO:26) (27) CD22 (B-cell receptor CD22-B isoform, Genbank accession No. NP-001762.1); StamenkovicJ. and Seed^., 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

ΜΗΜΛΡΗΙιΙ^νΐ,ΕΥΙ^βΟδδΚΚνΡΕΗΡΕ^ΥΆΜΕΟΑίΛ^ΙΡζΤΐΎΗΑΕΟαπΙ^βΡΙίΕΗ ΗΡΕΪΝΧϊΤΓδΚΡΙΧίΤΚΙΥΕΒΤίαΧίΚνΡδΕΟΒΚνΟΡΙΧ^ΚΚΚΝΟΤΜΙΗΡνΗΙΛΟδαοΐ,αΐΚ MESin^ramERIHLNVSERPFPPHIQIfPBIQESQEmi^mUtFSCYGYPXQlXlWLLEG VPMRQAAVTSTSLTIKSVFTRSKÏKFSPQWSHHGKIVTCQIXJDADGKFLSNIXrVQLNVKÎ TPKLEIKVTPSDArVREGDSVTMTCÆVSSSNPETrTVSWLKDGTSIiKKQHTFTLimREVT KDQSGKYCCQVSNDVGPGRSEEVFLQVQYTLPEPSTVQILHSPAVSGSQVEFLCMSÏAlfPL· PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTOQRGPGAELDVQYPPK

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IWTTVIQNPMPIRSGiyrVTI^anMSSNPSVTRYBWKPHGAWEEPSLGVLKI^iVGWDNT

TIACARCNSWCSWASPVALNVQÏAPRDVRVRKIKPbSElHSGblSVSLQCDFSSSHPKEVQ

ΡΡ»ΕΚΝΟΗΐ4ΐΧ^®50ΕΝΡΙ)ΞΙ5ΡΕηί«ΐ3Υ8α«νΝΝβΐααΤΑ5ΚΑ»ΤΙχΕ^ΥΑΡΚΑΙιΚνΞΜ

SPGDQVMEGJKSATIiTCESDANPPVSHY’IWFDTOINQSLPHHSQKIiRLEPVKVQHSGAYWCQ

GTNGVGKGRfiPLSTLTVYYSPETIGRRVAVGLGSCIAILILAICGLKLQRRWKRTQSQQG

LQENSSGQSyPVRNKKVRRAM.SBGPHSLGCïmtMmiSYTTLRFPEMWIPRTGDABS SaiQRPPRTCDDTVTYSALHXRQVODYKNVIPDFPEDEGrHYSBI&lt;IQFGVGERPQAQENV DYVILKH (SEQ ID NO:27) (28) CD79a (CD79A, CD79O, 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), pt 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 (daim 13, Fig 16); WO9207574 (Fig 1); US5644033; Ha et aL (1992) J. Immunol. 148(5):1526-1531; MuelleretaL (1992)Eur. J. Biochcm. 22:1621-1625; Hashimoto etaL (1994) Immunogenetics 40(4):287-295; Preud’homme et aL (1992) Gin. Exp. Immunol. 90(l):14M46; Yu etaL (1992)1 Immunol. 148(2) 633637; Sakaguchi et aL (1988) EMBO J. 7(11):3457-3464;

226 aa MPGGPGVLQALPATIFLLFIASAVYLQPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSN NAimimVUIGimWPPEFWP6EDPHaTI.IiaNVNKSHaGrYVCRVQEGNESYQQSCG τΥίΛνκύΡΡΡηΡΕί.ι^ΒΒΤΚΗηιιτΑΕΟίιΐιηροΑννρστηΐΛΡΗκκΜΟΝΒΚΕαηηΑαο ΕΥΕηΕΝΙιΥΕθυΠι0ΣΧ^ΜΪΒ0Ι8καΧι00Τϊ0ηνθΞηΝΙβονΌυΕΚΡ (SEQ ID JKH28) (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 nmypltl...atslttf (1..372; 372 aa), pt 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 1 lq23.3, Genbank accession Na NP_001707.1;

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W02004040000; W02004015426; US2003105292 (Example 2); US6555339 (Example 2); W0200261087 (Fig 1); W0200157188 (daim 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, cd 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, Hg 5); Dobner et aL (1992) Eur. J. Immunol. 22:27952799; Barella et aL (1995) Biochem. J. 309:773-779;

372 aa ΜΝΥΡΙΤΙΕΜΟΙΕΝΙιΒΟΒΡΝΕΙιΟΒΣΟΝΥΙΓυΤδηνΕΝΗΒσΡΑΤΕΟΡΙιΜΑΒΡΚΑνρνΡνΑΥδΙ» IFIiGyiGNVLVLVniERKRQTRSSiI^TFLFHIAVAIÆlmVPILPFAVAEGSVGWVLGTF Ι₁0ΚΠΗ1«ΛΚνΝΓΥα58Ι2*ΙΑεΐΑνηΚΥΙΑΐνΗΑνΗΑΥβΗΗΚΙΛδΙΗΙΤσ0ΤΙΪΠ.ναΡΤΒ ΑΙιΡΒΐηΡΑΚν80&lt;ΜΗΝΝ3Ι»ΡΚσΤΒ80ΒΜ0ΑΒ1ΗΑΗΡΤ8βΡΙ»ϊΗνΑΒΡΙιΙΛ·ΜηνΜΟΗσΥνθ ννΗΚΙβζ^ΟΜΙΡΟΚΟΚΑνκνΆΐηνΚΙΡΡησίδΡΥΗΐνίΡΙΟΤΙΛΚΙιΧΑνηΗΤΟΚΙιΝδεί. ΡνΜΤΜ0ΕΡΙΧ»ΙΛΗΚΙιΝΡ»ΛΥΤΡλ0νΚΡΗ8ΟΕ3ΐαΐιΤΚΙΧ3αΤΟΡΑ3Ι₁Ο0Ι₁ΡΡ8ΝΚβ351 SESENA.TSt.TTF (SEQ ID ΝΟ: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, pt 6.56 MW: 30820 TM: 1 [Pj 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 et aL (1992) J. Mol. Biol. 228:433-441; Strausberg et aL (2002) Proc. Natl. Acad. Sd USA 99:16899-16903; Servenius et aL (1987) J. Biol. Chem. 262:8759-8766; Beck et aL (1996) J. Mol. Biol. 255:1-13; Naruse et aL (2002) Tissue Antigens 59:512-519; WO9958658 (daim 13, Hg 15); US61534O8 (Col 35-38); US5976551 (cd 168-170); US6011146 (cd 145-146); Kasahara et aL (1989) Immunogenetics 30(1):66-68: Larhammar etaL (1985) J. Biol. Chem. 260(26):1411114119;

273 aa MGSGWVPWWALLVNLTRIiDSSMtlQGTDSPEDFVIQAKADCYFTNGTEKVQFWRFIFNL· KKYVRFDSDV(2<FVALTKl<K)PMEC^iNSRLDLLERSRty\VDGVCRHNYRLQAPPTVGRK νΟΡΒΤΤνΥΡΕβΤΡΙιΙιΗΟΒίαΧΗΟβνΤΟΡΥΡΟΟΙΚΙΚΗΡΙΝΟΟΒΒΡΑσνΜδΤΟΡΙΚΝσΟΝΤ FQTVVMIiEMTPKLGH5nfTCLVDHSSIir>SPVSVENRAQSEYSWRKMI>SGIAAFIiIiGt>IFLI> VGIVIQLRAQKSYVRTQNSGNBVBRXVLLPQSC

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WO 2005/081711 PCT/US2004/038392 (SEQ ID »O:3O) (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-Jephrst (1 ..422; 422 aa), pt 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17pl3.3, Genbank accession No. NPJJ02552.2;

Le et al. (1997) FEBS Lett 418(1-2):195-199: W02004047749; W02003072035 (claim

10); Touchman et aL (2000) Genome Res. 10:165-173; W0200222660 (claim 20); W02003093444 (claim 1); W02003087768 (claim 1); W02003029277 (page 82);

422 aa

Μ30ΆΰΟΚΙΪΙΖΧ5ΕΕΌΥΚΤΕΚΥνΐΑΚΝΚΚνΤ3^ΥΒΙΖίιΟΑ3ΙΙΑΥΐνν»νΚ)ΙΚΚΟΥΰηνΠΤ SI^SAVITKVKGVAFTNTSDI^RIWDVADYVIPAQGENVFFVVTimiVTPHQRQNVCAE

NEGIPEX3ACSKDSDCHAGEAVIAGNGVXTGRCLRRSNLARGTCBIFAWCPIjSTSSRPEEP

FIJOïAEDPTIFIKNHIRFPKi'NFSKSNVMDVKDRSFLKSCHFGPKHHYCPIFRIÆSVIRW

AGSDFQDIALEGGyiGIKIEXNCDLDKAASECHPHYSPeRLDNKLSKSVSSGYNFRPARY

YRDAAGVEFRTLMKAYGXRFDVMVNGKGAFFCDLVT.IYLIKXREPYRDKKYEKVRGLEDS SQEAEDBASGLGLSEQLTSGPGLLGMPBQQEI.QEPPKAKRGSSSQKGNGSVCPQLLEPHR ST (SEQ ID NO:31) (32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full maeaity...tafrfpd (L.359; 359 aa), pt 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 etaL (1990) L Immunol. 144(12):4870-4877: Strausberg et aL (2002) Proc. Nail Acad. Sci USA 99:16899-16903;

359 aa

MAEAITYADLRFVKAPtiKKBISSRIiGQDPGADMXSBITYENVQVPAVlGVPSSIASSVIXS

Ο1ΟΛνΚ5Ε0ΡΤΑ5ϊΠΛνΚΡΑνθΗΐηΡαΚΤΤαΛΥ1Ι₁ΙΧ3ηΐ»ητα^ιΙι(3νΤΑΐ(3ΛνΚΥΐΧ2

VSQQIiQQTHKVIiEVTNSSl«RQQIiRI>KITQIX3QSREDLQGSRREIAQSQBALQVEQRAHQA AEGQljQACQRDRQKTKETIjQSEBQQRRAI»EQKIjS]iME»RLKPFFTCGSAM^,CCPSGWIMH QKSCFYISLTSKNWQESQRQCBTLSSKIATPSEIYPQSHSYYFIirSIiLPNGGSmiSYWTG Ι£5Μ®ΝκηΤ0ηΤΰΚΤΗΤΥΑ0Β5ΚαΗΧνΗΧΤ1»5ΝΗΤΙ)ΕΒΕΒαΐ35Ι>ΡΥΙΟΒΜ7Μ’ΚΡΡΟ (SEQ ID »0:32)

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PC17US2004/038392 (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), pt 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5ql2, Genbank accession No. NP_005573.1; US2002193567; WO97Û7198 (claim 11, pages 39-42); Miura et aL (1996) Genomics 38(3):299-304-, Miura et aL (1998) Blood 92:2815-2822; W020Q3083047; WO9744452 (claim 8, pages 57-61); W0200012130 (pages 24-26);

661 aa

MAFDVSCFFWVVLFSAGCKVITSKDQMCIKKEANKTYNCENLGI^BIPDTLPNTTEFLEF

3ΡΗΓΙ»ΡΤΪΗΝΚΤΡ3ΜιΜΝηΤΓΙ₁Ο1Τηθ0ΙΝΗΙΗΒΟΤΡ08ΗΗ0η8^,Π.νΐΤ(3ϊΡΙιΙΡΜΑΕΤ8

ΙΛΟΡΚ3υαα.Ρηΐ0Τσΐ5ΝηΕΡΙΡνΗΝηΕ!Π₁Ε8ηΥΙ₁Ε5ΝΗΙ33ΙΚΡΡΚΠΡΡΑΗΝηκνυ3Ρ

QNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIKljGAFDSTVFQSLNFGGTPNLSVIFN GI^NSrTOSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEIIRFSDISSTTFQCFTQL 0ΚΤ·ητ·ΤΑΤΗΖΚΏηΡ8<^θηΝΙΧΣΚηνΐ_>3νΝΗΡΡΟΙ^ΟΙ5ΑΑΝΡΡ8Ι.ΤΗηΥΙΕΟΝνΚΚίΗ îjqyrarT,BXTZMT^ynj>LSHNDIEASDCCSLOI>KNI»SHI>OTI»NI»SHNEPXÆLQSQAFKBCP ρτ.ΒΤ.τ.ΤΊΤ.ΑΡΤΒΐ.ΗΤΜΑΡη5ΡΡΟΝΙΉΡΒ0νΐ.Ν1ΤΥΓΡΕΡΤ3Ν0ΗΐηΑΰΕΡνίΗΗΙιΝΕΚ0ΝΗ FQXXjriTKTNLaXJWGSLEVLILSSCGLLSIDQQAFHSIÆKMSHVDLSHNSLTCDSIDSL ΒΗΙΛΟΙΥωΠΑΑΝδΙΝΙΙδΡΚΙΛΡΠώΟΟεΤΙΝΒδΗΝΡηοαΤΟδΝΙΗΡΙ,ΤΚΥΚΕΝΙιΗΚΙιΕ Ο3ΒΕΤΤΟΑΝΡΡ3ΙιΗ0νΚ15θνκη806ΙΤΑΙ6ΙΡΡηΐνκηηηΐΑΐηηΡΡΑνΚΥυιΚΜΚΥ0Η

I (SEQ ID NOt33) (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ΤΓΑΜ domains, may have a role in Blymphocyte differentiation) PROTEIN SEQUENCE Full mlprlll...vdyedam (1..429; 429 aa) , pt 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 et aL (2001) Proc.

Natl. Acad. Sci USA 98(17): 9772-9777; W02003089624 (claim 8); EP1347046 (claim

l) ; W02003089624 (claim 1)\

429 aa

ΜΤ.ρητ.Τ·τ.Τ.ΤΠΑΡΤ·(!ΒΡΑπ₁ΡηΐΆ8Ρ8ΗΡΤΒα8ΡνΠ_ίΤαΚΜΡΡη038αΑ0ΡΟΡΟΡΡΕΡΤΚΆ

ΙιΟΡβΚ885ΡΚΙιΟΙΑΑΜΜΚΕηΤβ3ΥϊίαΕΑΟΤΜΑ3ΚνΐιΚ5ΚΚ30ΙΝνΗΚνΡνΜΐν3ΙιΕΤ0ΡΡ

G<XlVMEOTRLVLI<aVAMGTGDITFIiWYKGaVGLNI>QSKrQRSLTABYBIPSVRESDAEQ

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YYCVAENGYGPSPSCTVSITVRIPVSRPIU&RAPRAQAAVEüVLELHCEAIaRGSPPILY νϊΡΥΗΕΠΓΠΧ35ΚβΑΐ>8ββ0ΑδΕΙΠ₍δΙιΤΒΕΗ8(®ϊ3ΟΒΜηϊ0Ι₎θΑ0Κ5ΕΆνΠ>ΝΒ^,ΤνΡΤαΑ ΚβΝΗ&Τ8σνΐΕσΐΛ8ΤΙ_ιβΡΑΤνΑΙΛΡσΥαωαϋα6Β»5Αΐωΐ»ΙΛ3ΓΡ8ΡΙ^ΕΕΤΥΙ>1ί18Ρ ΤΡΟςίΧΪΡΙΥΕΙ^ίννδαϋΚνΥδΣΑΥΥΝΟΡΕΟΕΗνΆΑΒΤΙΧΙΓΗΜΕϋΚνδωΓΥδΚΙιβΚΑΝΙ TDVDYEDAM (SEQ id Ν0·34) f (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunorcceptor 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), pt 6.88 MW: 106468 IM: 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, Hg 18B-1-18B-2); 977 aa

MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGKRVTLTCKGFRFYSPQKTKWYHR YT,nin;TT.RTmm>m.CTQggCa?yRCQaQGSPLSSPVHimFS£ASLIIÆAPI^FEGDSVV ΣΗθυυίΆΕνΤΣΝΝΤΙΥΚΝ0ΝνΐΑΡηΝΚΕΤϋΡΗΙΡΗΑίΖΣΚηΝ6ΑΥΚ0ΤΰΥΚΕ300Ρν35ΝΤ νκΐ0ν2ΕΡ?τκρνηκΑ35Ρ0Ρΐ80ΝΡντι»ταΕΤοη8ηΕΚ5ονρηκΡΗΡκκηοζ)ηισιχΐΗ3 LSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTÎiSPKKAMtFB GTKVTIiHCETQEDSI>RTI<YWYHEGVPI«RHKSVRCERGASISFSLTTENSGNYYCTAEMG DGAKPSKAVSI>SV*rVPVSHPVUrLSSPEDIiIFEGAK7TIiHCEAQRGSLPIl>YQFHHEDAA Ι_<ΕΚΚ5ΑΝ3Αί3σνΑΙδΡ8Ι.^ιΕΑΕΗ5σΗ^ΓΥΥΟΤΜ>ΝΟΡσΡ0Κ5ΚΑν5η5ΙΤνρν8ΗΡνΊ>Τ185Α EALTFSGATVTLHCEVQRG3PQILYQFÏHBDMPLWSSSTPSVGRVSPSPSLTEGHS®TYY σΤΑΓΝΟΡΟΡΟΚδΕννΕΣΡντνΡνδΚΡΙΙΤΣΚνΡΚΑΟΑννσΟΣΣΚΣΗΩΕΑΡΚσΞΡΡΙΣΥΗΡ ▼m3DVTT.zasSAPSGGBA£FNI^TAEHSCTlYSCBMUKa.VADHSPTISI>SVIVPVSRPI &ΤΡΕΛΜ»0Ανν®1ΛΕΙιΗαΕΆΙΛβ85ΡΙΜΝΡΥΗΕ0νΤΙΛΚΙ8ΑΡ80<3βΑ8ΕΝΪί8Ι»ΤΤΕ ΗβΟΓΥε€ΕΑΕΗΟΡΚΛΟΚ5ΕΜνΤΣκνΑνΡν3ΛΡνΐ>ΤΚΚΑΡαΤΗΑΑν<ΪΟΣΣΕηΗΓΈΑΣΚ55Ρ τ·ττ,γρρτπη^ντΓπ^.ς.^ρηππΑβΐιπ^ΣΤΆκΗ3αΝΥ30ΕΑΐ^αΣαΑ0Κ3ΕτντΣΥΐτση TANRSGPFA1GVAGGLLSIÀGLAAGALLLYCWLSRXAGRKPASDPARSPPDSDSQEPTYH NVPAWKEhQPVYTNANPRGENVVYSSVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKVA STPYSGSLELASSAPHR (SEQ ID NO :35)

Sec also: W004/045516 (03 Inn 2004); W003/000113 (03 Jan 2003); W002/016429 (28 Feb 2002); WO02/16581 (28 Feb 2002); W003/024392 (27 Mar 2003); W004/016225 (26 Feb 2004); and W001/40309 (07 Jun 2001).

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In an embodiment, the Ligand-Lmker-Drug Conjugate has Formula IHa, 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. Exemplary Conjugates of Fonnula Uta include where R¹⁷ is -(CHOs-· Also included are such Conjugates of Formula IHa in which D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Fonnula Ma containing about 3 to about 8, in one aspect, about 3 to about 5 Drag moieties D, that is, Conjugates of Fonnula 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 Fonnula Uta, 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 Ma in which R¹⁷is -(CHz);-. Also included are such Conjugates of Fonnula IHa in which W is -VaLCit-, and/or where D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Formula IHa 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-Dnig Conjugate has Formula IHa, where the Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Y antigen, w«=l, y=l, and D has Fonnula lb. Included are Conjugates of Formula IHa in which Rⁿ is -(CHOs-. Alsa included are such Conjugates of Formula IHa where: W is Val-Cit-; Y has Fonnula 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 Drag 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”).

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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 patienl fbr 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.

43.1 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 which involves the 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.

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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. 77-96). 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 (Sec, e.g., Clackson et al., 1991, Nature352:624; HaneetaL, 1997 Proc. NatL Acad. ScL 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 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 sulfbydyl 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

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PCT/ÜS2004/038392 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. Nad. Acad. Sci. 81:851-855; Neuberger et aL, 1984, Nature 312:604-608; Takeda et ai, 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, eg., 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’)j fragments.

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).

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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 (eg., 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 (Poecking et aL, 198, Gaie 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 immunoglobulin molecule in situ. These include, but are not limited to, microorganisms such as bacteria (eg., £ coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (eg., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (eg., baculovinis) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (eg., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (eg., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (eg., 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 (eg., the adenovirus late promoter; the vaccinia virus 75K promoter).

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being

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PCT/US2004/038392 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 die £ 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 Adds Res. 13:3101-3109; Van Heeke&amp;Schuster, 1989, J. BioL Chem. 24:55035509); and the like. pGEX Vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferasc (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 cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Auiographa californien nuclear polyhedrosis virus (AcNPV) or the analogous virus from Drosophila Melanogaster is used as a vector to express foreign genes. The virus grows in Spodopterafivgiperda 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 promot»' 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 81:355359). 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

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WO 2005/081711 PCT/US2004/038392 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 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 transcripU 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, WD8, 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, \9ΊΊ, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &amp; Szybalski, 192, Proc. NatL Acad. Sei USA 48:202), and adenine phospboribosyltransferase (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:

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DHFR, which confers resistance to methotrexate (Wigler et aL, 1980, Proc. Natl. Acad. ScL USA 77:357; O’Hare et aL, 1981, Proc. Natl. Acad. ScL USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan &amp; Berg, 1981, Proc. NatL Acad. ScL USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (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):155215) and hygro, which confers resistance to hygromycin (Santerrc 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.; ColberreGarapin etaL, 1981, J. 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 amplifîable, 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 et aL, 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 rhain 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 (Proudfixtf, 1986, Nature 322:52; Kohler, 1980, Proc. NatL Acad. ScL USA 77:2197). The 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

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WO 2005/081711 PCT/US2004/038392 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.

45.2 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 aT 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 die extracellular 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 et aL Proc. NatL Acad. ScL 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

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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 thatis immunogenic in the species to be immunized, e.g._t 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), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC1₂, or R*N=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. Otic 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.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, Î.&amp;, 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

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WO 2005/081711 PCI7ÜS2004/038392 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 unfitted, 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 highlevel 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 MOPC-21 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 heteromyelomacell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. ImmunoL, 133:3001 (1984); and Brodeur et aL, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

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

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CA 02841741 2014-02-03 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 DNA encoding die antibody include Skerra et aL, Curr, Opinion in Immunol., 5:256-262 (1993) and Plückthun, 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, Biotechnology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse er cd, 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 die 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 non-immunoglobulin polypeptide.

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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 antigencombining site having specificity for a different antigen.

(ill) 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 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) ice the humanized antibody (Sims et aL, J. ImmunoL, 151:2296 (1993); Chothia eiai, 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

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WO 2005/081711 PCT/US2004/038392 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. Thrce-dimensioDal immunoglobulin models are commonly available and are familiar to those skilled in the ait Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, Le., the analysis of residues that influence the ability of foe candidate immunoglobulin to bind its antigen, hi this way, FR residues can be selected and combined from the recipient and import sequences so that foe desired antibody characteristic, such as increased affinity for foe target antigen(s), is achieved. In general, foe hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Various forms of the humanized antibody are contemplated. For example, foe 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, 7IH and 73H utilizing the variable domain numbering system set forth in Rabat 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 («.#., mice) that are capably 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 (In) gene in

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CA 02841741 2014-02-03 . chimeric and germ-line mutant mice 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. ScL USA, 90:2551 (1993);

Jakobovits et aL, Nature, 362:255-258 (1993); Bmggermann et aL, Year in Immuno., T33 (1993); and U.S. Patent Nos. 5591,669,5589,369 and 5545,807.

Alternatively, phage display technology (McCafferty et aL,Nature 348552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized 10 donors. According to this technique, antibody V domain gates are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 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 15 selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-ccll. Phage display can be performed in a variety of formats; for their review see, eg., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can bé used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a 20 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 etdL.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 inU.S. Patent No. 7,387,776.

(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

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24:107-117 (1992); and Brennan etaL, 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 Fiab’Jj 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”, eg., as described in U.S. Patent No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

(vi) B is pedfic antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two différait 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), FcyRH (CD32) and FcyRIB (CD16) 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 (MiDstein et aL, Nature, 305:537-539 (1983)). Because of foe 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 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 Tiaunecker 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. Ή» fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2,

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WO 2005/081711 PCT/ÜS2004/038392 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 erne 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 cotransfected 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 ann, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in tire other arm. ft 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).

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 (eg., 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 (eg., alanine or threonine). This provides a mechanism for increasing tire 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

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WO 2005/081711 PC17US2004/038392 procedure wherein intact antibodies are protcolytically cleaved to generate Ffab'h fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent mtermolecular 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 et aL, J. Exp. Med., 175:217-225 (1992) describe the production of a fully humanized bispecific antibody I^ab^ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

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 etaL, J. ImmunoL, 148(5):1547-1553 (1992). The leucine zipper peptides from the 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. ScL 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 (Vl) by a linker which is too short, to allow pairing between the two domains on the same chain. Accordingly, the Vh and Vl domains of one fragment are forced to pair with the complementary Vl and V_Hdomains of another ίτ^ιηεηζ 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, 1525368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt etaL J. ImmunoL 147:60 (1991).

(vii) Other amino add sequence modifications

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Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may ba 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 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 (eg., 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 ammo 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 carboxyl-terminal 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 Ν-termînal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertion^ 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 arid residue in the antibody molecule replaced by a

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WO 200S/081711 PCT/ÜS2004/038392 different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.

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-conscrvative 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) arc 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 ΠΙ product of M13 packaged within each particle. The phage-displayed 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 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

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WO 2005/081711 PCT/US20Ü4/D38392 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 cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDQ of the antibody. This may be achieved by introducing one or more ammo 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 etaL 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 Be regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al Anti-Cancer 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 ILS. 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, IgGz, IgGj. or IgG*) 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. 65:111128; Wright and Morrison, (1997) TibTECH 15:26-32). The 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 three-dimensional surface of the glycoprotein (Hcfferis and Lund, supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7:409416). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based

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WO 2005/081711 PCI7US2004/038392 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 N-acetylglucosamine residues become available to bind mannose binding protein (Malhotra et al, (1995) Nature Med. 1:237-243). Removal by giycopeptidase 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) ceils resulted in a complete reduction in complement mediated lysis (CMCL) (Boyd et aL, (1996) Mol. Immunol. 32:1311-1318), while selective removal of sialic add residues using neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCQ. In particular, CHO ceils with tetracycline-regulated expression of p(l,4)-N-acetyigJucosaminyltransferase HI (GnTDI), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et aL (1999) Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. Nlinked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-Xserine and asparagine-Xthreonine, 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 Naceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-bydroxylysme 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).

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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. Chcm. 272:9062-9070. In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media fornmlation, culture density, oxygenation, pH, purification schemes and the like. Various methods 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 endoglycosidasc H (Endo H). hr 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-giycosidase F/endo-βgalactosidasc), elimination using harsh alkaline environment to release mainly O-linked

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WO 20054)81711 PCT/IJS2004/038392 structures, and chemical methods using anhydrous hydrazine to release both N- and Olinked oligosaccharides.

452a SCREENING FOR ANTIBODY-DRUG CON.TUGATES (ADO

Transgenic animals and ceil lines are particularly useful in screening antibody drag 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 effects) of the ADC on the disease or disorder being evaluated. Alternatively, or additionally, the drag can be 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 screened for utility for prophylactic or therapeutic treatments of diseases or disorders is limited only by the rate of synthesis or screening methodology, including detectin^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 tbe 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 tbe non-human animal and (c) determining the ability of the candidate to

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WO 2005/081711 PCT/ÜS2004Æ38392 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 HER2 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) 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, hmg, 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 drag 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.

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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 suitable injection, depending on the chemical nature of die compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-fectors 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 (ΕΛΒ2) receptor (e.g., in conjugation with another ErbB receptor with which the ErbB receptor of interest forms an ErbB hetero-oligomer) may be determined. For example, cells isolated from the transgenic animal overexpressing HER2 and transfected to express another ErbB receptor (with which HER2 forms heterooligomer) 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 non-human 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 IC» value (cytotoxic activity) may be calculated for the compound of interest

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Alternatively, (X additionally; the ability of an ADC to block ErbB ligandstimulated tyrosine phosphorylation of an ErbB receptor present in an ErbB heterooligomer 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 anti-phosphotyrosine 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 ΓΗΚθβππ-Μ* 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 pl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Eabb sample (25 pl) may be electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane. Antipbosphotyrosine (at 1 pg/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 Pham, and Biomed. Anal. Some of the well established monoclonal antibodies against HER2 that are known to inhibit HRG stimulation of pl 80 tyrosine phosphorylation can be used as positive control in this assay. A dose-response curve for inhibition of HRG stimulation of pl80 tyrosine phosphorylation as determined by reflectance densitometry may be prepared and an IC» for the compound of interest may be calculated.

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

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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 F12 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 mlsZ35mm 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 20100% 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 compound. The cells are incubated for a 3-day time period. Following each treatmenL 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 CaCfo) aliquoted into 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 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 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

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WO 2005/081711 PCT/DS2004/038392 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 |Xg/ml of the antibody drag 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 V-FTTC) (1 |ig/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those compounds which induce statistically significant levels of armexin binding relative to control are selected as apoptosis-inducing compounds.

4.5.3 IN V777?O 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 15 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), 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 20 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 25 present, an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth.

160:81-88, U.S. Patent No. 6602677). The CellTiter-Glo* Assay was conducted in 96 well fonnati making it amenable to automated high-throughput screening (HTS) (Cree et aL (1995) AntiCancer Drags 6:398-404). The homogeneous assay procedure involves aiding the single reagent (CellTiter-Glo* Reagent) directly to cells cultured in serum30 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

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ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, Le. 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 signa! proportional to the amount of ATP present The amount of ATP is directly proportional to the number of cells present in culture. Hie CellTiter-Glo® Assay generates a glow-type luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than 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-hfe 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 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 + (¾---------► Oxyludferin 4- AMP + PPi + COg + 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 (IC») 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; TrastuzumabMC-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-PABMMAF, 3.7 MMAF/Ab did not inhibit the proliferation of MCF-7 cells (figure 9).

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Antibody drag conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; Trastuzumab-MC-vcPAB-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 Trastnzumab-MC5 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-HER2 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 (gg 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>

<td> 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab</td><td> 0.01</td>

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<td> 4D5 Fab-MC-vc-PAB-MMAF, 15 MMAF/Ab</td><td> 0.02</td>

<td> Anti-ΊΈ 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> ICso(pgADC/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-v&gt;PAB-MMAF, 4.4 MMAF/Ab</td><td> 0.008</td>

<td> HgMC-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 5 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 non-glycosylated.)

Anti-TF Fab = anti-tissue factor antibody fragment * activity against MDA-MB-468 cells

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In a surprising and unexpected discovery, the in vitro ceil proliferation activity results of the ADC in Tables 2a and 2b show generally that ADC with a low average number of drag moieties per antibody showed efficacy, e.g., ICjo &lt; 0.1 pg ADC/ml. The results suggest that at least for trastuzumab ADC, the optimal ratio of drag 5 moieties per antibody may be less than 8, and may be about 2 to about 5.

4.5.4 IN V7VÛ PLASMA CLEARANCE AND STABILITY

Pharmacokinetic plasma clearance and stability of ADC were investigated in rats and cyuomolgus monkeys. Plasmaconcentration was measured overtime. Table 10 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

2 mg/kg dose except where noted

<td> Sample dose mg/kg</td><td> AUCinf day* pg/mL</td><td> CL mUday/kg</td><td> Cmax pg/mL</td><td> Th Term, days</td><td> % Conj.</td>

<td> H-MC-vc-PAB-MMAE (Total Ab H-MC-vc-PAB-MMAE (Conj.)</td><td> 78.6 31.1</td><td> 26.3 64.4</td><td> 39.5 33.2</td><td> 5.80 3.00</td><td> 40.6</td>

<td> H-MC-vc-PAB-MMAF (Total Ab) H-MC-vc-PAB-MMAF (Conj.)</td><td> 170 83.9</td><td> 12.0 24.0</td><td> 47.9 44.7</td><td> 8.4 4.01</td><td> 50.0</td>

<td> H-MC-MMAE (Total Ab) H-MC-MMAE (Conj.) 5 mg/kg</td><td> 279 90.6</td><td> 18.9 62.9</td><td> 79.6 629</td><td> 7.65 4.46</td><td> 33</td>

<td> H-MC-MMAF (Total Ab) H-MC-MMAF (Conj.)</td><td> 299 110</td><td> 6.74 18.26</td><td> 49.1 50.2</td><td> 11.6 454</td><td> 37</td>

<td> H-MC-vc-MMAF, wo/PAB, (Total Ab) H-MC-vc-MMAF, wo/PAB, (Conj.)</td><td> 306 59.9</td><td> 6.6 33.4</td><td> 78.7 828</td><td> 11.9 2.1</td><td> 19.6</td>

<td> H-Mc-vc-PAB-MMAF (Total Ab)</td><td> 186</td><td> 10.8</td><td> 46.9</td><td> 8.3</td><td> 453</td>

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<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) H-Me-vc-PAB-MMAE (Conj.)</td><td> 135 31.9</td><td> 15.0 63.8</td><td> 44.9 45.2</td><td> 11.2 3.0</td><td> 23.8</td>

<td> H-MC-vc-MMAF, wo/PAB, (Total Ab) H-MC-vc-MMAF, wo/PAB, (Conj.)</td><td> 306 59.9</td><td> 6.6 33.4</td><td> 78.7 82.8</td><td> 11.9 2.1</td><td> 19.6</td>

<td> H-MC-(D)val-(L)cit-PABMMAE (Total Ab) H-MC-(D)val-(L)cit-PABMMAE(Conj.)</td><td> 107 40</td><td> 192 50.4</td><td> 30.6 33.7</td><td> 9.6 3.98</td><td> 38.1</td>

<td> H-MC-(Me)-vc-PAB-MMAE, Total Ab H-MC-(Me)-vc-PAB-MMAE, Conj.</td><td> 135.1 31.9</td><td> 15.0 63.8</td><td> 44.9 45.2</td><td> 11.2 2.96</td><td> 23.8</td>

<td> H-MC-(D)val-(D)cit-PABMMAE, Total Ab H-MC-(D)val-(D)cit-PABMMAE, Conj.</td><td> 88.2 33.6</td><td> 22.8 59.8</td><td> 33.8 36.0</td><td> 10.5 4.43</td><td> 38.3</td>

<td> H-MC-vc-PAB-MMAE, Total</td><td> 78.6</td><td> 26.3</td><td> 395</td><td> 5.8</td><td> 40.6</td>

<td> Ab H-MC-vc-PAB-MMAE, Conj. H linked to MC by lysine Pys]</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> 0224</td><td></td>

<td> MMAE 2O6gg/kg</td><td> 3.71</td><td> 62.6</td><td> 649</td><td> 0.743</td><td></td>

<td> HER F(ab’h-MC-vc-MMAE, Total Ab HER F(ab’&gt;2-MC-vc-MMAE, Conj.</td><td> 9.3 8.8</td><td> 217 227</td><td> 34.4 36.9</td><td> 0.35 0.29</td><td> 95</td>

<td> 4D5-H-Fab-MC-vc-MMAF, Total Ab 4D5-H-Fab-MC-vc-MMAF, Conj.</td><td> 43.8 29.9</td><td> 46.2 68.1</td><td> 385 34.1</td><td> 1.49 1.12</td><td> 68</td>

<td> 4D5-H-Fab-MC-vc-MMAE,</td><td> 71.5</td><td> 70.3</td><td> 108</td><td> 1.18</td><td> 59</td>

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<td> Total Ab 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> 343</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 5 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 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 10 Experimental Pharmacology, Vol. 110, Springer-Vedag. The % Conj. calculation is simply AUCinf 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 drag conjugates: H-MC-vc-PAB-MMAF-TEG and H-MC15 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.

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IN VIVO EFFICACY

The in vivo efficacy of the ADC of the invention was measured by a high expressing HER2 transgenic expiant 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 the downstream authentic initiation codon of HER2 (for example, see Child etaL (1999) J. Biol. Chem. 274:2433524341). 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, pCI-neo 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).

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Table 2d

Tumor measurements in allograft mouse model - MMTV-HER2 Fo5 Mammary Tumor, athymie nude mice single dose at day 1 (T=0) except where noted H=trastuzumab linked via a cysteine (cysj except where noted

Sample

Drugs per antibody

Dose Pn IpR

Vehicle

H-MC-vc-PAB-MMAE 8.7MMAEZAb H-MC-vc-PAB-MMAF 3.8MMAF/Ab H-MC(Me)-vc-PABMMAF________

H-MC-MMAF 4.8MMAF/Ab

1250 gg/m² 5/5 4/7

555 gg/m² 12/5 2/7

H-MC-MMAF

4.8MMAF/Ab

H-MC-vc-PAB-MMAF

5.9MMAF/Ab

H-MC-vc-PAB-MMAF

5.9MMAF/Ab

H-MC-vc-PAB-MMAF

5.9MMAF/Ab

H-(L)val-(L)cit-MMAE 8.7MMAE/Ab H-MC-MMAB 4.6MMAE/Ab H-(D)val-(D)citMMAB 4.2MMAE/Ab

9.2 mg/kg I 7/7 I 6/7 Ab

550 gg/m² atO, 7,14 and 21 days || mg/kg Ab I 5/5 5/7

840 gg/m² I I at 0,7,14 and 21 days ||

3.5mg/kg |5/6 11/7 Ab

300 gg/m² II at 0,21, and I days ||

4.9 mg/kg 14/7 2/7 Ab

425gg/m² at 0,21, and days______j

6.4 mg/kg 3/61/7

Ab

550 gg/m² II at 0,21, andI days I|

TOmg/kg [7/71/7

10mg/kg [7/710/7

TÔ mg/kg 17/70/7

CR on sn on

2Π

3Π

5/7

6n on

Ô/7 on

<img file="CA2841741C_D0071.tif" />

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<td> H-(D)val-(L)cit-MMAE 3.2 MMAE/Ab</td><td> 13 mg/kg</td><td> 7/7</td><td> 0/7</td><td> on</td><td> 9</td><td> 0.6</td>

<td> H-MC(Mc)-vc-MMAE 3.0 MMAE/Ab</td><td> 13mg/kg</td><td> ΊΠ</td><td> 3/7</td><td> on</td><td> 17</td><td> 1.2</td>

<td> H-(L)val-(D)cit-MMAE 3.5 MMAE/Ab</td><td> I2mg/kg</td><td> in</td><td> 0/7</td><td> on</td><td> 5</td><td> 0.2</td>

<td> H-vc-MMAE 8.7 MMAE/Ab</td><td> 10mg/kg</td><td> in</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> in</td><td></td><td></td><td> 3</td><td></td>

<td> H-cys-vc-MMAF 3.8 MMAF/Ab_______.</td><td> 3mg/kg</td><td> in</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> 4Π</td><td> 4/7</td><td> 3Π</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> 6Π</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> in</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«</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 550pg/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>

<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> in</td><td> 17</td><td> 2.3</td>

<td> H-vc-MMAE 75 MMAE/Ab</td><td> 75 mg/kg at 0,7,14.21, 28 days</td><td> 5Π</td><td> 377</td><td> 3Π</td><td> 69</td><td> 10</td>

<td> anti IL8-VC-MMAE 75 MMAE/Ab</td><td> 75 mg/kg at 0,7,14,21, 28 days</td><td> in</td><td> on</td><td> on</td><td> 5</td><td> 05</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> on</td><td> 3</td><td> 02</td>

<td> H-fk-MMAE 75 MMAE/Ab</td><td> 75 mg/kg at 0,7,14,21, 28 days</td><td> 7/7</td><td> 1/7</td><td> on</td><td> 31</td><td> 4.4</td>

<td> H-fk-MMAE 75 MMAE/Ab</td><td> 3.7 mg/kg at 0,7,14,21, 28 days</td><td> in</td><td> 0/7</td><td> on</td><td> 8.3</td><td> 0.9</td>

<td> anti IL8-fk-MMAE 75 MMAE/Ab</td><td> 75 mg/kg at 0,7,14,21, 28 days</td><td> in</td><td> 0/7</td><td> on</td><td> 6</td><td> 05</td>

<td> anti IL8-fk-MMAE 75 MMAE/Ab</td><td> 3.7 mg/kg at 0,7,14.21, 28 days</td><td> in</td><td> on</td><td> on</td><td> 3</td><td> 0.1</td>

<td> Trastuzumab</td><td> 75 mg/kg at</td><td> in</td><td> on</td><td> on</td><td> 5</td><td> 0.4</td>

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<td colspan="7"> 0,7,14,21, 28 days </td>

<td> H-vc-MMAE 8.7MMAE/Ab</td><td> lOmg/kg 1250 pg/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 pg/m²atO, 7, and 14 days</td><td> 7/7</td><td> 5Π</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> ΊΠ</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 days</td><td> 7/7</td><td></td><td></td><td> 6</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 Odays</td><td> 6/7</td><td> 3/8</td><td> 5/8</td><td> 56</td><td> 5.1</td>

<td> Fc8-MC-vc-PAB- MMAF 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 Odays</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 Odays</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> 1 IDlO-vc-PAB-MMAB</td><td></td><td> 9/9</td><td></td><td></td><td> &gt;14 days</td><td></td>

<td> llDlO-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

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Hg = “Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in j£ coti (therefore non-glycosyiated.)

2H9 = Anti-BphB2R HD10 = Anti-0772P

The term Ti is the number of animals in the study group with tumor at T= 0 + total animals in group. The term PR is the number of animals attaining partial remission of tumor +- animals with tumor at T = 0 in group. The term CR is the number of animals attaining complete remission of tumor+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 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:

++++ £ 3.4 (highly active) +++ =15-3.4 ++ = 1.7-2.4 + = 1.0-1.6 inactive = 0

Figure 13 shows the mean tumor volume change over time in athymie 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-vcPAB-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 athymie nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660 gg/m²) of TrastuzumabMC-MMAE and 1250 gg/m² Trastuzumab-MC-vc-PAB-MMAE. Figure 15 shows the mean tumor volume change over time in athymie nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed with 650 gg/m² frastuzumab-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 anti-tumor activity in this model (Erickson el aL US. Patent No. 6632979). As illustrated in Figures 13-15, the

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WO 2005/081711 PCT/US2004/038392 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 drag moieties per antibody showed efficacy, e.g„ tumor doubling time > 15 days and mean log cell kill > 1.0. Figure 16 shows that for the antibody drag conjugate, trastirzumab-MC-vc-PAB-MMAF, the mean tumor volume diminished and did not progress where the MMAFztrastuzumab ratio was 2 and 4, whereas tumor progressed at a ratio of 5.9 and 6, but at a rate lower than Vehicle (buffer). The rate of tumor progression in this mouse xenograft model was about the same, Le. 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.

455 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. Hematolympboid toxicity was observed by depletion of leukocytes, primarily granuloctyes (neutrophils), and/or platelets, and lymphoid organ involvement Le. 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)

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

-Li verPlasma ratio of 7000:1

-T1/2:17 hrs

ALT (alanine aminotransferase)

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

-LiverPlasma ratio of 3000:1

-Tl/2:42 hrs; diurnal variation

GGT (g-glotamyl transferase)

-Localization: plasma membrane of cells with high secretary 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, trastuzumabMC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-citPAB-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)-val-cit-PABMMAF, and 10.17 &amp; 25.50 mg/kg trastnzumab-MC-MMAF, respectively) gained weight during the study. Animals in groups 5 and 8 (26.78 mg/kg trastuzumab-MC(Me)-va]-citPAB-MMAF and 21.85 mg/kg trastuzumab-MC-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; Le. 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-PAB-MMAF conjugate (group 8). The platelet counts on day 5 were at approximately 30% of baseline values in animals of group 3

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PCT/US2004/038392 (high dose trastuzumab-MC-val-cit-MMAF) compared with 15% in animals of group 8 (high dose trastuzumab-MC-val-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_r the platelet count remained essentially unchanged in all animals. Morphological changes were mild; animals treated at the 4210gg/m² dose 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/m² dose 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 m Group 3. Also, extramedullary hematopoiesis was seen in spleen and liver.

EphB2R is a type 1TM 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 2H9val-cit-MMAE killed EphB2R expressing cells and showed efficacy in a mouse xenograft model using CXF1103 human colon tumors (Mao etal (2004) Cancer Res. 64:781 -788). 2H9 and 7C2 are both mouse IgGl anti-HER2 antibodies. The toxicity profiles of 2H9MC-val-cit-PAB-MMAF (3.7 MMAF/Ab), 7C2-MC-val-cit-PAB-MMAF (4 MMAF/Ab), and trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) were compared. The differences in the structure of each immunoconjugate or the drug portion of the

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immnnoconjugate 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 TOXICTTY/S AFETY

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 10 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 1 I00mg/m2 with complete recovery 14 days post-dose. The antibody drug conjugate H-MC-vc-PAB15 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.

4Λ> SYNTHESIS OF THE COMPOUNDS OF THE INVENTION

The Exemplary Compounds and Exemplary Conjugates can be made using 20 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 Drag 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 Ligand, such as but not limited to an antibody. Useful 25 nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The beteroatom 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 30 In another embodiment, a Linker has a reactive site which has a nucleophilic group that is reactive to an electrophilic group present cm an antibody.

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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. Schrfidcr and K. LQbke, “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 I .inker unit having a val-cit group, an electrophilic maleimide group and a PAB selfimmolative 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, fix' example but not limited to, 2 or 4 drags 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

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WO 2005/081711 PCT/US2004/038392 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 &amp; 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.

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. Schrdder 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/dolastaiin drug moieties may be prepared according to the general methods of: U.S. Patent No. 5635483; U.S. Patent No. 5780588; Pettit et ak (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; and Pettit et aL (1996) J. Chem. Soc. Perkin Trans. 15:859-863.

In one embodimenL a Drug is prepared by combining about a stoichiometric équivalent 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.

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Scheme 5

<img file="CA2841741C_D0072.tif" />

<img file="CA2841741C_D0073.tif" />

E F

As illustrated in Scheme 5, a protected amino acid A (where PG represents an amine protecting group, R⁴ is selected from hydrogen, Cj-Cg alkyl, Cj-Cg carbocycle, CHCrCs alkyl), -aryl, alkyl-aryl, alkyl-(C3-Cg carbocycle), CyCg heterocycle, alkyl-(C_rCg heterocycle) wherein R⁵ is selected from H and methyl; or R⁴ and R⁵ join, have the formula -(CR’R^n- wherein R* and R^b are independently selected from hydrogen, Ci-Cg alkyl and Ca-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 r-butyl ester B (where R⁶ is selected from -H and -Ci-Cg alkyl; and R⁷ is selected from hydrogen, Ci-Cg alkyl, CrCg carbocycle, -O^Cj-Cg alkyl),-aryl, alkyl-aryl, alkyHCj-Q carbocycle), CyCg heterocycle and alkyl-(Cj-Cg heterocycle)) under suitable coupling conditions, eg., in the presence of PyBrop and diisopropylethylamine, or using DCC (see, for example, Miyazaki, K. et. aL Chan. Pharm. Bulk 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, eg., 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 PG-He and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PGcyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-l-carboxyiic acid, PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert

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WO 2005/081711 PC17US2004/038392 butylglycine. Z is an exemplary protecting group. Fmoc is another exemplary protecting group. An exemplary r-butyl ester B is dolaisoleuine r-butyl 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 add BB (wherein R¹ is selected from -H, -C)-Cg alkyl and -Gj-Cg carbocycle; and R² is selected from -H and -CjCg alkyl; or R* and R² join, have the formula -(CR*R^b)0- wherein R* and R^b are independently selected from -H, -Cj-Cg alkyl and -C3-Cg 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, Cj-Cg alkyl, CyCg carbocycle, -O-(Ci-Cg alkyl), -aryl, alkyl-aryl, alkyl-(C3-Cg carbocycle), Cj-Cg heterocycle and alkyl-(CyCg heterocycle)). MlV-Dialkyl amino acids are exemplary amino adds for BB, such as commercially available jV,V-dimethyl valine. Other ft Mdialkyl amino adds 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 andFmoc-Me-L-glycme are two exemplary amino adds BB useful for the synthesis of 2V-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 Gâterai Procedure C.

General Procedure A: Peptide synthesis using DEPC. The//-protected 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 tripejXide E which is dried under vacuum overnight

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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 Zprotected amino acid A (1.1 eq.) is added as a solid in one portion, then PyBrop (12 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. Zprotectcd 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 thick-walled 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 th 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.

Scheme 6 shows a method useful for making a C-tcrminal 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.

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Scheme 6

<img file="CA2841741C_D0074.tif" />

<img file="CA2841741C_D0075.tif" />

(Π>)

The dipeptide K can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, G JR., el al. Synthesis, 1996,719725), with an amine of formula H using condensing agente well known for peptide chemistry, such as, for example, DEPC in the presence of triethylaminc, 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 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 tripeptidc 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

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PCI7US2004/038392 hours at O°C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is azcotropically 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 5 such as triethylamine or diisopropylethylamine (13 eq.) is then added, followed by either

PyBrop (1.2 eq.) or DEPC (12 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. AnFmocprotected 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).

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<img file="CA2841741C_D0076.tif" />

<img file="CA2841741C_D0077.tif" />

HCVdioxanc

<img file="CA2841741C_D0078.tif" />

<img file="CA2841741C_D0079.tif" />

(lb) where Z is -0- (nd R is -H

The dipeptide 0 can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, GJl, et al. Synthesis, 1996,719725), 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 0 can then be coupled with a tripeptide of formula F using General Procedure D to make the Fmoc-protected MMAF compounds of formula P which can be subsequently deprotected using General Procedure E in order to provide the MMAF drug compounds of formula (lb).

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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 ~Aq-Ww—Yy—-| 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 Aq-V^—Reactive Site 1 when the Spacer unit (-Y-) is absent

The Linker can also have the stmcture:

Reactive Site 2 —W*— Reactive Site 1 when both the Stretcher unît (-A-) and the Spacer unît (-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 Stretcher. If a Spacer unit is not present then Reactive site 1 is present at the C-tenninus 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.

5—COOH

In one aspect of the invention, Reactive Site No. 1 is &lt;

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In another aspect of the invention. Reactive Site No. 1 is

<img file="CA2841741C_D0080.tif" />

In still another aspect of the invention, Reactive Site No. 1 is a pnitrophenyl carbonate having the formula

<img file="CA2841741C_D0081.tif" />

NOg

In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups having the formula wherein X represents a leaving group, preferably O-mesyl, O-tosyl, -Cl, Br, or -I; or a maleimide group having the formula

<img file="CA2841741C_D0082.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.

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<img file="CA2841741C_D0083.tif" />

Ο

Scheme 8

<img file="CA2841741C_D0084.tif" />

<img file="CA2841741C_D0085.tif" />

Et₃N, CHgC^

<img file="CA2841741C_D0086.tif" />

<img file="CA2841741C_D0087.tif" />

wherein X is -CHr or -CH2OCH2-; and n is an integer ranging either from 0-10 when X is-CH2-; ot 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 (see for example, Walker, M.A. J. Org. Chan. 1995,60,5352-5), followed by installation of a pnitrophenyl carbonate Reactive Site group.

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PCT/ÜS2004/038392 .gchgipp.9.

<img file="CA2841741C_D0088.tif" />

<img file="CA2841741C_D0089.tif" />

PPh*.DIAD

THF

<img file="CA2841741C_D0090.tif" />

<img file="CA2841741C_D0091.tif" />

wherein E is -CHj- or -CH2OCH2-; and e is an integer ranging from 0-8; Alternatively, PEG-maleiraide and PEG-haloacetamide stretchers can be prepared as described by Frisch, et al., Bioconjugate Chem. 1996,7,180-186.

Scheme 10 illustrates a genera] synthesis of an illustrative Linker unit containing a malcimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.

Scheme 10

<img file="CA2841741C_D0092.tif" />

1. NaHCC^, DME/HgO

2. EEDQ,

<img file="CA2841741C_D0093.tif" />

<img file="CA2841741C_D0094.tif" />

1. dettiylamina, CHzCk

R, CHEA, CHjCt,

3.bte(4nitropnanyt)carton*te

DEACHzCfe

1, NaHCQg, DME/HjO

2. diethylamlna, CHjQj i oompond R.DMF

<img file="CA2841741C_D0095.tif" />

<img file="CA2841741C_D0096.tif" />

R’=tenzyl; RM^NHMtr (U)

R¹«4sopropyt; R^CHzJgNHCONHa (V)

<img file="CA2841741C_D0097.tif" />

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PCT/ÜS2004/038392 wherein Q is -Ci-Ce alkyl, -O-(Ci-Ce alkyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and n is an integer ranging from 0-10.

Usefill Stretchers may be incorporated into a Linker using the « commercially available intermediates from Molecular Biosciences (Boulder, CO) 5 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-tenninus of an Amino Acid unit as depicted in Schemes 11 and 12:

<img file="CA2841741C_D0098.tif" />

where n is an integer ranging from 1-10 and T is -H or -SOjNa;

<img file="CA2841741C_D0099.tif" />

where n is an integer ranging from 0-3;

<img file="CA2841741C_D0100.tif" />

<img file="CA2841741C_D0101.tif" />

<img file="CA2841741C_D0102.tif" />

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<img file="CA2841741C_D0103.tif" />

Stretcher units of formula (Ulb) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:

<img file="CA2841741C_D0104.tif" />

<img file="CA2841741C_D0105.tif" />

<img file="CA2841741C_D0106.tif" />

<img file="CA2841741C_D0107.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

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<img file="CA2841741C_D0108.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="CA2841741C_D0109.tif" />

<img file="CA2841741C_D0110.tif" />

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-R¹⁷-C(Oywherein -R¹⁷- is selected from -Cj-Cjo alkylene-, -C3-C3 carbocyclo-, -O-(C|-C₈ alkyl)-, -arylene-, -Ci-C_I0 alkylene-arylene-, -arylene-Cj-Cw alkylene-, -Ci-Cjo alkylene-(CyC8 carbocyclo)-, -(C3-C8 carbocyclo)-C]-Cjo alkylene-, -C3-Q heterocyclo-, -Cj-Cio alkyIcne-(C3-C8 heterocyclo)-, -(Cs-Cg heterocyclo)-CrCio alkylene-, -(CHzCHzOjr, (CH₂CH₂O)rCH2-; and r is an integer ranging from 1-10;

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Isothiocyanate Stretchers of the formula shown below may be prepared from isothiocyanatocarboxyiic acid chlorides as described in Angew. 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 self-immolative Spacer.

Scheme 11

<img file="CA2841741C_D0111.tif" />

p-rttrophenytOCOOiHiltrophenyl

DlEA(1.5eq.), DMF

<img file="CA2841741C_D0112.tif" />

p^i^jhanyi-OCOO^>rttrophwiy< DIEA(15eq.), DMF

<img file="CA2841741C_D0113.tif" />

wherein Q is -Ci-C₈ alkyl, -O-(Ci-C8 alkyl), -halogen, -nitro or -cyano;

and m is an integer ranging from 04.

Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyl self-immolative 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.

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<img file="CA2841741C_D0114.tif" />

p-rttropbenyWXCOO-p-ntrophenyt (2.0 eq.)

DIEA(1.5eq.),DMF

AH

<img file="CA2841741C_D0115.tif" />

<img file="CA2841741C_D0116.tif" />

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

As shown in Scheme 13, a Linker can be reacted with an amino group of a

Drag Compound of Formula (lb) to form a Drag-Linker Compound that contains an amide or carbamate group, linking the Drag 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 HATH or PyBrop and an appropriate amine base, resulting in a Drag-Linker

Compound AK, containing an amide bond between the Drug unit and the Linker unit When Reactive Site No. 1 isacarbonate, as in Linker AL, the Linker can be coupled to

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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 Drag via a nucleophilic substitution process to provide a Drug-Linker Compound having an amine linkage (AO) between the Drag unit and the Linker unit

Illustrative methods useful for linking a Drug to a Ligand to form a DrugLinker Compound are depicted in Scheme 13 and are outlined in General Procedures G-H.

HATU

Drug-NH-C(O)-Linker

AK

<img file="CA2841741C_D0117.tif" />

HOBt

NO₂ -----base *- Drag-NH—C“O-Linker

AM

Drag—N-Linker

AO

G: Amide formation using HATU. ADrog(Ib)

Drag + Linker-COOH (lb) AJ

O

II

Drag + Linker-O^-C^—O(lb) AL

Drug + Linker-X (lb) AN

General Procei (1.0 eq.) and an N-protected Linker containing a carboxylic acid Reactive site (1.0 eq.) arc diluted with a suitable organic solvent, such as dichloromethane, and the resulting solution is treated with HATH (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

T .inker AT. having a p-nitrophenyl carbonate Reactive site (1.1 eq.) and Drag (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

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PCT/US2004/038392 (2.0 eq.) and placed under an inert atmosphere, preferably argon. Tbe 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.

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Scheme 14

<img file="CA2841741C_D0118.tif" />

Diethylamine •π

<img file="CA2841741C_D0119.tif" />

<img file="CA2841741C_D0120.tif" />

AS R=

<img file="CA2841741C_D0121.tif" />

wherein Q is -Ci-Cg alkyl, -O-(Cj-Cg 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.

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Scheme 15

<img file="CA2841741C_D0122.tif" />

NaH.THF

<img file="CA2841741C_D0123.tif" />

1.1 MHO, THF

2. Raney Nl, hydrazine

MeOH-THF

<img file="CA2841741C_D0124.tif" />

<img file="CA2841741C_D0125.tif" />

Scheme 15 illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-bydroxymethyl)styrene (BUMS) unit The synthesis 5 of the BHMS intermediate (AW) has been improved from previous literature procedures (see International Publication No, WO 9813059 to Firestone et aL, and Crozet MP.; Ardiaimbault G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. (1985) 26:5133-5134) and utilizes as starting materials, commercially available diethyl (4-nitrobenzyl)phosphonate (AT) and commercially available 2,2-dimethyl-13-dioxan-5-one (AU). Linkers AY and

BA can be prepared from intermediate AW using the methodology described in Scheme 9.

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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 drag to antibody, Le. loading, which is related to the potency of the Drug-Linker-Ligand Conjugate. Thus, where a cysteine engineered antibody bears only one reactive cytsteine thiol group, a multitude of drag 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="CA2841741C_D0126.tif" />

<img file="CA2841741C_D0127.tif" />

<img file="CA2841741C_D0128.tif" />

(CHjCHsCOî

<img file="CA2841741C_D0129.tif" />

O

II

CHzOCHzCHgCNHCHgCYa

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4.6.4 CONJUGATION OF DRUG MOETIES TO ANTIBODIES

Scheme 16 illustrates methodology useful for making Drug-Linker-Ligand 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 (-CILSH). The partially reduced antibody thus reacts with drug-linker compounds, or linker reagents, with electrophilic functional groups such as maleimide or α-halo carbonyl, according to the conjugation method at page 766 of Kiussman, et aL (2004), Bioconjugate Chemistry 15(4):765-773.

Scheme 16

Drug-Linker

DTTT Coi0iXHi&amp;d

Antibody--------►- Partially Reduced Antibody-----^Ύ · » Drug-Linker-Ligand Conjugate with Reduced Drug Load

For example, an antibody, e.g., AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation al 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 drag linker, e.g., MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and water at known concentration, is added to the chilled 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., AC10-MC-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.

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<td> Target (antigen)</td><td> ADC</td><td> isolated amount (mg)</td><td> drug/Ab ratio</td>

<td> O772P</td><td> 10E12-MC-VC-PAB-MMAE</td><td> 1.75</td><td> 4</td>

<td> 0772P</td><td> 1 IDlO-MC-vc-PAB-MMAE</td><td> 46.8</td><td> 4.4</td>

<td> 0772P</td><td> 1 IDlO-MC-vc-PAB-MMAF</td><td> 545</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> 23</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>

<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-PABtMMAF</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> 75</td>

<td> EphB2R</td><td> 2H9-MC-VO-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>

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<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> DL-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> 10Hl-vc-MMAE</td><td> 4.5</td><td> 4.8</td>

<td> Napi3b</td><td> lOHI-vc-MMAF</td><td> 6.5</td><td> 4</td>

<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> 562</td><td> 6.4</td>

<td> PSCA</td><td> PSCA-fk-MMAE</td><td> 51.7</td><td> 85</td>

<td> PSCA</td><td> PSCAvc-MMAE</td><td> 61.1</td><td> 8.6</td>

<td> Napi3b</td><td> 10Hl-MC-vc-PAB-MMAE</td><td> 75</td><td> 42</td>

<td> Napi3b</td><td> lOHl-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> EpbB2R</td><td> 2H9-MC-MMAF</td><td> 92</td><td> ’ 45</td>

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<td> 0772P</td><td> 1 lD10(Fc chîmera)-MC-vc-PAB- MMAE</td><td> 79</td><td> 4.3</td>

<td> 0772P</td><td> 1 lD10(Fc chimera)-MC-vc-PAB- MMAF</td><td> 70</td><td> 43</td>

<td> 0772P</td><td> 1 1DI0(Fc chimera)-MC-MMAF</td><td> 23</td><td> 43</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> 036</td><td> 43</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> 103</td><td> 4.1</td>

<td> El 6</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>

<td> E16</td><td> 3B5-MC-VC-PAB-MMAF</td><td> 8</td><td> 3.1</td>

<td> 0772P</td><td> HD10(Fc chimera)-MC-vc-PAB- MMAE</td><td> 340</td><td> 3.9</td>

<td> Steapl</td><td> (Steapl-92)-MC-vc-PAB-MMAE</td><td> 33</td><td> 4</td>

<td> Steapl</td><td> (Steapl-92&gt;MC-vc-PAB-MMAF</td><td> 4.7</td><td> 4</td>

<td> Steapl</td><td> (Steapl-120)-MC-vc-PAB MMAE</td><td> 2</td><td> 4</td>

<td> Steapl</td><td> (Steapl-120)-MC-vc-PAB-MMAF</td><td> 2.3</td><td> 4</td>

<td> B16</td><td> 3B5-MC-vc-PAB-MMAF</td><td> 52.2</td><td> 43</td>

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4.7 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-tnmor, 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.

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 nontoxic 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 carricrfs) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carriers) can be gaseous or particulate, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

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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, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent

When the composition is in the form of a capsule, eg., a gelatin capsule, it . 15 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, eg., 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 20 one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. Ina composition for administration by injection, one or more of a 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 25 like form, can also include one or more of the foil owing: 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 cthylenediaminetetraacetic 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

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WO 2005/081711 PCT/US2004/038392 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.

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. Id 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 axe 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’.·; 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

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WO 2005/081711 PCT/ÜS2004/038392 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 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 pre-neoplastic 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 agenl or via perfusion in a fluorocarbon or synthetic pulmonary surfoctant

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

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PCTZUS2004/038392 embodiment, a controlled-ielease system can be placed in 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 controlledrelease 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, hi one embodiment, when administered to a patieirt, 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 fonn of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustainedrelease 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 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

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PCT/DS2004/038392 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 peppeimint, 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 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.

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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.

4.8 THERAPEUTIC USES OF THE

WLARY CONJUGATES

The Exemplary Compounds and/or Exemplary Conjugates are useful for treating cancer, an autoimmune disease or an infectious disease in a patient

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-Linker-Ligand 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 tumor-cell-associated antigen, and the Exemplary Conjugate can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis. The antigen

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WO 2005/081711 PCT/US2004/038392 can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumen* 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 DrugLinker 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 ceil 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 unit can be useful for treating antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Exemplary Conjugates having an Anti-CD30 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 lymphangioendotbeliosarcoma synovioma mesothelioma

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<td></td><td> WO 2005/081711 PCI7US2004/038392 Ewing’s tumor leiomyosarcoma rhabdomyosarcoma colon cancer</td>

<td> . 5</td><td> colorectal cancer kidney cancer pancreatic cancer bone cancer breast cancer</td>

<td> 10</td><td> ovarian cancer prostate cancer esophogeal cancer stomach cancer oral cancer</td>

<td> 15</td><td> nasal cancer throat cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma</td>

<td> 20</td><td> sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma</td>

<td> 25</td><td> medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma</td>

<td> 30</td><td> choriocarcinoma seminoma embryonal carcinoma Wilms’ tumor cervical cancer</td>

<td> 35</td><td> uterine cancer testicular cancer small cell lung carcinoma bladder carcinoma lung cancer</td>

<td> 40</td><td> epithelial carcinoma glioma glioblastoma multiforme astrocytoma medulloblastoma</td>

<td> 45</td><td> craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma</td>

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PCT/US2004/038392 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 acute nonlymphocyctic leukemia acute undifferentiated leukemia chronic myelocytic leukemia “CML” chronic lymphocytic leukemia “CLL” hairy cel] 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 tumor-specific proteases within the cell, liberating a Drug.

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4.82 MULTI-MODALTTY 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 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

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PCT7US2004/038392 being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending cm 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 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.

4,83 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, idaiubicin, daunorubicin, dactinomycin, piicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In one aspect, the anticancer agent includes, but is not limited to, a drug listed in Table 4.

TABLE4

<td colspan="2"> Alkylating agents</td>

<td> Nitrogen mustards:</td><td> cyclophosphamide ifosfamide rofosfamide chlorambucil melphalan</td>

<td> Nitrosoureas:</td><td> carmustine (BCNU) [omustine (CCNU) -</td>

<td> Alkylsulphonates</td><td> &gt;usulfan</td>

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<td></td><td> treosulfan</td>

<td> Triazenes:</td><td> lecarbazine</td>

<td> Platinum containing compounds:</td><td> cisplatin carboplatin</td>

<td> Plant Alkaloids</td><td></td>

<td> Vinca alkaloids:</td><td> vincristine vinblastine vindesine vinorelbine</td>

<td> Taxoids:</td><td> jaclitaxel locetaxol</td>

<td> DNA Topoisomerase Inhibitors</td><td></td>

<td> Epipodophyllins:</td><td> etoposide teniposide topotec an 9-aminocamptothecin camptothecin crisnatol</td>

<td> mitomycins:</td><td> mitomycin C</td>

<td> Anti-metabolitcs</td><td></td>

<td> Anti-folates:</td><td></td>

<td> DHFR inhibitors:</td><td> methotrexate rimetrexate</td>

<td> IMP dehydrogenase Inhibitors:</td><td> mycophenolic acid tiazofiirin ribavirin EICAR</td>

<td> Ribonucleotide reductase Inhibitors:</td><td> lydroxyurea deferoxamine</td>

<td> Pyrimidine analogs:</td><td></td>

<td> Uracil analogs</td><td> 5-Fhiorouracil</td>

<td></td><td> floxuridine Joxifluridine ratitrexed</td>

<td> Cytosine analogs</td><td> cytarabine (ara C) cytosine arabinoside fludarabine</td>

<td> Purine analogs:</td><td> mercaptopurine hioguanine</td>

<td> Hormonal therapies:</td><td></td>

<td> Receptor antagonists:</td><td></td>

<td> Anti-estrogen</td><td> tamoxifen raloxifene tnegestrol</td>

<td> LHRH agonists:</td><td> «oscrctin leuprolide acetate</td>

<td> Anti-androgens:</td><td> Qutamide ncalutamide</td>

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<td colspan="2"> Retinoids/Deltoids</td>

<td> Vitamin D3 analogs:</td><td> EB 1089 CB1093 KH1060</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 tevamid Dial amid</td>

<td> Isoprenylation inhibitors:</td><td> &gt;o vas ta tin</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> iactinomycin</td>

<td> Bleomycins:</td><td> jleomycin A2 jlcomycin B2 peplomycin</td>

<td> Anthracyclines:</td><td> iaunorubicin Doxorubicin (adriamycin) idarubicin spirubicin pirarubicin rorubicin fntoxantrone</td>

<td> MDR inhibitors:</td><td> verapamil</td>

<td> Ca^2tATPase 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 5 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 Exemplary Conjugate is then taken up inside a target-cell 10 through receptor-mediated cndocytosis. Once inside the cell, one or more specific

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In one embodimenL 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 embodimenL 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 die Exemplary Conjugates include, but are not limited to, Th2 lymphocyte related disorders (e.g._t 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

Allergic Reaction

Allergic Rhinitis

Alport’s Syndrome

Anaphlaxis

Ankylosing Spondylitis

Anti-phosholipid Syndrome

Arthritis

Ascariasis

Aspergillosis

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<td></td><td> WO 2005/081711 PC17US2004/038392 Atopic Allergy Atropic Dermatitis Atropic Rhinitis Behcet’s Disease</td>

<td> 5</td><td> Bird-Fancier’s Long Bronchial Asthma Caplan’s Syndrome Cardiomyopathy Celiac Disease</td>

<td> 10</td><td> Chagas’Disease Chronic Glomerulonephritis Cogan’s Syndrome Cold Agglutinin Disease Congenital Rubella Infection</td>

<td> 15</td><td> CREST Syndrome Crohn’s Disease Cryoglobulinemia Cushing’s Syndrome Dermatomyositis</td>

<td> 20</td><td> Discoid Lupus Dressier’s Syndrome Eaton-Lambert Syndrome Ecbovirus Infection Encephalomyelitis</td>

<td> 25</td><td> Endocrine opthalmopathy Epstein-Barr Virus Infection Equine Heaves Erythematosis Evan’s Syndrome</td>

<td> 30</td><td> Felty’s Syndrome Fibromyalgia Fuch’s Cyclitis Gastric Atrophy Gastrointestinal Allergy</td>

<td> 35</td><td> Giant Cell Arteritis Glomerulonephritis Goodpasture’s Syndrome Graft v. Host Disease Graves’ Disease</td>

<td> 40</td><td> Guillain-Barre Disease Hashimoto’s Thyroiditis Hemolytic Anemia Henoch-Schonlein Purpura Idiopathic Adrenal Atrophy</td>

<td> 45</td><td> Idiopathic Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases Insulin-dependent Diabetes Mellitus Juvenile Arthritis</td>

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Juvenile Diabetes Mellites (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 Sampler’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 Thyroiditis

Thrombocytopenia Thyrotoxicosis Toxic Epidermal Necrolysis Type B Insulin Resistance Type I Diabetes Mellitus Ulcerative Colitis Uveitis

Vitiligo

Waldenstrom’s Macroglobulemia Wegener’s Granulomatosis

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4.8.5 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

4.8.6 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 Dmg-Linker-Iigand 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.

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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

Bacteria] 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 Epididymitis Gonorrhea Syphilis Listeriosis

Anthrax

Nocardiosis Salmonella Typhoid Fever Dysentery Conjunctivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic Plague Tetanus

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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:

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

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Entamebiasis Giardiasis Cryptosporidiasis Isosporiasis Cyclosporiasis Microsporidiosis Asc ariasis

Whipworm Infection

Hookworm Infection Threadworm Infection Ocular Larva Migrans

Trichinosis

Guinea Worm Disease

Lymphatic FiJariasis

Loiasis

River Blindness

Canine Heartworm Infection

Schistosomiasis

Swimmer’s Itch

Oriental Lung Fluke

Oriental Liver Huke Fascioliasis

Fasciolopsiasis

Opisthorchiasis Tapeworm Infections

Hydatid Disease

Alveolar Hydatid Disease

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-Fbot-and-Mouth Disease Bornholm Disease

Genital Herpes

Genital Warts

Aseptic Meningitis

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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 pharyngoconjnnctival 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 California Encephalitis St. Louis Encephalitis

Yellow Fever

Dengue

Lymphocytic choriomeningitis

Lassa Fever

Hemorrhagic Fever Hantvirus Pulmonary Syndrome Marburg Virus Infections Ebola Virus Infections

Smallpox

4.8.7 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 antiinfectious disease agent is, but not limited to, agents listed in Table 8.

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TABLE 8 β-Lactam Antibiotics:

Penicillin G Penicillin V Cloxacilliin Dicloxacillin Methicillin Nafcillin Oxacillin Ampicillin Amoxicillin Bacampidllin Azlocillin Carbeniciilm Mezlocillin Piperacillin Ticarcillin

Aminoglycosides:

Amikacin Gentamicin Kanamycin Neomycin Netilmicin Streptomycin Tobramycin

Macrolides:

Azithromycin Clarithromycin Erythromycin Lincomycm Clindamycin

Tetracyclines:

Demeclocycline Doxycycline Minocycline Oxytetracycline Tetracycline

Quinolones:

Cinoxacin Nalidixic Add

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Fluoroquinolones:

Ciprofloxacin Enoxacin Grepafloxacin Levofloxacin Lomefloxacin Norfloxacin Ofloxacin Sparfloxacin Trovafloxicin

Polypeptides:

Bacitracin Colistin Polymyxin B

Sulfonamides:

Sulfisoxazole Sulfamethoxazole Sulfadiazine Sulfamethizole Sulfacetamide

Miscellaneous Antibacterial Agents:

Trimethoprim Sulfamethazole Chloramphenicol Vancomycin Metronidazole Quinupristin Dalfbpristin Rifampin Spectinomycin Nitrofurantoin Antiviral Agents:

General Antiviral Agents:

Idoxuradine Vidarabine Trifluridine Acyclovir Famcicyclovir Pencicyclovir Valacyclovir Gancicyclovir

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<td></td><td> WO 2005/081711 PCT/US2004/038392 Foscarnet Ribavirin Amantadine Rimantadine</td>

<td> 5</td><td> Cidofovir · Antisense Oligonucleotides Immunoglobulins Inteferons Drugs for HIV infection:</td>

<td> 10</td><td> Tenofbvir Emtricitabine Zidovudine Didanosine Zalcitabine</td>

<td> 15</td><td> Stavudine Lamivudine Nevirapine Delavirdine Saquinavir</td>

<td> 20</td><td> Ritonavir Indinavir Nelfinavir 5. EXAMPLES</td>

<td> 25</td><td> Example 1 - Preparation of compound AB NHj ⁰ Γ γ\</td>

AB

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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 el aL, (1993) Bioconjugate Chcm. 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 ail 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 mà 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].

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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 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%).

<img file="CA2841741C_D0130.tif" />

Phenylalanine i-butyl ester HC1 salt (868 mg, 3 mmol), N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 gL, 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.) NaHCOj (2 x 10 mL), saturated aq. NaCI (2 x 10 mL). The organic layer was separated and concentrated. The resulting residue was resuspended 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 r-butyl ester, the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 mL). 4M HCl/dioxane (960 gL, 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 t~ butyl ester cleavage. The mixture was cooled down on an ice bath, and triethylamine (500 gL) was added. After 10 min., the mixture was removed from the cooling bath, diluted

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WO 2005/081711 PCT/ÜS2004/038392 with dichloromethane (20 mL), washed successively with saturated aq. NaHCQj (2 x 10 mL), saturated aq. NaCl (2 x 10 mL). Hie 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-r-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 witb dichloromethane (5 mL) and added to the deprotected dipeptide (287 mg, 0.73 mmol), followed by DÏEA (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. NaHCOj (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-dap-phe-O-rBu as a white solid: 533 mg (71 %). R_f 0.4 (EtOAc). ES-MS mk 1010.6 [M+HJ*.

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/CHjCh). ES-MS ink 788.6 [Μ+Η]⁺.

Example 3 - Preparation of compound 2

<img file="CA2841741C_D0131.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

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Example 4 - Preparation of compound 3

<img file="CA2841741C_D0132.tif" />

Fmoc-Meval-val-dil-dap-phe-O-i-Bu (Example 2,50 mg) was treated with 4M HCl/dioxaue (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 dilated 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 / 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 r-butyiamine (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 (10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was diluted with dichloromethanc 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 %). Rf 0.75 (10 % McOH/CHiCh).

Fmoc protected intermediate was diluted with dichloromethanc (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 %). R_f 0.2 (10 % MeOH/CH₂C12). ES-MS n/z 787 [Μ+ΗΓ, 809 [M+Na]⁺.

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<img file="CA2841741C_D0133.tif" />

Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and tricthylencglycol 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 %). R_f 0.5 (EtOAc). ES-MS m/z 434 [M+Naf.

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 room temperature. The solvent was removed m a vacuum, the residue was dried in a vacuum to give a white solid.

The HC1 salt of Phenylalanine-triethylenegiycol monomethyl ether ester (236 mg, 0.458 mmol, leq.) and IV-Boc-Dolaproine (158 mg, 055 mmol, 12 eq.) were diluted with dichloromethane (3 mL). DEPC (125 pL, 15 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. NaHCCb (2x10 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 %). Rr 025 (EtOAc). ES-MS m/z 581.3 [M+H]⁺.

Boc deprotection was done in dichloromethane (2 mL), TFA (05 mL) at room temperature for 2 h. Solvent was removed in vacuum, and the residue was coevaporated with toluene (3 x 25 mL), then dried in vacuum to give 138 mg of dipeptide TFA salt

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Fmoc-Meval-val-dil-OH (Example 2,147 mg, 033 mmol, 1 eq.), and dipeptide TFA salt (138 mg) were diluted with dichloromethane (2 mL). To the mixture DEPC (63 gL, 15 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 5 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 %). Rf 0.4 (10 % MeOH/CHjClî). ES10 MS m/z 1100.6 [M+H]⁺, 1122.4 [M+Naf.

Fmoc protecting group was removed by treatment with diethylamine (2 mL) in dichloromethane (6 mL). After 6 h at room temperature solvent was removed in vacuum, product was isolated by flash chromatography on silica gel m a step gradient 0-.. 10 % MeOH in dichloromethane. The relevant fractions were combined and concentrated. 15 After evaporation from dichloromethane/hexane, 1:1, Compound 4 was obtained as a white foam: 133 mg (80 %). RfO.15 (10% MeOH/CHzCk). ES-MS m/z 878.6 [M+H]\

Example 6 - Preparation of compound 5

<img file="CA2841741C_D0134.tif" />

Fmoc-Meval-val-dil-OH (Example 2,0.50 g, 0.78 mmol) and dap-pheOMe-HCl (0.3 g, 0.78 mmol, prepared according to Pettit, GJL, et al Anti-Cancer Drug

Design 1998,13,243-277) were dissolved in CH2CI2 (10 mL) followed by the addition of diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (030 mL, 1.17, U eq.) was added and the contents stood over Ar. Reaction was complete according to HPLC in

h. The mixture was concentrated to an oil and purified by SiOj chromatography (300 x mm column) and eluting with 100 % EtOAc. The product was isolated as a white foamy solid. Yield: 0.65 g (87%). ES-MSmà 968.35 [M+Hf, 991.34 [M+-Na]*;UV Xqu 215,265 urn.

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The Fmoc-protected peptide (0.14 g, 0.14 nunol) 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 (C_!2-RP column, 5 μ, 100 Â, 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: 0.126 g (98%). Rf 0.28 (100 % EtOAc); ES-MS m/z 746.59 [M+HJ⁺, 76851 [M+Na]⁺; UV 215 nm.

Example 7 - Preparation of compound 6

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 μιηοΐ, 0.2 eq.) were suspended in 15 DMF/pyridinc (2 mL/0.5 mL, respectively). Diisopropylethylamine (225 pL, 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 (CjrRP . 20 column, 5 μ, 100 Â, 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 mJz 1344.32 [M+H]⁺, 136629

[M+Na]*; UV U 215,248 nm.

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Example 8 - Preparation of compound 7

<img file="CA2841741C_D0135.tif" />

<img file="CA2841741C_D0136.tif" />

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 b. The reaction mixture was diluted with dicbloromethane (20 mL), washed successively with 10 % aq. citric acid (2 x 10 mL), water (10 mL), saturated aq. NaCI (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in dicbloromethane 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 %). Rf 0.15 (10 % MeOH/CHzClz). ES-MS m/z 1476.94 [M+H]⁺.

Example 9 - Preparation of MC-Val-Cit-PAB-MMAF t-butvl ester 8

<img file="CA2841741C_D0137.tif" />

Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12 nunol, 1.1 eq.), and HOBt (2.8 mg, 21 fimol, 0.2 eq.) were taken up in dry DMF (15 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

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DMSO 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 of25mL/min) to provide Compound 8 as a white solid. Yield: 103 mg (71%).

ES-MS mJz 1387.06 1409.04 (M+Na]*; 205,248 nm.

Example 10 - Preparation of MC-val-cit-PAB-MMAF 9

Compound 8 (45 mg, 32 pmol) 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 vacao and purified by prep-HPLC (CirRP column, 5 μ, 100 A, linear gradient of McCN in water (containing 0.1% TFA) 10 to 100 % in 40 min followed by 20 min at 100 %, at a flow rate of 25 mUmin). The desired fractions were concentrated to provide maleimidocaproyl-valine-citnilline-pbydroxymethylaminobenzene-MMAF (MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MS mfz 1330.29 (M+Hf, 1352.24 [M+Naf; UV 205,248 nm.

Example 11 - Preparation of MC-val-cit-PAB-MMAP tert-butyl amide 10

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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 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 (0-510% methanol in DCM) to provide MC-val-cit-PAB-MMAF tert-butyl amide 10. Yield: 172 mg (45 %); ES-MS m/z 1386.33 1408.36 [M+Naf; UV 215,248 nm.

Example 12 - Preparation of AC10-MC-MMAE by conjugation of AClOand MCMMAE

AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 °C for 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 m 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 AC10 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 02 pm filters under sterile conditions, and frozen for storage.

Example 13 - Preparation of AC10-MC-MMAF by conjugation of AC10 and MCMMAE

AC10-MC-MMAF was prepared by conjugation of AC10 and MC-MMAF following the procedure of Example 12.

Example 14 - Preparation of AC10-MC- val-cit-PAB-MMAE bv conjugation of AC10 and MC-val-cit-PAB-MMAE

AC10-MC-val-cit-PAB-MMAE was prepared by conjugation of AC10 and MC-val-cit-PAB-MMAE following the procedure of Example 12.

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Example 15 - Preparation of AC10-MC- val-cit-PAB-MMAF by conjugation of AC10 and MC-val-dt-PAB-MMAF(9)

AClQ-MC-val-cit-PAB-MMAF was prepared by conjugation of AC10 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 cAC 10 Conjugates of Compounds 710, assayed as described in General Procedure I on a CD30+ ceil 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 cAClO-val-cit-MMAE.

TABLE 10

<td> Conjugate</td><td> ICso(ng/mL)</td>

<td> cAC10-val-cit-MMAE</td><td> 6</td>

<td> cACtO-7</td><td> 1.0</td>

<td> cAClO-8</td><td> 15</td>

<td> cAClQ-9</td><td> 05</td>

<td> cAClO-10</td><td> 20</td>

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In other experiments, BR96-val-cit-MMAF was at least 250 fold more potent than the free MMAF.

General Procedure I - Cytotoxicity détermination. 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 pl 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 of535nm 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 - In 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 10* cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each well of a 96-well, opaquewalled 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.

5. A volume of CeUTitcr-do 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.

Examplel9 - 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

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WO 2005/081711 PCT/US2004/038392 each). Animals were dosed by bohis tail vein injection (IV Push). Approximately 300 μΐ whole blood was collected through jugular cannula, or by tail stick, into lithium/beparin 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 FT ISA - ECD/GxhuFc-HRP. Antibody drug conjugate was measured by ELISA MMAE/MMAF/EOD-Bio/SA-HRP.

Example 20 - Plasma clearance in monkey

Plasma clearance pharmacokinetics of antibody drug conjugates and total antibody can be studied in cynomolgns monkeys. Figure 12 shows a two-stage plasma concentration clearance study after administration of H-MC-vc-MMAE to Cynomolgns monkeys at different doses: 05,13,25, 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 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 129ZBL6 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-butvl ester

Synthesis 1:

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<img file="CA2841741C_D0138.tif" />

MeVal-V«M»O»p4*h«O1Bu, 1001

<img file="CA2841741C_D0139.tif" />

UGMeVat-YMMOBi^PheOIBu

<img file="CA2841741C_D0140.tif" />

MC44MAF

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-diisopropylcarbodiimidc (0.0505 mL, 0326 mmol) were added followed by pyridine (0.500 mL). Reaction mixture was allowed to stir for 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 CHjCh. A total of 96 mg of pure MC-MeVal-Val10 Dil-Dap-Pbe-OtBu (12) (60% yield) was recovered. BS-MS mk. 98126 [M+H]*; 1003.47 [M+Naf; 979.65 [M-Hf.

MC-MeVal-Val-Dil-Dap-Pbe-OtBu (Compound 12,74 mg, 0.0754 mmol) was suspended in CH2CI2 (2.0 mL) and TFA (1 mL) at room temperature. After 23 hr, HPLC analysis indicated complete consumption of starting material. Volatile organics 15 were evaporated under reduced pressure, and the product was isolated via preparatory RP-HPLC, using a Phenomenex™ C12 Synergi Max-RP 80Â 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 92533 [M+H]*; 94730 [M+NaJ⁺; 923.45 [Μ-ΗΓ.

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Example 23a - Synthesis of MC-MMAF fl 1) via dimethox vbenzyl ester

Synthesis 2:

<img file="CA2841741C_D0141.tif" />

envoya.

Rnoc^feVat-VaHMOap-Phe-OOMB

<img file="CA2841741C_D0142.tif" />

MeV*VaM>Oa|M>t»ODMB

<img file="CA2841741C_D0143.tif" />

MGJJeVa»-V«H>W<»-Rie-ODMB

<img file="CA2841741C_D0144.tif" />

MOMMAF

Preparation of Fmoc-L-PhenylaIanine-2,4-dimethoxybcnzyl ester (FmocPbe-ODMB)

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A 3-ncck, 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-bntyl 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 punfied through a short plug of silica gel (25 cm x 25 cm, CH2C12) to give a colorless foam (250 g). MeCN (1L) 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 (Phe-ODMB) A 500-mL round-bottom flask was charged with Fmoc-L-phenylalanine2,4-dimethoxybenzyl ester (26.00g, 48.3 mmol), CH2C12 (150 mL) and diethylamine (75 mL, Acres). 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, 1HNMR.

Preparation of Fmoc-Dolaproine (Fmoc-Dap) Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HC1 in 1,4dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC analysis indicated the reaction was complete (10% MeOH/CH2C12) and the mixture was concentrated to ncar-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, 1223

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WO 2005/081711 PCT/ÜS2004/038392 mol, Acres) was added ova: one minute, causing an exotherm from 205 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 copions white precipitate to fonm. 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 5925 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 Et2O (50 mL) keeping the solids with the organic layer. The organic layer was filtered and concentrated to give 3233 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 (35 L), then 3% MeOH/ CH2C12 (9 L) to give 68.23 g of Fmoc-dolaproine as a white foam (81%, 975% purity by HPLC (AUC)).

Preparation of Fmoc-Dap-Phe-ODMB

Crude Pbe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 mL, Acres) 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. NJSf-diisopropylethylaimne (253 mL, 145.0 mmol, Acres) was added via syringe over 3 min. After Ih, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (1L) 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 dibenzofutvene). 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).

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Data: HPLC, 1H NMR, TLC (1:1 EtOAc/Heptane Rf= 0.33, red in vanillin stain).

Preparation of Dap-Phe-ODMB

A1-L round bottom flask was charged with Fmoc Dap-Pho-ODMB (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 CH2CI2 (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-Dy-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 ( 13.81g, 43.0 mmol, Matrix Innovations) was added. Ν,Ν-Diisopropylethylamine (20.5 mL, 117.3 mmol, Acros) was added via syringe over 2 minutes. After 1 hour, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (15 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 Fmoc-MeVal-Val-Dil-Dap-PheODMB as a white foam (73%, 93% purity by HPLC (AUQ).

Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0.21, red in vanillin stain).

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Preparation of MeVal-Val-Dil-Dap-Phe-ODMB

A1-L, round-bottom flask was charged with Fmoc-MeVal-Val-Dil-DapPhe-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 purified by flash chromatography (15 cm x 8 cm dia column; 2% MeOHZDCM (2 L), 3% MeOH/DCM (1L), 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

A 50-mL, round-bottom flask was charged with MeVal-Val-Dil-Dap-PheODMB (750 mg, 0.85 mmol), anhydrous DMF (4 mL), maleimidocaproic acid (180 mg, 0.85 mmol), and TBTU (300 mg, O.93mmol, Matrix Innovations) at room temperature. NJN-Diisopropylethylamine (450 pL, 2.57 mmol) was added via syringe. After 15 hours, the mixture was poured in waler (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: 1HNMR, HPLC.

Preparation of MC-MeVal-Val-Dü-Dap-Phe-OH (MC-MMAF) (11) A 50-mL, round-bottom flask was charged with MC-MeVal-Val-Dil-DapPhe-ODMB (Compound 13,428 mg, 0.39 mmol) and dissolved in 25% 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 MCMMAF (73%, 92% purity by HPLC) as a white solid.

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Example 23b - Synthesis of analog of mc-MMAF

<img file="CA2841741C_D0145.tif" />

<img file="CA2841741C_D0146.tif" />

MB-Me^VaHM-Dapflie -OtBu

MB-MMAF

MeVal-Val-Dil-Dap-Phe-OfBu (compound 1,35 mg, 0.044 nunol) was suspended in DMF (0250 mL). 4-(254Xoxo-25-dihydro-pyirol-l-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 10 indicated complete consumption of starting compound 1.

Product was isolated via preparatory RP-HPLC, using a Phenomenex C₁₂Synergi Max-RP 80Â Column (250 x 2120 mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCNÆL05% TFA (aq) for ' an additional 12 minutes. A total of 20 mg of pure product (14) was isolated (0.02 mmol, 15 46% yield). ES-MS mfz 987.85 [M+Hf; 1019.41 [M+NaJ⁺; 985.54 [M-Hf.

MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14,38 mg, 0.0385 mmol) was suspended in CH2CI2 (1 mL) and TFA (1 mL). Mixture was stirred for 2.0 hr, and then volatile organics were evaporated under reduced pressure. Product was purified by

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PCT/US2004/038392 preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80Â 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 m/z 930.96

[M+H]⁺ 952.98 [M+Na]⁺; 929.37 [M-H]‘.

Example 23c - Preparation of MC-MeVaLCit-PAB-MMAF (16)

NH,

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 CH2O2 (80 mL) was added DIEA (4.48 mL, 25.71 mmol). Ulis 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 L-citrulline (0.85 g,

4.84 mmol) and NaHCOj (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 tertBUOH/CHCI3/H2O, acidified to pH=2-3 with 1M HQ. The 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-Cit-COOH which was used without further purification.

The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1 CHaClj/MeOH (100 mL), and to it was added p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ

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WO 2005/081711 PCT/US2004/038392 (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/CHjCla. Pure Fmoc-MeValCit-PAB-OH (0.55 g, 0.896 mmol, 18.5 % yield) was recovered. ES-MS m/z 616.48

To a suspension of Fmoc-MeVal-Cit-ΡΑΒΌΗ (0.55g, 0.896 mmol) in CH₂C1₂ (40 mL) was added STRATOSPHERES^fpiperizine-resm-bound) (>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, MeValCit-PAB-OH, was suspended in CH2CI2 (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% NaHCQj (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/CHCh gradient, 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)cart)onate (2.59 g, 8.52 mmol) in CH2CI2 (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-MeVal-C5t-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 mixture was stirred for 16 hr at room temperature, and then purified by preparatory RP-HPLC (x3), using a Pbenomenex C₎₂Synergj Max-RP 80Â Column (250 x 2120 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 fM+H]⁴; 1366.94 [M+Na]⁺.

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Example 23d - Preparation rf succinimide ester of suberyl-Val-Cit-PAB-MMAF (17)

<img file="CA2841741C_D0147.tif" />

Compound 17

Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 037 mmol, 1.5 eq.) were suspended in anhydrous pyridine, 5 mL HOBt (10 mg, 0.076 mmol, 0.2 eq.) was added followed by DEA (199 μΐ, 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 re-suspended in CHjCk· Mixture was separated by silica gel flash chromatography in a step gradient of MeOH, from 0 to 10%, in CHjCLProduct 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 CH2CI2 (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% MeOH/CEhCh· Product was eluted with 30% MeOH/CH2Ch. After drying in vacuum ovemigtrt, Fmoc-Val-Cit-PABMMAF 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) diethyiamine (2 mL) and DMF (2 mL). Mixture was stirred for 2 hrs at room temperature. Solvent was removed under reduced pressure. Residue was co-evaporated 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-Cit-PABMMAF, was suspended in pyridine (2 mL), and added to a solution of disuccinimidyl suberate (74 mg, 02 mmol, 4 eq.), m pyridine (1 mL). Reaction mixture was stirred at

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WO 2005/081711 PCT/US2004/038392 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/CHzCfo, filtered trough a pad of silica gel with 30% MeOH/CHzCbas an eluent. Compound 17 was obtained as white solid, 20 mg (29% yield). ES-MS mJz 1388.5 [Μ-ΗΓ

Example 24 - In vivo Efficacy of mcMMAF AntibodyJtaig Conjugates

Efficacy ofcAClO-mcMMAF in Karpas-299ALCL xenografts: To evaluate the in vivo efficacy of cACIO mcMMAF with an average of 4 drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL cells were implanted subcutaneously into immunodeficient CJB-17 SCID mice (5xl0⁶ cells per mouse). Tumor volumes were calculated using the formula (OJxLxW²) 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 >1,000 mm³ within 7 days of the start of therapy. In contrast, all of the cAC10-mcF4 treated tumor showed 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 ofcBR96-mcMMAF in 12987NSCLC 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 athymie 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/injcction 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

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WO 2005/081711 PCT/ÜS2004/038392 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 L54OCY. 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 cfcAClO-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 cAClO-mcMMAF (Figure 4a) or cAClO-vcMMAF (Figure 4b) for 96 hours. The cultures were labeled for 4 hours with 50 μΜ resazurin [7-hydroxy-3H-phenoxazin-3-one 10-oxide] and the fluorescence measured. The data were reduced m GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC» values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration was tested in quadruplicate.