Monomethylvaline compounds capable of conjugation to ligands

The invention relates to a drug compound, and more specifically to drug-linker-ligand conjugates, drug-linker compounds and drug-ligand conjugates, and compositions comprising the above.

The description also describes their methods of use for treating cancer, an autoimmune disease or an infectious disease. The invention also relates to antibody-drug conjugates and compositions comprising the above. The description also describes methods of using them for the treatment of cancer, autoimmune disease or infectious disease. Also described herein are methods of using antibody-drug conjugate compounds for diagnosis or treatment, in vitro, in situ and in vivo, mammalian cells, or related pathological conditions.

2. BACKGROUND OF THE INVENTION [0002] Improving the delivery of drugs and other agents to target cells, tissues and tumors, in order to achieve maximum efficacy and minimal toxicity, has been the subject of numerous studies for many years. Although many efforts have been made to develop effective methods for delivering biologically active molecules to cells, both in vivo and in vitro, none of them has been completely satisfactory. Optimizing drug binding to its intracellular target, while minimizing intercellular redistribution of the drug, e.g. to adjacent cells, is often difficult or ineffective.

[0003] Most of the agents currently administered to the patient parenterally are not targeted, which leads to systemic delivery of the agent to the cells and tissues of the body where this is unnecessary and often undesirable. This can result in a side effect of the drug, and often limits the dose of drug (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitors and anti-viral or antimicrobial agents) that can be administered. For comparison, although oral administration of drugs is considered to be a convenient and inexpensive mode of administration, it combines with the same problems regarding non-specific toxicity to unchanged cells after the drug enters the systemic circulation. Further complications concern problems with the bioavailability of oral medications and the presence of the drug in the intestine, which leads to additional intestinal exposure to the drug and subsequently to the risk of intestinal toxicity. Accordingly, the main goal was to develop methods for specifically targeting agents to cells and tissues. The benefits of such treatment include avoiding the general physiological effects of improper delivery of such agents to other cells and tissues, such as unchanged cells. Intracellular targeting can be achieved by methods, compounds and formulations that allow the accumulation or retention of biologically active agents i.e. active metabolites inside cells.

[0004] Monoclonal antibody therapy has an established position in the targeted treatment of patients with cancer, immune and angiogenic disorders.

[0005] Use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, e.g. drugs for killing or inhibiting cancer cells in the treatment of cancer (Syrigos and Epenetos (1999) AntiCancer Research 19: 605614; Niculescu-Duvaz and Springer (1997) Adv Vibrations Del. Rev. 26: 151-172; US Patent No. 4975278) theoretically allows targeted delivery of a drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as cancer cells that are to be removed (Baldwin et al ., 1986, Lancet str. (Mar. 15, 1986): 603-05; Thorpe, 1985, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Thus, maximum efficacy with minimal toxicity is sought. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21: 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., 1986, supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8 (6): 781-784; Mandler et al . (2000) Jour. Of the Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13: 786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamycin (Lode et al. (1998) Cancer Res. 58: 2928; Hinman et al. (1993) Cancer Res. 53: 3336-3342). Toxins can exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding or topoisomerase inhibition (Meyer, DL and Senter, PD "Antibody Drug Conjugates for Cancer Therapy" in Annual Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229237). Some cytotoxic drugs appear to be inactive or less active when coupling to large antibodies or protein receptor ligands.

[0006] ZEVALIN® (ibritumomab thiuksetan, Biogen / Idec) means a radioisotope antibody conjugate composed of the murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and the radioisotope ¹¹¹In or ⁹⁰Y-linked 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 cytopenia in most patients. MYLOTARG ™ (gemtuzumab ozogamycin, Wyeth Pharmaceuticals), an antibody conjugate consisting of hu [human] CD33 antibody combined with calicheamycin, 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. 4,970,198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantantumab Mertansine (Immunogen, Inc.), an antibody conjugate drug consisting of huC242 antibody linked via an SPP disulfide linker with a maytansinoid drug moiety, DM1, is proceeding to phase II clinical trials for the treatment of cancers that express CanAg, such as colon cancer , pancreas, stomach, and others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), ah antibody conjugate drug consisting of a monoclonal antibody directed against a prostate-specific membrane antigen (PSMA) connected to the maytansinoid drug moiety, DM1, is under development for potential treatment prostate tumors. The same maytansinoid drug moiety, DM1, was linked via a non-disulfide linker, SMCC, to a murine monoclonal antibody, TA.1 (Chari et al. (1992) Cancer Research 52: 127-131). This conjugate has been reported to be 200 times weaker than the corresponding disulfide linker conjugate. The SMCC linker was considered there as "indissoluble".

[0007] Several short peptide compounds have been isolated from the sea mollusk Dolabella auricularia and have been shown 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). Analogues of these compounds have also been obtained and some of them have been shown to have biological activity (for a review, see Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277). For example, auristatin E (US Patent No. 5635483) is a synthetic analogue of the natural marine product dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anti-cancer drug vincristine (GR Pettit, (1997) Prog. Chem Org. Nat. Prod. 70: 1-79). Dolastatin 10, auristatin PE and auristatin E are linear peptides having four amino acids, three of which are unique to the class of dolastatin compounds, and the amide at the C-terminus.

[0008] The peptides of auristatin, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, have been coupled to: (i) chimeric monoclonal antibodies cBR96 (Lewis Y specific [antigen] on cancers); (ii) cAC10, which is specific for CD30 on hematological tumors (Klussman, et al. (2004), Bioconjugate Chemistry 15 (4): 765-773; Doronina et al. (2003) Nature Biotechnology 21 (7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands"; Francisco et al. (2003) Blood 102 (4): 1458-1465; US Publication 2004/0018194; (iii) anti-CD20 antibodies such as RITUXAN® (WO 04/032828) for the treatment of CD-20 expressing cancers and immune disorders; (iv) anti-EphB2 2H9 and anti-IL-8 antibodies for the treatment of colorectal cancer (Mao, et al. (2004) Cancer Research 64 (3): 781-788); (v) E-selectin antibodies (Bhaskar et al. (2003) Cancer Res. 63: 6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583).

[0009] Aurystatin E conjugated to monoclonal antibodies is disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, abstract no.

623, presented on March 28, 2004.

[0010] Despite in vitro data for compounds of the class dolastatin and its analogues, significant general toxicity at the doses required to achieve a therapeutic effect reduce their effectiveness in clinical studies. Accordingly, there is a clear need in the art for dolastatin / auristatin derivatives having significantly lower toxicity, with useful therapeutic efficacy. These and other known limitations and problems are solved by the invention.

[0011] The ErbB family of tyrosine kinase receptors are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p 185^neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies was characterized using a human breast cancer cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol. 9 (3): 1165-1172. Maximum inhibition was obtained for the antibody called 4D5, which inhibited cell proliferation by 56% Other panel antibodies reduced cell proliferation to a lesser extent in this assay. In addition, 4D5 antibody has been found to sensitize ErbB2 overexpressing breast cancer cell lines to the cytotoxic effects of TNF-α (US Patent No. 5,677171). Anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. (1990) Cancer Research 50: 1550-1558; Kotts et al. (1990) In vitro 26 (3): 59A; Sarup et al. (1991) Growth Regulation 1: 72-82; Shepard et al. J. (1991) Clin. Immunol. 11 (3): 117-127; Kumar et al. (1991) Mol. Cell. Biol. 11 (2): 979-986; Lewis et al. (1993) Cancer Immunol. Immunother. 37: 255-263; Pietras et al. (1994) Oncogene 9: 18291838; Vitetta et al. (1994) Cancer Research 54: 5301-5309; Sliwkowski et al. (1994) J. Biol. Chem. 269 ​​(20): 14661-14665; Scott et al. (1991) J. Biol. Chem. 266: 14300-5; D'souza et al. Natl. Acad. Sci. (1994) 91: 7202-7206; Lewis et al. (1996) Cancer Research 56: 14571465; and Schaefer et al. (1997) Oncogene 15: 1385-1394.

[0012] Other anti-ErbB2 antibodies with different properties are described in Tagliabue et al. Int. J. Cancer 47: 933-937 (1991); McKenzie et al. Oncogene 4: 543-548 (1989); Maier et al. Cancer Res. 51: 5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3: 350-362 (1990); Stancovski et al. Natl. Acad. Sci. USA 88: 8691-8695 (1991); Bacus et al. Cancer Research 52: 2580-2589 (1992); Xu et al. Int. J. Cancer 53: 401-408 (1993); WO94 / 00136; Kasprzyk et al. Cancer Research 52: 2771-2776 (1992); Hancock et al. (1991) Cancer Res. 51: 4575-4580; Shawver et al. (1994) Cancer Res. 54: 1367-1373; Arteaga et al. (1994) Cancer Res. 54: 3758-3765; Harwerth et al. (1992) J. Biol. Chem. 267: 15160-15167; U.S. Patent No. 5,783,186; and Klapper et al. (1997) Oncogene 14: 2099-2109.

[0013] Homology screening led to the identification of two other members of the ErbB receptor family; ErbB3 (US Patent No. 5,183,884; US Patent No. 5,480,968; Kraus et al. (1989) Proc. Natl. Acad. Sci. USA 86: 9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1746-1750; and Plowman et al. (1993) Nature 366: 473-475). Both of these receptors display increased expression on at least some breast cancer cell lines.

[0014] HERCEPTIN® (Trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to high affinity in a cellular assay (Kd = 5 nM) with the extracellular domain of human epidermal growth factor receptor 2 protein, HER2 (ErbB2) (US Patent No. 5821337; US Patent No. 6054297; US Patent No. 6407213; US Patent No. 6639055; Coussens L, et al. (1985) Science 230: 1132-9; Slamon DJ, et al. (1989) Science 244: 707-12). Trastuzumab is an IgG1 kappa antibody that contains human framework regions with complementarity determining regions of the murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen and thus inhibits the growth of cancer cells. Because Trastuzumab is a humanized antibody, it minimizes any HAMA response in patients. Humanized anti-HER2 antibody is produced by suspension culture of mammalian cells (from Chinese hamster ovary, CHO). The HER2 (or c-erbB2) proto-oncogen encodes a transmembrane receptor protein of 185kDa, which is structurally associated with the epidermal growth factor receptor. HER2 protein overexpression is observed in 25% -30% of primary breast cancers and can be determined using immunohistochemistry evaluation of fixed tumor blocks (Press MF, et al. (1993) Cancer Res 53: 4960-70. Trastuzumab has been shown to inhibit the proliferation of human tumor cells that overexpress HER2 in both in vitro and animal assays (Hudziak RM, et al. (1989) Mol Cell Biol 9: 1165-72; Lewis GD, et al. (1993 ) Cancer Immunol Immunother; 37: 255-63; Baselga J, et al. (1998) Cancer Res. 58: 2825-2831). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling TE, et al. (1996) [summary]. Proc. Annual Meeting Am Assoc Cancer Res; 37: 471; Pegram MD, et al. (1997) [abstract]. Proc Am Assoc Cancer Res; 38: 602). Trastuzumab-mediated ADCC has been shown in vitro to preferentially HER2 overexpressing cancer cells compared to HER2 overexpressing cancer cells. HERCEPTIN® as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 and who have received one or more chemotherapy regimens in connection with their metastatic disease. HERCEPTIN® in combination with paclitaxel is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 and who have not received chemotherapy for their metastatic disease. HERCEPTIN® is clinically active in patients with ErbB2 overexpressing metastatic breast cancer who have received extensive prior cancer therapy (Baselga et al, (1996) J. Clin. Oncol. 14: 737-744).

[0015] Mouse anti-HER2 monoclonal antibody inhibits the growth of a breast cancer cell line that overexpresses HER2 at levels 2+ and 3+ (1-2 x 10⁶ HER2 receptors per cell), but has no activity on cells that express lower HER2 levels (Lewis et al., (1993) Cancer Immunol. Immunother. 37: 255-263). Based on this observation, the 4D5 antibody (huMAb4D5-8, rhuMAb HER2, US Patent No. 5821337; Carter et al., (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289) and was tested on patients with breast cancer whose tumors overexpressed HER2 but who had progressed after conventional chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17: 2639- 2648).

[0016] Although HERCEPTIN represents a breakthrough in the treatment of patients with ErbB2 overexpressing breast cancers who have received extensive prior cancer therapy, some patients in this population do not respond or respond only poorly to HERCEPTIN treatment.

[0017] WO01 / 18032 A2 describes dolastatin peptides of formula (I)

<img file="PL1725249T3_D0001.tif" />

[0018] WO2004 / 073656 A2 describes anti-CD70 antibodies and their derivatives conjugated to cytotoxic, immunosuppressive or other therapeutic agents for the treatment of CD70-expressing cancers and immune disorders.

[0019] WO2004 / 010957 A2 describes drug-linker-ligand conjugates in which the drug is linked to the ligand via a peptide-based linker unit. The ligand may be an antibody. Methods for treating cancer, an autoimmune disease or an infectious disease are described.

[0020] Therefore, there is a significant clinical need to develop further HER2-targeted cancer therapies for those patients with HER2-overexpressing tumors or other HER2-related diseases who do not respond, or respond poorly to HERCEPTIN treatment.

[0021] Citation of any reference in the application does not mean that the publication cited is the prior art with respect to this application.

3. BRIEF DESCRIPTION OF THE INVENTION [0022] In one aspect, the invention provides drug compounds of formula Ib:

<img file="PL1725249T3_D0002.tif" />

and independently for each location

R² selected from hydrogen and -C₁-C₈ alkyl;

R³ selected from hydrogen, -C₁-C₈ alkyl, -C₃-C₈ carbocycle, aryl, -C₁-C₈alkylaryl, -C₁-C₈ alkyl- (C₃-C₈ carbocycle), -C₃-C₈ heterocycle and -C₁-C₈ alkyl- (C₃-C₈a heterocycle);

R⁴ selected from hydrogen, -C1-C8 alkyl, -C3-C8 carbocycle, -aryl, -C1-C8 alkylaryl, -C1-C8 alkyl- (C3-C8 carbocycle), -C3-C8 heterocycle and -C1-C8 alkyl - (C3-C8 heterocycle), wherein R⁵ selected from -H and -methyl; or R⁴ and R⁵ together form a carbocyclic ring and have the formula - (CR^andR^b) n- where R^and and R^b are independently selected from -H, -C₁-C₈ alkyl and -C₃-C₈ carbocycle, and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from H and -C₁C₈ alkyl;

R⁷ selected from hydrogen, -C₁-C₈ alkyl, -C₃-C₈ carbocycle, aryl, -C₁-C₈alkylaryl, -C₁-C₈ alkyl- (C₃-C₈ carbocycle), -C₃-C₈ heterocycle and -C₁-C₈ alkyl- (C₃-C₈a heterocycle);

₈ each R is independently selected from H, -OH, -C₁-C₈ alkyl, -C₃-C₈ carbocycle and O- (C 1 -C 8 alkyl);

R⁹ is selected from H and -C₁-C₈ alkyl;

R¹⁰ is selected from aryl or -C₃-C₈ heterocyclic;

Z is -O-; and R¹¹ means H.

[0023] The compounds of formula (Ib) are useful in the treatment of 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.

[0024] In a further aspect, the invention provides a conjugate as defined in claim 2, with the formula:

L- (LU-Dp) p or a pharmaceutically acceptable salt or solvate thereof, wherein:

L- is the ligand unit,

LU is a linker unit which may be present or absent, p ranges from 1 to 20, and

<img file="PL1725249T3_D0003.tif" />

[0025] The conjugate defined in claim 2 may be an antibody-drug conjugate of formula Ia ':

<img file="PL1725249T3_D0004.tif" />

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antibody

-AND_and-IN_in-Y_s- means the linker unit,

A is a stretch unit, and is 0 or 1, each W is independently an amino acid unit, w is an integer in the range of 0 to 12,

Y is the distance unit, y is 0, 1 or 2, and p is in the range of 1 to 20.

[0026] In a further aspect, the invention provides a conjugate as defined in claim 3, with the formula:

LU-Df or a pharmaceutically acceptable salt or solvate thereof, wherein:

LU- means a linker unit containing a functional group capable of linking to a ligand unit, and

<img file="PL1725249T3_D0005.tif" />

containing effective amounts of the antibody-drug conjugate of the invention and a pharmaceutically acceptable carrier or excipient.

[0028] Also described herein are compositions comprising effective amounts of a drug-ligand conjugate having a cleavable drug unit from a drug-ligand conjugate and a pharmaceutically acceptable carrier or excipient.

[0029] Also described herein are methods for killing or inhibiting the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0030] Also described herein are methods for killing or inhibiting the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0031] Also described herein are methods for killing or inhibiting the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable drug unit from the Drug-Ligand Conjugate.

[0032] Also described herein are methods for treating cancer including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0033] Also described herein are methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0034] Also described herein are 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.

[0035] Also described herein are methods for killing or inhibiting 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.

[0036] Also described herein are methods for killing or inhibiting 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.

[0037] Also described herein are methods for killing or inhibiting 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 drug unit from the drug-ligand conjugate.

[0038] Also described herein are methods for treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0039] Also described herein are methods for treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a Drug-Linkerligand Conjugate.

[0040] Also described herein are methods for treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable drug unit from the Drug-Ligand Conjugate.

[0041] Also described herein are methods for treating an infectious disease, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0042] Also described herein are methods for treating an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0043] Also described herein are methods for treating an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable drug unit from the Drug-Ligand Conjugate.

[0044] Also described herein are methods for preventing the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0045] Also described herein are methods for preventing the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a drug-linker-ligand conjugate.

[0046] Also described herein are methods for preventing the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a drug-ligand conjugate having a cleavable drug unit from the drug-ligand conjugate.

[0047] Also described herein are methods for preventing cancer, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0048] Also described herein are methods for preventing cancer, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0049] Also described herein are 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 Drug-Ligand Conjugate.

[0050] Also described herein are methods for preventing the proliferation of cells that express an autoimmune antibody, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0051] Also described herein are methods for preventing the proliferation of cells that express an autoimmune antibody, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0052] Also described herein are methods for preventing the proliferation of cells that express an autoimmune antibody, including administering to a patient in need thereof an effective amount of a drug-ligand conjugate having a cleavable drug unit from the drug-ligand conjugate.

[0053] Also described herein are methods for preventing an autoimmune disease, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0054] Also described herein are methods for preventing an autoimmune disease, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0055] Also described herein are methods for preventing an autoimmune disease, including administering to a patient in need thereof an effective amount of a drug-ligand conjugate having a cleavable drug unit from the drug-ligand conjugate.

[0056] Also described herein are methods for preventing an infectious disease, including administering to a patient in need thereof an effective amount of a drug-linker compound.

[0057] Also described herein are methods for preventing an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.

[0058] Also described herein are methods for preventing an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable drug unit from the Drug-Ligand Conjugate.

[0059] Also described herein is a drug compound that can be used as an intermediate for the synthesis of a drug-linker compound having a cleavable drug unit from a drug ligand conjugate.

[0060] Also described herein is a drug-linker compound that can be used as an intermediate for the synthesis of a drug-linker-ligand conjugate.

[0061] In one embodiment of the compounds of formula Ia ', Ab is not an antibody that binds to the ErbB receptor or that binds to one or more of the receptors (1) (35):

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(15) CD79b (IGb (associated with immunoglobulin beta), B29, Genbank accession number NM_000626);

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

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20Ra (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) Ephb2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763);

(26) BAFF-R (Genbank accession number NP_443177.1);

(27) CD22 (Genbank accession number NP-001762.1);

(28) CD79a (CD79A, CD79a, linked to immunoglobulin alpha, a B cell specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, causes transduction of a signal involved in B cell differentiation, Genbank accession number NP_001774.1);

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

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

(31) P2X5 (P2X purinergic receptor, ligand-gated ion channel 5, extracellular ATP-gated ion channel, may participate in synaptic conduction and neurogenesis, deficiency may contribute to pathophysiology of spontaneous detrusor instability, Genbank accession number NP_002552.2);

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

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein from the leucine rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, accession number Genbank NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, putative receptor for an immunoglobulin Fc domain that contains Ig-like and ITAM-like C2 domains, may play a role in B cell differentiation, Genbank accession number NP_443170.1); or (35) IRTA22 (superfamily of immunoglobulin receptors associated with translocation 2, putative immunoreceptor with a potential role in B cell development and lymphoma formation; gene deregulation by translocation occurs in certain B cell tumors, Genbank accession number NP_112571.1).

[0062] Also described herein are pharmaceutical compositions comprising effective amounts of a drug-link er-antibody conjugate and a pharmaceutically acceptable carrier or excipient.

[0063] Also described herein are compositions comprising effective amounts of a drug-antibody conjugate having a cleavable drug unit (moiety) from the antibody-conjugate conjugate and a pharmaceutically acceptable carrier or excipient.

[0064] Also described herein are methods for killing or inhibiting the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0065] Also described herein are methods for killing or inhibiting the proliferation of a cancer 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.

[0066] Also described herein are methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0067] Also described herein are methods for treating cancer including administering to a patient in need thereof an effective amount of a drug-antibody conjugate having a cleavable drug unit from the drug-antibody conjugate.

[0068] Also described herein are methods for killing or inhibiting 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.

[0069] Also described herein are methods for killing or inhibiting 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.

[0070] Also described herein are methods for treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a Drug-Linker Antibody Conjugate.

[0071] Also described herein are methods for treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a drug-antibody conjugate having a cleavable drug unit from the drug-antibody conjugate.

[0072] Also described herein are methods for treating an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Linker Antibody Conjugate.

[0073] Also described herein are 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.

[0074] Also described herein are methods for preventing the proliferation of a cancer cell or cancer cell, including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0075] Also described herein are methods for preventing the proliferation of a cancer 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.

[0076] Also described herein are methods for preventing cancer, including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0077] Also described herein are 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.

[0078] Also described herein are methods for preventing the proliferation of cells that express an autoimmune antibody, including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0079] Also described herein are methods for preventing the proliferation of cells that express 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.

[0080] Also described herein are methods for preventing an autoimmune disease, including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.

[0081] Also described herein are methods for preventing an autoimmune disease, including administering to a patient in need thereof an effective amount of a drug-antibody conjugate having a cleavable drug unit from the drug-antibody conjugate.

[0082] Also described herein are methods for preventing an infectious disease, including administering to a patient in need thereof an effective amount of a Drug-Linker Antibody Conjugate.

[0083] Also described herein are methods for preventing an infectious disease, including administering to a patient in need thereof an effective amount of a drug-antibody conjugate having a cleavable drug unit from the drug-antibody conjugate.

[0084] As described herein, a drug compound is provided that can be used as an intermediate for the synthesis of a drug-linker compound having a cleavable drug unit from an antibody-conjugate conjugate.

[0085] As described herein, a drug-linker compound is provided that can be used as an intermediate for the synthesis of a drug-linker-antibody conjugate.

[0086] In drug-linker-antibody conjugates (also referred to as antibody-drug conjugates) described herein [0087] Ab can be an antibody that binds to one or more of the antigens (1) (35):

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(15) CD79b (IGb (associated with immunoglobulin beta), B29, Genbank accession number NM_000626);

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

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20Ra (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) Ephb2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763);

(26) BAFF-R (Genbank accession number NP_443177.1);

(27) CD22 (Genbank accession number NP-001762.1);

(28) CD79a (CD79A, CD79a, linked to immunoglobulin alpha, a B cell specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, causes transduction of a signal involved in B cell differentiation, Genbank accession number NP_001774.1);

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

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

(31) P2X5 (P2X purinergic receptor, ligand-gated ion channel 5, extracellular ATP-gated ion channel, may participate in synaptic conduction and neurogenesis, deficiency may contribute to pathophysiology of spontaneous detrusor instability, Genbank accession number NP_002552.2);

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

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein from the leucine rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, accession number Genbank NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, putative receptor for an immunoglobulin Fc domain that contains Ig-like and ITAM-like C2 domains, may play a role in B cell differentiation, Genbank accession number NP_443170.1); or (35) IRTA22 (superfamily of immunoglobulin receptors associated with translocation 2, putative immunoreceptor with a potential role in B cell development and lymphoma formation; gene deregulation by translocation occurs in certain B cell tumors, Genbank accession number NP_112571.1).

[0088] In another aspect, the antibody from the antibody-drug conjugate (ADC) of the invention specifically binds to a receptor encoded by the ErbB2 gene.

[0089] In another aspect, the antibody from the antibody-drug conjugate is a humanized antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5 ).

[0090] Also described is a factory article comprising the antibody-drug conjugate compound of the invention; container; and a package insert or label indicating that the compound can be used to treat cancer characterized by ErbB2 receptor overexpression.

[0091] Also described herein is a method for treating cancer in a mammal in which the cancer is characterized by overexpression of the 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 .

[0092] In some cases, a significant amount of the drug moiety is not cleaved from the antibody until the antibody-drug conjugate compound enters the cell via an antibody-specific receptor on the cell surface of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug conjugate enters the cell.

[0093] In some cases, the bioavailability of an antibody-conjugate compound or intracellular metabolite of a compound in a mammal is improved compared to a drug compound containing the drug moiety of the antibody-drug conjugate, or compared to an analog of the compound not having the drug moiety.

[0094] The drug moiety may be intracellularly cleaved in a mammal from the compound antibody or intracellular metabolite of the compound.

[0095] In another aspect, the invention includes a pharmaceutical composition comprising effective amounts of an antibody-drug conjugate compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. The composition may additionally contain a therapeutically effective amount of a chemotherapeutic agent such as a tubulin-forming inhibitor, topoisomerase inhibitor and DNA binding agent.

[0096] Also described herein is a method for killing or inhibiting the proliferation of cancer cells or cancer cells, comprising treating cancer cells or cancer cells with an amount of an antibody-drug conjugate compound of the invention or a pharmaceutically acceptable salt or solvate thereof, effective to kill or inhibit cell proliferation cancer or cancer cells.

[0097] Also described herein is a method for inhibiting cell proliferation comprising exposing mammalian cells in cell culture medium to an antibody-drug conjugate compound of the invention, wherein the antibody-drug conjugate compound enters the cells and the drug is excised from the rest of the compound antibody-drug conjugate; thanks to which cell proliferation is inhibited.

[0098] Also described herein is a method for treating cancer comprising administering to a patient an antibody-drug conjugate compound formulation of the invention and a pharmaceutically acceptable diluent, carrier or excipient.

[0099] Also described herein is an assay for detecting cancer cells, including:

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

[0100] The invention may best be understood by reference to the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings, figures and diagrams. The following is descriptive, illustrative and exemplary and should not be construed as limiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0101]

Figure 1 shows an in vivo assay for the efficacy of a single dose of cAC10mcMMAF in subcutaneous Karpas-299 ALCL xenografts.

Figure 2 shows in vivo assay of single dose efficacy of cAC10mcMMAF in subcutaneous L540cy. In this study there were 4 mice in the untreated group and 10 in each of the treatment groups.

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

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

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

Figures 6a and 6b show in vitro activity of c1F6 antibody-drug conjugates against CD70 + renal cell carcinoma cell lines.

Figure 7 shows an in vitro cell proliferation assay with SK-BR-3 cells treated with antibody drug conjugates (ADC):

- • - Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF / Ab, -O- Trastuzumab-MC-MMAF,

4.1 MMAF / Ab and -Δ- Trastuzumab-MC-MMAF, 4.8 MMAF / Ab, measured in relative fluorescence units (RLU) versus ADC concentration in pg / ml. H = Trastuzumab, in which H is connected through cysteine ​​[cys].

Figure 8 shows an in vitro cell proliferation assay with ADC-treated BT-474 cells: - · - 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 -Δ- Trastuzumab-MCMMAF, 4.1 MMAF / Ab.

Figure 10 shows an in vitro cell proliferation assay with ADC-treated MDA-MB468 cells: - · - Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE / Ab, -O20

Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE / Ab and -Δ- Trastuzumab-MC-vc-PABMMAF, 3.7 MMAF / Ab.

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

Figure 12 shows a plasma clearance test after H-MC-vcMMAE administration to Cynomolgus monkeys at various doses: 0.5, 1.5, 2.5 and 3.0 mg / kg administered on day 1 and day 21. Total antibody concentrations and ADC were measured over time. (H = Trastuzumab).

Figure 13 shows the mean change in tumor volume over time in Nude MMTV-HER2 Fo5 mice with breast tumor allografts that were dosed on day 0: vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 pg / m²) and TrastuzumabMC-vc-PAB-MMAF (555 pg / m²). (H = Trastuzumab).

Figure 14 shows the mean change in tumor volume over time in nude Nude mice with MMTV-HER2 Fo5 with breast tumor allografts that were dosed on day 0: 10 mg / kg (660 pg / m²) Trastuzumab-MC-MMAE and 1250 pg / m² TrastuzumabMC-vc-PAB-MMAE.

Figure 15 shows the mean change in tumor volume over time in nude Nude mice with MMTV-HER2 Fo5 with breast tumor allografts that were dosed on Day 0 with vehicle and 650 pg / m² Trastuzumab-MC-MMAF.

Figure 16 shows the mean change in tumor volume over time in nude Nude mice with MMTV-HER2 Fo5 with breast tumor allografts that were dosed on Day 0 with vehicle and 350 pg / m² four Trastuzumab-MC-MMAF conjugates, with the MMAF / Trastuzumab (H) ratio being 2, 4, 5.9 and 6.

Figure 17 shows the mean lesion group, with error bars, in animal (rat) body weights (mean ± SD) after vehicle administration, Trastuzumab-MC-valcit-MMAF, Trastuzumab-MC (Me) -val-cit-PAB-MMAF, Trastuzumab-MC-MMAF and Trastuzumab-MC-val-cit-PAB-MMAF.

Figure 18 shows the group of 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-MC-vcMMAF, 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 Trastuzumab cysteine ​​for each conjugate.

Figure 19 shows the mean lesion group, with error bars, in the body weights of Sprague-Dawley rats (mean ± SD) after administration of Trastuzumab (H) -MC-MMAF at 2105, 3158, and 4210 pg / m². The MC linker is attached via cysteine

Trastuzumab, for each conjugate.

4. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

4.1 DEFINITIONS AND ABBREVIATIONS [0102] Unless otherwise stated, the following terms and phrases as described herein are intended to have the following meanings:

When the trade names as used herein are used, the applicant is by definition independently encompassing the formulation of a product with a registered trade name, a generic drug and the active pharmaceutical ingredients of a product with the registered trade name.

[0103] The term "antibody" as used herein is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, as long as they exhibit the desired biological activity. An antibody is a protein generated by the immune system that is able to recognize and bind to a specific antigen. Described with respect to its structure, the antibody typically has a Y-shaped protein, consisting of four amino acid chains, two heavy and two light. Each antibody has essentially two regions: a variable region and a constant region. The variable region, located at the ends of the Y arms, binds and interacts with the target antigen. This variable region includes a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region, located on the "tail" of 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 ). The target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on a number of antibodies. Each antibody that specifically binds to different epitopes has a different structure. Thus, one antigen may have more than one corresponding antibody.

[0104] The term "antibody" as used herein also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunologically binds to the antigen of the target object or part thereof, such targets include non-limiting method of cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD and IgA), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass of immunoglobulin molecules. Immunoglobulins can be from any species. In one aspect, however, the immunoglobulin is of human, mouse or rabbit origin. In another aspect, the antibodies are polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F (ab ') fragments, F (ab') 2 fragments, fragments produced by expression of the Fab library, antibodies anti-idiotype (anti-Id), CDRs, and epitope-binding fragments of any of the above that bind immunologically to cancer cell antigens, viral antigens, or microbial antigens.

[0105] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies forming the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being targeted to a single antigenic site. In addition, unlike polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized unpolluted by other antibodies. The "monoclonal" modifier indicates the nature of the antibody as derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of antibodies in any particular manner. For example, monoclonal antibodies for use in accordance with the invention may be obtained by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or can be obtained by recombinant DNA methods (see U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in, e.g., Clackson et al. (1991) Nature, 352: 624-628 and Marks et al. (1991) J. Mol. Biol., 222: 581-597.

[0106] Monoclonal antibodies as described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) are identical or homologous to the corresponding sequences in the antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (US Patent mp / 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 68516855).

[0107] Various methods have been used to produce monoclonal antibodies (MAbs). The hybridoma technique, which relates to a cloned cell line that produces a single type of antibody, uses cells from a variety of species, including mice (mouse), hamsters, rats and humans. Another way to obtain MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies produced by these techniques include, inter alia, chimeric and humanized antibodies. A chimeric antibody combines DNA coding regions from more than one type of species. For example, the chimeric antibody may use a mouse variable region and a human constant region. The humanized antibody is mainly of human origin, even if it contains non-human fragments. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike the chimeric antibody, the variable region may be partially derived from man. Non-human synthetic parts of the humanized antibody often come from CDRs in mouse antibodies. In any case, these regions are crucial in enabling the antibody to recognize and bind to specific antigen.

[0108] As noted, murine antibodies can be used. While useful for diagnosis and short-term therapies, mouse antibodies cannot be administered to humans long-term without the increasing risk of a harmful immune response. This response, called human anti-mouse antibodies (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. The HAMA response can cause toxic shock or even death.

[0109] Chimeric and humanized antibodies reduce the likelihood of HAMA responses by minimizing non-human antibody administered parts. In addition, chimeric and humanized antibodies have the added benefit of activating secondary human immune responses such as antibody dependent cellular cytotoxicity.

[0110] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising an antigen binding or variable region [intact antibody]. Examples of antibody fragments include Fab, Fab ', F (ab') fragments₂ and Fv; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0111] An "intact" antibody is one that contains an antigen binding variable region as well as the light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Constant domains can be native constant domain sequences (e.g., human native constant domain sequence) or a variant of their amino acid sequence.

[0112] An intact antibody may have one or more "effector functions" that refer to those biological activities attributed to the Fc region (native sequence Fc region or variant amino acid sequence Fc region) of the antibody. Exemplary antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; reducing the level of cell surface receptor expression (e.g. B cell receptor; BCR), etc.

[0113] 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 main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), e.g. IgG1, 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. Structure subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

[0114] The expressions "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al., Proc. Natl. Acad. Sci. USA, 82: 6497-6501 (1985) and Yamamoto et al., (1986) Nature, 319: 230-234 (Genbank accession number X03363). The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to the gene encoding rat p185neu. Preferred ErbB2 means native human ErbB2 sequence.

[0115] Antibodies to ErbB receptors are commercially available from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.

[0116] By "ErbB ligand" is meant a polypeptide that binds to and / or activates the ErbB receptor. The ErbB ligand may be a native sequence human ErbB ligand, such as epidermal growth factor (EGF) (Savage et al. (1972) J. Biol. Chem., 247: 7612-7621); transforming growth factor alpha (TGF-α) (Marquardt et al. (1984) Science 223: 10791082); Amphiregulin, also known as schwanoma or autocrine keratinocyte growth factor (Shoyab et al. (1989) Science 243: 1074-1076; Kimura et al., Nature, 348: 257260 (1990); and Cook et al., Mol. Cell. Biol., 11: 2547-2557 (1991)); betacellulin (Shing et al., Science, 259: 1604-1607 (1993); and Sasada et al., Biochem. Biophys. Res. Commun., 190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science, 251: 936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem., 270: 7495-7500 (1995); and Komurasaki et al., Oncogene, 15: 2841-2848 (1997)); heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al., Nature, 387: 512-516 (1997)); neuregulin-3 (NRG3) (Zhang et al., Proc. Natl. Acad. Sci., 94: 9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al., Oncogene, 18: 2681-89 (1999)) or cripto (CR-1) (Kannan et al., J. Biol. Chem., 272 (6): 3330-3335 (1997)). ErbB ligands that bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands that bind ErbB3 include hegulins. ErbB ligands capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulin. The ErbB ligand may also be a synthetic ErbB ligand. The synthetic ligand may be specific for a particular ErbB receptor, or may recognize specific ErbB receptor complexes. An example of a synthetic ligand is the synthetic heregulin / chimera EGF biregulin (see for example Jones et al., (1999) FEBS Letters, 447: 227-231, which is incorporated by reference).

[0117] "Heregulin" (HRG) refers to a polypeptide encoded by the heregulin gene product as disclosed in US Patent No. 5641869 or Marchionni et al., Nature, 362: 312318 (1993). Examples of heregulins include heregulin-α, heregulin-31, heregulin-32 and heregulin-33 (Holmes et al., Science, 256: 1205-1210 (1992); and US Patent No. 5641869); neu differentiation factor (NDF) (Peles et al., Cell 69: 205-216 (1992)); activity inducing acetylcholine receptor (ARIA) (Falls et al. (1993) Cell 72: 801-815); glial growth factors (GGFs) (Marchionni et al., Nature, 362: 312-318 (1993)); a factor derived from sensory and motor neurons (SMDF) (Ho et al., J. Biol. Chem., 270: 1452314532 (1995)); γ-heregulin (Schaefer et al., Oncogene, 15: 1385-1394 (1997)). The term includes biologically active fragments and / or variants of the amino acid sequence of a native HRG polypeptide sequence, such as an EGF-like domain of a fragment thereof (e.g., HRGp 1177-244).

[0118] "ErbB hetero-oligomer" means a non-covalently linked oligomer containing at least two different ErbB receptors. "ErbB dimer" means a non-covalently linked oligomer that contains two different ErbB receptors. Such complexes can form when a cell expressing two or more ErbB receptors is exposed to an ErbB ligand. ErbB oligomers, such as ErbB dimers, can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described e.g. in Sliwkowski et al., J. Biol. Chem., 269 (20): 14661-14665 (1994). Examples of such ErbB hetero-oligomers include EGFR-ErbB2 complexes (also referred to as HER1 / HER2), ErbB2-ErbB3 (HER2 / HER3) and ErbB3-ErbB4 (HER3 / HER4). In addition, an ErbB hetero-oligomer may contain two or more ErbB2 receptors linked to different ErbB receptors, such as ErbB3, ErbB4 or EGFR (ErbB1). Other proteins, such as the cytokine receptor subunit (e.g., gp130) may be contained in the hetero-oligomer.

[0119] A "native sequence" polypeptide means one that has the same amino acid sequence as the polypeptide, eg, a tumor associated antigen receptor that is naturally derived. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, the native sequence polypeptide may have the amino acid sequence of a naturally occurring human polypeptide, mouse polypeptide or polypeptide from any other mammalian species.

[0120] The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Typically, amino acid sequence variants will have at least about 70% homology with at least one native ligand receptor binding domain, or with at least one native receptor ligand binding domain, such as tumor associated antigen, and preferably, they will be at least about 80 %, more preferably, at least about 90% homologous to such a receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence.

[0121] "Sequence identity" is defined as the percentage of residues in an amino acid sequence variant that are identical after sequence alignment and introduction of gaps, if necessary, to achieve maximum percent sequence identity. Methods and computer programs for matching are well known in the art. One such computer program is "Align 2," developed by Genentech, Inc., which was filed with user documentation at the United States Copyright Office, Washington, DC 20559, December 10, 1991.

[0122] "Antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cellular reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils and macrophages) recognize bound antibody on target cell and consequently cause lysis of the target cell. Primary ADCC-mediating cells, NK cells, express only Fc? RIII, while monocytes express Fc? RI, Fc? RII and Fc? RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, (1991) Annu. Rev. Immunol, 9: 457-92. To assess the ADCC activity of the subject molecule, an in vitro ADCC assay can be performed, such as described in US Patent No. 5,500,362 or US 5,823,377. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the subject molecule can be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al. Natl. Acad. Sci. USA, 95: 652-656 (1998).

[0123] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. Preferred FcR means native sequence human FcR. In addition, preferred FcR is one that binds to IgG (gamma receptor) and includes receptors for the FcyRI, Fc? RII and Fc? RIII subclasses, including allelic variants and alternative spliced ​​forms of these receptors. Fc? RII receptors include Fc? RII ("receptor activating") and Fc? RIIB ("receptor inhibiting"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. Activating FcyRIIA receptor contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor Fc? RIIB contains an inhibitory motif based on tyrosine immunoreceptor (ITIM) in its cytoplasmic domain. (See review of M. in Daeron, Annu. Rev. Immunol., 15: 203234 (1997)). FcRs are discussed 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 for future identification, are encompassed by the term "FcR" as described herein. The term also includes the neonatal receptor, FcRn, which is responsible for transmitting maternal IgGs to the fetus. (Guyer et al., J. Immunol., 117: 587 (1976) and Kim et al., J. Immunol., 24: 249 (1994)).

[0124] "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 binding the first complement component (C1q) to a molecule (e.g., antibody) complexed with the appropriate antigen. To assess complement activation, a CDC assay can be performed, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

[0125] The term "variable" refers to the fact that some parts of the variable domains differ significantly in sequence between antibodies, and are used in relation to the binding and specificity of each specific antibody to its particular antigen. However, the variability is not evenly distributed between antibody variable domains. It is focused on three segments called hypervariable regions, both in the light and heavy chain variable domains. The more conservative parts of the variable domains are called framework regions (FRs). Each of the variable domains of native heavy and light chains contains four FR regions adopting largely the β sheet configuration, connected by three hypervariable regions that form connecting loops, and in some cases forming part of the β sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, with hypervariable regions from another chain, contribute to the formation of an antigen binding site for antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition. Public Health Service, National Institutes of Health, Bethesda, MD). Constant domains are not directly involved in binding the antibody to the antigen, but display various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity (ADCC).

[0126] The term "hypervariable region" as used herein refers to antibody amino acid residues that are responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Supra) and / or those residues from the "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain heavy chain; Chothia and Lesk (1987) J. Mol. Biol., 196: 901-917). "Framework" or "FR" residues mean those variable domain residues other than hypervariable region residues as defined herein.

[0127] Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and the remaining "Fc" fragment, whose name reflects its ability to easily crystallize. Pepsin treatment results in an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-attaching the antigen.

[0128] "Fv" is the minimal antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen binding site on the surface of the VHV dimer_L. In total, six hypervariable regions confer an antibody's ability to specifically bind antigen. However, even a single variable domain (or half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, although with lower affinity than the full binding site.

[0129] The Fab fragment also contains the light chain constant domain and the first heavy chain constant domain (CH1). Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is an assay as described for Fab 'in which the cysteine ​​constant domain residues have at least one free thiol group. F (ab ') fragments₂ the antibodies were originally produced as pairs of Fab 'fragments that had hinge cysteines between them. Other chemical combinations of antibody fragments are also known.

[0130] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0131] "Single-chain Fv" or "sFv" antibody fragments contain V antibody domains_H and V_L, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V domains_H and V_Lwhich allows sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0132] The term "diabody" refers to small antibody fragments, with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (V_L) in the same polypeptide chain (V_h-V_L). Using a linker that is too short to allow pairing between two domains on the same chain, pairing domains with complementary domains on another chain is imposed and creates two antigen binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

[0133] "Humanized" forms of non-human antibodies (eg, rodents) are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the hypervariable region residues of the recipient are replaced with hypervariable region residues from non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity , affinity and fitness. In some instances, human immunoglobulin framework (FR) residues are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications have been made to further refine antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those having a human immunoglobulin sequence. The humanized antibody will also optionally contain at least a portion of an immunoglobulin (Fc) constant region, typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature, 321: 522-525; Riechmann et al. (1988) Nature 332: 323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2: 593-596.

[0134] Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN® patent) as described in the patent U.S. Patent No. 5821337 is explicitly incorporated herein by reference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies as described below.

[0135] An "isolated" antibody is one which has been identified and extracted and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that would typically interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of the antibody, as found by the Lowry method, and most preferably to more than 99% by weight, (2) sufficiently to obtain at least 15 N-terminal residues or internal amino acid sequence, by using a spinning cup protein sequencer or (3) for homogeneity, by SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue or, preferably, silver staining. An isolated antibody includes the antibody in situ in recombinant cells, as at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody will be obtained by at least one purification step.

[0136] An antibody "which binds" to an object antigen means one that is capable of binding the antigen with sufficient affinity, such that the antibody is useful in targeting a cell that expresses the antigen.

[0137] An antibody that "induces apoptosis" means that which induces programmed cell death as determined by annexin V binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum enlargement, cell fragmentation and / or membrane vesicle formation (called apoptotic bodies) ). Cell means a cancer cell e.g. breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder cell. Various methods are available for assessing cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by stepped DNA bands; and nuclear / chromatin condensation along with DNA fragmentation can be assessed by any increase in the number of hypodiploid cells.

[0138] "Disorder" means any condition that would benefit from a treatment according to the invention. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose the mammal to the disorder. Non-limiting examples of disorders to be treated as described herein include benign and malignant tumors; leukemia and malignant neoplasms of the lymphatic system, in particular cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney of the colon, thyroid, pancreas, prostate or bladder; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocell disorders; and inflammatory, angiogenic and immunological disorders.

[0139] 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, a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e. slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e. slow to some extent and preferably stop) tumor metastasis; inhibit the growth of cancer to some extent; and / or relieve to some extent one or more of the symptoms associated with cancer. As to the extent to which the drug may prevent the growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. In the treatment of cancer, efficacy can be measured, e.g., by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0140] The term "significant amount" refers to the majority, i.e.&gt; 50% of the population, set or sample.

[0141] The term "intracellular metabolite" refers to a compound resulting from a metabolic process or reaction within a cell against an antibody drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process, such as proteolytic cleavage of the ADC peptide linker, 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 that has experienced intracellular cell cleavage, diffusion, entry or transport into the cell.

[0142] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or intracellular reaction against a drug-ligand conjugate, a druglinker-ligand conjugate, an antibody drug conjugate (ADC) or the like through which covalent binding, e.g. linker, is broken. between the drug (D) moiety and the antibody (Ab) resulting in the formation of a free drug disconnected from the antibody within the cell. The cut moieties of drug-ligand conjugate, drug-linker-ligand conjugate or ADC are therefore intracellular metabolites.

[0143] 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 concept that indicates the measurement of both the time (speed) and total amount (range) of a drug that achieves general circulation from the administered dosage form.

[0144] The term "cytotoxic activity" refers to cell killing, cytostatic effect or anti-proliferation of an antibody-drug conjugate compound or intracellular metabolite of an antibody-drug conjugate compound. Cytotoxic activity can be expressed as an IC50 value, which is the concentration (molar or weight) per unit volume at which half the cells survive.

[0145] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. "Cancer" contains one or more cancer cells. Examples of cancer include, but are not limited to, malignant tumor, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphatic malignancies. More specifically, examples of such cancers include squamous cell carcinoma (e.g. squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, stomach cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer, rectal cancer, penile cancer as well as head and neck cancer.

[0146] "ErbB2 expressing cancer" means one that produces sufficient levels of ErbB2 on the surface of its cells, such that an anti-ErbB2 antibody can bind to it and have a therapeutic effect on cancer.

[0147] Cancer "characterized by excessive activation" of the 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-cancer cells of the same type of tissue. Such excessive activation may be due to overexpression of the ErbB2 receptor and / or greater than normal levels of the ErbB2 ligand available for activation of ErbB2 receptors in cancer cells. Such excessive activation can cause and / or be caused by a malignant state of the cancer cell. In some embodiments, the cancer will undergo diagnostic or prognostic assays to determine if there is amplification and / or overexpression of the ErbB2 receptor that results in such excessive activation of the ErbB2 receptor. Alternatively, or in addition, the cancer may be subjected to diagnostic or prognostic assays to determine if the ErbB2 ligand is amplified and / or overexpressed in the cancer that results in such excessive receptor activation. In a subset of such cancers, excessive receptor activation may result from an autocrine stimulatory pathway.

[0148] Cancer that "overexpresses" the ErbB2 receptor is one that has significantly higher levels of the ErbB2 receptor on its cell surface compared to a non-cancerous cell from the same type of tissue. Such overexpression may be caused by gene amplification or by increased transcription or translation. ErbB2 receptor overexpression can be determined in a diagnostic or prognostic assay by assessing elevated levels of ErbB2 protein present on the cell surface (e.g., by immunohistochemical assay; IHC). Alternatively, or in addition, levels of ErbB2 encoding nucleic acid in the cell can be measured, e.g., by fluorescent in situ hybridization (FISH; see WO 98/45479), Southern hybridization, or polymerase chain reaction (PCR) techniques, such as quantitative real-time PCR (RTPCR). ErbB2 ligand overexpression can be determined diagnostically by assessing the patient's ligand (or nucleic acid encoding) levels, e.g., in tumor biopsy, or through various diagnostic assays such as IHC, FISH, Southern hybridization, PCR or in vivo assays described above. Receptor overexpression can also be tested

ErbB2 by measuring "shedding" antigen (e.g., extracellular domain of ErbB2) in a biological fluid such as serum (see e.g. US Patent No. 4933294; ​​WO 91/05264; US Patent No. 5401638; and Sias et al., (1990) J. Immunol. Methods, 132: 7380). In addition to the above assays, those skilled in the art have access to various other assays in vivo. For example, you can expose cells within a patient's body to an antibody that is optionally labeled with a detectable tag, e.g. isotope, and binding of the antibody to the patient's cells can be assessed, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.

[0149] HER2 overexpressing tumors are assessed by immunohistochemical scoring, corresponding to the number of HER2 molecules expressed per cell, and this can be determined biochemically: 0 = 0-10,000 copies / cell, 1+ = at least about 200,000 copies / cell, 2+ = at least about 500,000 copies / cell, 3+ = about 1-2 x 10⁶ copies / cell. HER2 overexpression at 3+, which leads to ligand independent activation of tyrosine kinase (Hudziak et al., (1987) Proc. Natl. Acad. Sci. USA, 84: 7159-7163), occurs in approximately 30% of cancers and in these patients, relapse-free survival and overall survival are reduced (Slamon et al., (1989) Science, 244: 707-712; Slamon et al., (1987) Science, 235: 177-182).

[0150] Conversely, cancer that is not "characterized by overexpression of the ErbB2 receptor" means one that does not express higher than normal ErbB2 receptor levels compared to a non-cancerous cell from the same type of tissue in a diagnostic assay.

[0151] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The concept is intended to include radioactive isotopes (e.g.²¹¹ at, ¹³¹AND, ¹²⁵AND, ⁹⁰Y ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³sm, ²¹²bi, ³²P ⁶⁰C, and radioactive isotopes Lu), chemotherapeutic agents and toxins, such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including their synthetic analogues and derivatives. In one aspect, the concept is not intended to include radioactive isotopes.

[0152] "Chemotherapeutic agent" means a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocarbone, meturedopa, and uredopa; ethyleneimine and methylamelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; TLK 286 (TELCYTA ™); acetogenins (in particular bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); Beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic topotecan analogs (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 (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornafazine, cholophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine and ranimnustine; bisphosphonates such as clodronate; antibiotics such as enediin antibiotics (e.g. calicheamycin, in particular calicheamycin gamma1l and calicheamycin omega1l (see e.g. Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX 100, idarubicin, KRN5500, menogaril, dynemycin, including dynemycin A, and esperamycin, chrominoferrin and bound chromoprotein enediine chromoprotein antibiotics, aclacinomizine, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholine-doxorubicin, cyanomorpholine-doxorubicin, 2-pyrrole-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), ezomomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrine, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; folic acid analogues such as denopterin, pteropterin and trimetrexate, purine analogues such as fludarabine, 6mercaptopurine, thiaprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenocortical hormone antagonists such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid (leucovorin); aceglatone; anti-folate anti-cancer agents such as ALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, anti-metabolites such as 5-fluorouracil (5- FU) and its prodrugs such as UFT, S1 and capecitabine and thymidyl glycine synthase inhibitors ribonucleotide formyltransferase such as raltitrexed (TOMUDEX^RM, TDX) pyrimidine dehydrogenase inhibitors such as eniluracil; aldophosphamide glycoside, aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptin acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins, mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxine; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (in particular T-2 toxin, veracurin A, roridine A and anguidine); urethane; vindesine (ELDISINE®, Fildesin®), dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;

arabinoside ("Ara-C"), cyclophosphamide; thiotepa; taxoids and taxanes, e.g. TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ™ Cremophor-free albumin nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; 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®); nowantron, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, topoisomerase inhibitor RFS 2000; difluoromethyl organnitine (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, abbreviation for combination treatment with cyclophosphamide, doxorubicin, vincristine and prednisolone, or FOLFOX , abbreviation for oxaliplatin (ELOXATIN ™) treatment regimen in combination with 5-FU and leucovorin.

[0153] Also included in this definition are antihormonal agents that act to regulate or inhibit the hormone's effect on cancer, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX ® tamoxifen), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxyphene, keoxyfen, LY117018, onapristone and FARESTON® toremifene; aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestan, fadrozole, RIVISOR® vorozole, FEMARA letrozole and ARIMIDEX® anastrozole, and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue) antisense oligonucleotides, in particular those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and growth factor receptor epidermis (EGF-R); vaccines such as those used in gene therapy, for example the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine and the VAPID® vaccine; PROLEUKIN® rIL2; LURTOTECAN® topoisomerase 1 inhibitor, ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0154] As used herein, the term "EGFR targeted drug" refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that 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. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®) and human 225 (H225) with changed shape (see WO 96/40210, Imclone Systems Inc.); antibodies that bind type II EGFR mutant (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5891996; and human antibodies that bind EGFR, such as ABX-EGF (see WO 98/50433, Abgenix). An anti-EGFR antibody can be conjugated to a cytotoxic agent, hence an immunoconjugate is generated (see e.g. EP 659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSA ™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA ™; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen).

[0155] "Tyrosine kinase inhibitor" means a molecule that inhibits, to some extent, tyrosine kinase activity against a tyrosine kinase, such as an ErbB receptor. Examples of such inhibitors include EGFR targeted drugs noted in the previous paragraph, as well as quinazolines such as PD 1530355,4- (3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGPol 62 , 4- (phenylamino) -7H-pyrrolo [2,3-d] pyrimidine, curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino) phthalimide), tyrifostine containing nitrotiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g. those that bind to ErbB-encoding nucleic acid); quinoxalines (US Patent No. 5,804,396); tryfostine (US Patent No. 5,804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-ErbB inhibitors such as CI1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: US Patent No. 5,804396; 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).

[0156] An "anti-angiogenic agent" refers to a compound that blocks, or interferes with, to some extent, the formation of blood vessels. An anti-angiogenic factor may be, e.g., a small molecule or antibody that binds to a growth factor or growth factor receptor that is involved in stimulating angiogenesis. In one embodiment, the anti-angiogenic factor is an antibody that binds to vascular endothelial growth factor (VEGF).

[0157] The term "cytokine" is a generic term for proteins released by one population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; a substance that inhibits the functions of Muller's ducts; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β;

insulin-like growth factors -I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -α, -β, and -γ; growth-promoting factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native cytokine sequences.

[0158] The term "prodrug" as used in the 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 undergoing enzymatic or hydrolytic activation or being transformed into a more active parent form . See. e.g. Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, p. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Humana Press (1985). Prodrugs of the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted / containing prodrugs phenoxyacetamide, or optionally substituted / phenylacetamide prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to a more active cytotoxic free drug. Examples of cytotoxic drugs that can be converted into a prodrug form for use in the invention include, but are not limited to, those chemotherapeutic agents described above.

[0159] "Liposome" means a small vesicle composed of different types of lipids, phospholipids and / or surfactant that is useful for drug delivery (such as, inter alia, anti-CD30, CD40, CD70 or Lewis Y antibodies and, optionally , a chemotherapeutic agent) to a mammal. The liposome components are commonly arranged in a bilayer system similar to the lipid system of biological membranes. The term 'package leaflet' refers to instructions normally found in commercial packaging of therapeutic products that contain information on the indications, use, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic products.

[0160] An "isolated" nucleic acid molecule means a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is customarily associated in a natural source nucleic acid antibody. An isolated nucleic acid molecule means a different form or environment from that found in nature. Isolated nucleic acid molecules are therefore distinguished from the nucleic acid molecule found in natural cells. An isolated nucleic acid molecule, however, includes a nucleic acid molecule contained in cells that typically express the antibody when, for example, the nucleic acid molecule is at a chromosomal location different from that of natural cells.

[0161] The expression "control sequences" refers to the DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotic organisms, for example, include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known for using promoters, polyadenylation signals and enhancers.

[0162] Nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, DNA encoding a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. In general, "operably linked" means that the DNA sequences linked together are contiguous and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to be direct neighbors in the sequence. Binding is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.

[0163] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such assays include progenes. Hence, the terms "transformants" and "transformed cells" include primary subject cells and cultures derived therefrom, regardless of the number of passages. It is also understood that all offspring need not be exactly identical in DNA content due to intentional or unintentional mutations. Mutated progeny that have the same biological function or activity as those sought for screening primary transformed cells are included. Where clear demarcations are intended, this will be clear from the context.

[0164] An "autoimmune disease" as used herein is a disease or disorder arising from and directed against the individual's own tissues or a co-segregation or manifestation thereof, or a condition resulting 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 autoimmune chronic urticaria, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and scleroderma, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), and IBD with colregation 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 collagen colitis, glomerulonephritis (GN) such as membrane GN, idiopathic membrane GN, membrane proliferative GN (MPGN), including type I and type II, and rapidly progressing GN, allergic conditions, eczema, asthma, conditions associated with T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such like cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), adolescent diabetes, multiple sclerosis (MS) such as optic spinal sm allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity regulated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, including Wegener's granulomatosis, agranulocytosis, vasculitis (including large vasculitis (including rheumatic poliomygia and giant cell) arterial (Takayasu)), inflammation of the medium vessels (including Kawasaki disease and nodular arteritis), CNS vasculitis and ANCA-associated vasculitis such as Churg-Strauss vasculitis or syndrome (CSS)), aplastic anemia, Coombs positive anemia, Blackfan-Diamond anemia, immunological hemolytic anemia, including autoimmune hemolytic anemia (AIHA) ), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, leukocyte migration diseases, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, diseases regulated by the antigen-antibody complex, glomerular basement membrane disease with antibodies directed against its components, anti-phospholipid antibody syndrome, allergic neuritis, disease Bechet, Castleman's syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Reynaud's syndrome, Sjorgen's, Stevens-Johnson's syndrome, solid organ transplant rejection (including pre-treatment for a panel of reactive high titers, IgA deposition in tissues, and rejection after kidney transplantation, liver transplantation, intestinal transplantation, heart transplantation, etc.), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (including ordinary, deciduous, and pemphigus pemphigoid), autoimmune poliendocrinopathies, Reiter's disease, generalized stiffness syndrome, nephritis with immune complexes, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thrombocytopenia (developing e.g. in patients with myocarditis), including autoimmune thrombocytopenia, autoimmune testicular and ovarian disease, including autoimmune testicular and ovarian inflammation, primary hypothyroidism;

autoimmune endocrine diseases, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves's disease, autoimmune multi-glandular syndromes (or multi-glandular endocrinopathy syndromes), also referred to as type I diabetes insulin dependent diabetes mellitus (IDDM), including childhood IDDM, and Sheehan's syndrome; autoimmune hepatitis, interstitial lymphatic pneumonia (HIV), bronchiolitis obliterans (not after transplant) vs NSIP, Guillain-Barry syndrome, Berger's disease (IgA nephropathy), primary biliary cirrhosis, celiac disease (gluten enteropathy), treatment-resistant celiac disease with co-segregation of herpetic dermatitis, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou-Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, myoclonus and opsoclonal syndrome (OMS), polio-cartilaginitis such as treatment-resistant multi-cartilaginitis, alveolar proteinosis, amyloidosis, giant cell hepatitis , scleritis, monoclonal gammopathy of unspecified / unknown meaning (MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis and CNS channelopathies; autism, inflammatory myopathy, and focal segmental gluing glomerulonephritis (FSGS).

[0165] "Alkyl" means hydrogen carbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH (CH3) 2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH (CH3) 2), 2-butyl (s-Bu, s -butyl, -CH (CH3) CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH3) 3), 1pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (- CH (CH3) CH2CH2CH3), 3-pentyl (CH (CH2CH3) 2), 2-methyl-2-butyl (-C (CH3) 2CH2CH3), 3-methyl-2-butyl (CH (CH3) CH (CH3)₂), 3-methyl-1-butyl (-CH₂CH₂CH (CH3)₂), 2-methyl-1-butyl (CH2CH (CH3) CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (CH (CH3) CH2CH2CH2CH3), 3-hexyl (-CH (CH2CH3) (CH2CH2CH3)), 2-methyl-2-pentyl (C (CH3) 2CH2CH2CH3), 3-methyl-2-pentyl (-CH (CH3) CH (CH3) CH2CH3), 4-methyl-2-pentyl (CH (CH3) CH2CH (CH3 ) 2), 3-methyl-3-pentyl (-C (CH3) (CH2CH3) 2), 2-methyl-3-pentyl (CH (CH2CH3) CH (CH3) 2), 2,3-dimethyl-2- butyl (-C (CH3) 2CH (CH3) 2), 3,3-dimethyl-2-butyl (CH (CH3) C (CH3) 3.

[0166] "Alkenyl" means C₂-C₁₈ a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturation site, i.e. carbon-carbon, a double bond sp². Examples include, but are not limited to: ethylene or vinyl (-CH = CH2), allyl (-CH2CH = CH2), cyclopentenyl (-C5H7) and 5-hexenyl (-CH2 CH2CH2CH2CH = CH2).

[0167] "Alkynyl" means C₂-C₁₈ a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturation site, i.e.

carbon-carbon, triple bond sp. Examples include, but are not limited to: acetylene (-C = CH) and propargyl (-CH₂C = CH).

[0168] "Alkylene" refers to a saturated, branched or straight chain, or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two centers of monovalent radicals obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH₂-) 1,2-ethyl (-CH₂CH₂-), 1,3-propyl (CH₂CH₂CH₂-), 1,4-butyl (-CH₂CH₂CH₂CH₂-) and similar.

[0169] "Alkenylene" refers to an unsaturated, branched or straight chain, or cyclic hydrocarbon radical with 2-18 carbon atoms, and having two centers of monovalent radicals obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH = CH-).

[0170] "Alkynylene" refers to an unsaturated, branched or straight chain, or cyclic hydrocarbon radical with 2-18 carbon atoms, and having two centers of monovalent radicals obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene (-C = C-), propargyl (-CH2C = C-) and 4-pentynyl (-CH2CH2CH2C = CH-).

[0171] "Aryl" means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms obtained by removing one hydrogen atom from a single carbon atom of the parent aromatic ring system. Some aryl groups are shown in exemplary structures as "Ar". Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl and the like.

[0172] "Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom or sp³ is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethene-1-yl, naphthylmethyl, 2-naphthylletan-1-yl, 2-naphthylethene-1yl, naphthobenzyl, 2-naphthophenylethane-1 -yl and similar. An arylalkyl group contains 6 to 20 carbon atoms, e.g., an alkyl moiety, including an alkanyl, alkenyl or alkynyl group of an arylalkyl group having 1 to 6 carbon atoms, and the aryl moiety has 5 to 14 carbon atoms.

[0173] "Heteroarylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom or sp³ it is replaced with a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl and the like. The heteroarylalkyl group contains 6 to 20 carbon atoms, e.g., an alkyl group, including an alkanyl, alkenyl or alkynyl group, of the heteroarylalkyl group have 1 to 6 carbon atoms, and the heteroaryl group has 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from O, P and S. The heteroaryl moiety of the heteroarylalkyl group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) , for example: the bicyclo system [4.5], [5.5], [5.6] or [6.6].

[0174] "Substituted alkyl", "substituted aryl" and "substituted arylalkyl" mean alkyl, aryl and arylalkyl, respectively, in which one or more hydrogen atoms, each independently, has been replaced with a substituent. Typical substituents include, but are not limited to: -X, -R, -O ', -OR, -SR, -S^-, -NR2, -NR3, = NR, -CXs, -CN, -OCN, -SCN, N = C = O, -NCS, -NO, -NO2, = N2, -N3, NC (= O) R, -C (= O) R, -C (= O) NR2, -SO3 ', -SO3H, S (= O) 2R, -OS (= O) 2OR, -S (= O) 2NR, -S (= O) R, -OP (= O) (OR) 2, -P (= O) (OR) 2, -PO'3, -PO3H2, C (= O) R, -C (= O) X, - C (= S) R, -CO2R, -CO2^-, -C (= S) OR, -C (= O) SR, -C (= S) SR, -C (= O) NR2, C (= S) NR2, -C (= NR) NR2, where each X is independently halogen: F, Cl, Br or I; and each R is independently -H, C₂-C₁₈ alkyl, C.₆-C₂₀ aryl, C.₃-C₁₄ heterocycle, protecting group or drug progroup. Alkylene, alkenylene and alkynylene groups as described above can also be similarly substituted.

[0175] "Heteroaryl" and "heterocycle" refer to a ring system in which one or more atoms in the ring is a heteroatom, e.g. nitrogen, oxygen and sulfur. The heterocycle radical contains 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. A heterocycle can be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: the bicyclo system [4.5], [5.5], [5.6] or [6.6].

[0176] Heterocycles are described in Paquette, Leo A .; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley &amp; Sons, New York, from 1950 until now), in particular volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82: 5566.

[0177] Examples of heterocycles include, by way of example and not limitation: pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, tetrahydrothiophenyl with the sulfur oxidant, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, indochilinylthinylnaphthalenyl -piperidinyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, thiantrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiol isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1Hindazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazinylphenyl phenanthrolinyl, phenynyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxaziazolyl, benzotriazinyl, benzotriazinyl, benzotriazinyl

[0178] By way of example and not limitation, carbon-related heterocycles are bonded to the pyridine 2, 3, 4, 5 or 6 position, pyridazine 3, 4, 5 or 6 position, pyrimidine 2, 4, 5 or 6 position 2, 3, 5 or 6 pyrazine, positions 2, 3, 4 or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, positions 2, 4 or 5 of oxazole, imidazole or thiazole, positions 3, 4 or 5 of isoxazole, pyrazole or isothiazole, positions 2 or 3 of aziridine, positions 2, 3 or 4 of azetidine, positions 2, 3, 4, 5, 6, 7 or 8 of quinoline or positions 1, 3, 4, 5, 6, 7 or 8 of isoquinoline. Even more typically, carbon-bound heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4 -pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

[0179] By way of example and not limitation, nitrogen-bound heterocycles are bonded to the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline , 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of isoindole or isoindoline, position 4 of morpholine and position 9 of carbazole or β-carboline. Even more typically, nitrogen-bound heterocycles include 1-aziridil, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0180] "Carbocycle" means a saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, even more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as a bicyclo [4.5], [5.5], [5.6] or [6.6] system, or 9 or 10 ring atoms arranged as a bicyclo [5.6] or [6.6] system . Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl and cyclooctyl.

[0181] "Linker", "linker unit", or "combination" means a chemical moiety containing a covalent bond or chain of atoms that covalently link the antibody to the drug moiety. In various embodiments, the linker is referred to as LU. Linkers include divalent radicals such as alkyl diyl, aryl diyl, heteroaryldiyl, moieties such as: - (CR2) nO (CR2) n-, repeating alkyloxy units (e.g. polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino, Jeffamine ™); and diacid esters and amides, including succinate, succinamide, diglycolate, malonate and caproamide.

[0182] The term "chiral" refers to molecules that have the property of not superimposing their mirror image, while the term "achiral" refers to molecules that are overlapping their mirror image.

[0183] The term "stereoisomers" refers to compounds that have identical chemical composition but differ in relation to the arrangement of atoms or groups in space.

[0184] "Diastereomer" refers to a stereoisomer with two or more chirality centers, and whose molecules are not mirror images of each other. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties and reactivity. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography.

[0185] "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0186] Stereochemical definitions and conventions used as described herein generally follow

SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley &amp; Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e. they have the ability to rotate the plane of polarized light. When describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule around its chiral center (s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of the plane of polarized light by a compound, where (-) or l means that the compound is left-handed. The relationship to the prefix (+) or d is clockwise. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate that may occur when no stereoselectivity or stereospecificity has occurred in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric forms, devoid of optical activity.

[0187] Examples of the "patient" include, but are not limited to, human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, fowl and poultry. In an exemplary embodiment, the patient is human.

[0188] "Aryl" refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. The carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or substituted with one or more groups, including, but not limited to, -C1-C8 alkyl, O- (C1-C8 alkyl), -aryl, -C (O) R ' , -OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', C (O) N (R') 2 -NHC (O) R ', -S (O) 2R ', - S (O) R', -OH, -halogen, -N3, -NH2, -NH (R '), -N (R') 2 and CN; wherein each R 'is independently selected from H, -C1-C8 alkyl and aryl.

[0189] The term "C₁-C₈ alkyl "as used herein refers to a straight or branched chain, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms.

Representative "C 1 -C 8 alkyl" groups include, but are not limited to, methyl, -ethyl,

-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkyls include, without limitation, -isopropyl, -sec-butyl, isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutyleneyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2- butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2hexyl, 3-hexyl, -acetyleneyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, - 3-methyl-1-butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, npentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl . 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl,

3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. Group C₁-C₈ alkyl may be unsubstituted or substituted with one or more groups including, without limitation, -C1-C8 alkyl, -O- (C_rC₈ alkyl), -aryl, -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH₂, C (O) NHR ', -C (O) N (R') 2 -NHC (O) R ', -SO3R', -S (O) 2R ', -S (O) R', -OH, -halogen, -N3, NH₂, -NH (R '), -N (R')₂ and -CN; each R 'is independently selected from H, -C₁-C₈alkyl and aryl.

[0190] "C₃-C₈ carbocycle "means a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C₃-C₈ carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, cyclohexyl, -cyclohexenyl, -1.3-cyclohexadienyl, -1.4-cyclohexadienyl, -cycloheptyl, 1,3-cycloheptadienyl, -1, 3,5-cycloheptatrienyl, -cyclooctyl and -cyclooctadienyl. Group C₃-C₈carbocyclic can be unsubstituted or substituted with one or more groups including, but not limited to, -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -aryl, -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH2, -C (O) NHR', -C (O) N (R ') 2 -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R ') 2 and -CN; each R 'is independently selected from H, -C₁-C₈ alkyl and aryl.

[0191] "C3-C8 carbocyclo" refers to a C3-C8 carbocyclic group as defined above, wherein one of the hydrogen atoms of the carbocyclic group is replaced with a bond.

[0192] "C1-C10 alkylene is a straight chain, saturated, hydrocarbon group with the formula - (CH₂)_1-10-. Examples C₁-C₁₀ alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decalene.

[0193] "Arylene" means an aryl group that has two covalent bonds and may have ortho, meta or para configurations as shown in the following structures:

<img file="PL1725249T3_D0006.tif" />

in which the phenyl group may be unsubstituted or substituted by one or up to four groups including, without limitation, -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), aryl, -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH2, -C (O) NHR', -C (O) N (R ') 2 -NHC (O) R', 45

S (O) 2R ', -S (O) R', -OH, -halogen, -N3, -NH2, -NH (R '), -N (R') 2 and -CN; wherein each R 'is independently selected from H, -C1-C8 alkyl and aryl.

[0194] "C3-C8 heterocycle" refers to an aromatic or non-aromatic C3-C8 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 the C3-C8 heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinylinyl, pyridinyl, pyrazinyl pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. The C3-C8 heterocycle may be unsubstituted or substituted with up to seven groups, including, without limitation, -C1-C8 alkyl, -O- (C1-C8 alkyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2 -NHC (O) R ', -S (ABOUT)₂R ', - S (O) R', -OH, -halogen, -N3, -NH₂, -NH (R '), -N (R')₂ and CN; wherein each R 'is independently selected from H, -C1-C8 alkyl and aryl.

[0195] "C3-C8 heterocyclo" refers to a C3-C8 heterocyclic group as defined above, wherein one of the hydrogen atoms of the heterocyclic group is replaced by a bond. The C3-C8 heterocyclo may be unsubstituted or substituted with up to six groups, including, without limitation, -C1-C8 alkyl, -O- (C1-C8 alkyl), -aryl, -C (O) R ', OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2 -NHC (O) R ', -S (O ) 2R ', - S (O) R', OH, -halogen, -N3, -NH2, -NH (R '), -N (R') 2 and -CN; wherein each R 'is independently selected from H, -C1-C8 alkyl and aryl.

[0196] "Exemplary compound" means a drug compound or a drug-linker compound.

[0197] "Exemplary conjugate" means a drug-ligand conjugate having a cleavable drug unit from a drug-ligand conjugate or a drug-linker-ligand conjugate.

[0198] In some embodiments, 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 after isolation the isolate contains at least 95%, and in a further aspect at least 98% by weight of an exemplary compound or exemplary isolate conjugate.

[0199] Examples of the "hydroxyl protecting group" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tri-ethylisil ether, triphenylmethylsilyl ester, acetate ester, substituted ester acetate, pivalate, benzoate, methanesulfonate and p-toluenesulfonate.

[0200] "Leaving group" refers to a functional group that can be substituted with another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate) and trifluoromethylsulfonate.

[0201] The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of an exemplary compound or exemplary conjugate. Exemplary compounds and exemplary conjugates contain at least one amino group, and accordingly acid addition salts can be formed with this amino group. Exemplary salts include, but are not limited to, salts such as sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tartrate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sugar, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and embonate (i.e. 1,1'metyleno-bis- (2-hydroxy-3-naphthoate)) salts. The pharmaceutically acceptable salt may include the inclusion of another molecule, such as acetate ion, succinate ion or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Cases in which numerous charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Hence, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0202] "Pharmaceutically acceptable solvate" or "solvate" refers to the 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.

[0203] The following abbreviations are used throughout the specification and have the definitions indicated: AE stands for auristatin E, Boc stands for N- (t-butoxycarbonyl), cit stands for citrulline, dap stands for dolaproin, DCC stands for 1,3-dicyclohexylcarbodiimide, DCM stands for dichloromethane, DEA stands for diethylamine, DEAD stands for diethylazodicarboxylate, DEPC stands for diethyl phosphodylenediisocyanate , DIEA means N, Ndiisopropylethylamine, dil means dolaisoleuine, DMAP means 4-dimethylaminopyridine, DME means ethylene glycol dimethyl ether (or 1,2-dimethoxyethane), DMF means N, N-dimethylformamide, DMSO means dimethylsulfoxide, doe means dolafenine, dov means N, N-dimethylvaline, DTNB means 5,5'-dithiobis (2-nitrobenzoic acid), DTPA means diethylenetriaminepentaacetic acid, DTT means dithiothreitol, EDCI means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EEDQ means 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS means electrospray mass spectrometry, EtOAc means ethyl acetate, Fmoc means N- (9-fluorenylmethoxycarbonyl), gly means glycine, HATU means O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate, HOBt means 1-hydroxybenzotriazole, HPLC means high pressure liquid chromatography , lys means lysine, MeCN (CH3CN) means acetonitrile, MeOH means methanol, Mtr means 4-anisyldiphenylmethyl (or 4-methoxytrityl), nor means (1S, 2R) - (+) norephedrine, PAB means p-aminobenzyl, PBS means phosphate buffered saline (pH 7.4), PEG means polyethylene glycol, Ph means phenyl, Pnp means p-nitrophenyl, MC means 6-maleimidocaproil, phe means L-phenylalanine, PyBrop means bromo-tris pyrrolidinophosphonium hexafluorophosphate, SEC means exclusion chromatography, Su means succinate, TBTU means O-benzotriazol-1-ylN, N, N, N-tetramethyluronium tetrafluoroborate, TFA means trifluoroacetic acid, TLC means thin layer chromatography, UV means ultraviolet and val means valine.

[0204] The following linker abbreviations are used herein and have the following definitions: Val Cit means valine-citrulline, a dipeptide site in a protease cleaved linker; PAB is p-aminobenzylcarbamoyl; (Me) vc is N-methyl-valine-citrulline in which the peptide bond in the linker has been modified to prevent its cleavage by cathepsin B; MC (PEG) 6-OH means maleimidocaproil-polyethylene glycol; SPP means N-succinimidyl-4- (2-pyridylthio) pentanoate; and SMCC is N-succinimidyl-4- (Nmaleimidomethyl) cyclohexane-1 carboxylate.

[0205] The terms "treat" or "treatment", unless the context otherwise indicates, refer to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or slow down (reduce) an undesirable physiological change or disorder such as onset or spread of cancer. For the purposes of the invention, beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowdown of disease progression, improvement or alleviation of the disease state and remission (or partial or full), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival when not receiving treatment. Those in need of treatment include those already having a disease state or disorder as well as those susceptible to the condition or disorder or those in which the disease or disorder is to be prevented.

[0206] In the context of cancer, the term "treatment" includes any or all of: preventing the growth of cancer cells, cancer cells, or cancer; preventing the replication of cancer cells or cancer cells, reducing the overall tumor burden or reducing the number of cancer cells, and improving one or more symptoms associated with the disease.

[0207] In the context of an autoimmune disease, the term "treatment" includes any or all of: preventing the replication of cells associated with an autoimmune disease including, but not limited to, cells that produce an autoimmune antibody, reducing the burden of autoimmune antibodies, and improving one or more symptoms of autoimmune disease.

[0208] In the context of an infectious disease, the term "treatment" includes any or all of: preventing the growth, multiplication or replication of a pathogen that causes an infectious disease and ameliorating one or more symptoms of an infectious disease.

[0209] The following abbreviations of cytotoxic drugs are used herein and have the definitions indicated: MMAE means auristatin E monomethyl (MW 718); MMAF means N-methylvaline-valine-dolaisoleuine-dolaproin-phenylalanine (MW 731.5); MMAF-DMAEA means MMAF with DMAEA (dimethylaminoethylamine) in an amide bond with a C-terminal phenylalanine (MW 801.5); MMAF-TEG means MMAF with tetraethylene glycol esterified to phenylalanine; MMAF-NtBu is Nt-butyl, attached as an amide to the C-terminus of MMAF; AEVB means valeryl benzylhydrazone aurystatin E, acid-sensitive linker via the C-terminus AE (MW 732); and AFP is phenylenediamine monoamide with aurystatin F C-terminal phenylalanine (MW 732).

4.2 COMPOUNDS ACCORDING TO THE INVENTION

4.2.1 COMPOUNDS WITH FORMULA (Ia) [0210] In one aspect, the invention provides drug-linker-ligand conjugates of formula Ia:

<img file="PL1725249T3_D0007.tif" />

Ia or a pharmaceutically acceptable salt or solvate thereof, wherein:

L- is the ligand unit;

-Aa-Ww-Yy- means the linker unit (LU), where the linker unit includes: -A means a stretch unit, and is 0 or 1, each -W- is independently an amino acid unit, w means an integer in the range from 0 to 12 .

-Y- is the distance unit, and y is 0, 1 or 2; p ranges from 1 to about 20; and -D is the drug unit of formula DF:

R '° Dp as defined in claim 2.

[0211] In another embodiment, the invention provides drug compounds of formula Ib:

<img file="PL1725249T3_D0008.tif" />

or a pharmaceutically acceptable salt or solvate thereof as defined in claim 1.

[0212] In yet another embodiment, the invention provides drug-linker-ligand conjugates of formula Ia ':

<img file="PL1725249T3_D0009.tif" />

Formula Ia 'or pharmaceutically acceptable salts or solvates thereof as defined in claim 4.

[0213] Ab is any antibody covalently attached to one or more drug units. Ab includes an antibody that binds to CD30, CD40, CD70 antigens, Lewis Y antigen. In another embodiment, Ab does not include an antibody that binds to an ErbB receptor or one or more of the receptors (1) - (35):

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(15) CD79b (IGb (associated with immunoglobulin beta), B29, Genbank accession number NM_000626);

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

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20Ra (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) Ephb2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763);

(26) BAFF-R (Genbank accession number NP_443177.1);

(27) CD22 (Genbank accession number NP-001762.1);

(28) CD79a (CD79A, CD79a, linked to immunoglobulin alpha, a B cell specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, causes transduction of a signal involved in B cell differentiation, Genbank accession number NP_001774.1);

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

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

(31) P2X5 (P2X purinergic receptor, ligand-gated ion channel 5, extracellular ATP-gated ion channel, may participate in synaptic conduction and neurogenesis, deficiency may contribute to pathophysiology of spontaneous detrusor instability, Genbank accession number NP_002552.2);

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

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein from the leucine rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, accession number Genbank NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, putative receptor for an immunoglobulin Fc domain that contains Ig-like and ITAM-like C2 domains, may play a role in B cell differentiation, Genbank accession number NP_443170.1); and / or (35) IRTA22 (superfamily of immunoglobulin receptors associated with translocation 2, putative immunoreceptor with a potential role in B cell development and lymphoma formation; gene deregulation by translocation occurs in certain B cell tumors, Genbank accession number NP_112571.1).

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

[0214] In another embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ means -H. In an exemplary embodiment, each R³ and R⁴ is isopropyl, R⁵is -H, and R⁷ means sec-butyl. In yet another embodiment, each R² and R⁶ is methyl and R⁹ means -H.

[0215] In yet another embodiment, each occurrence of R⁸ means -OCH₃.

[0216] In an exemplary embodiment, each R³ and R⁴ is isopropyl, each R² and R⁶is methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ means -OCH3 and R⁹ means -H.

[0217] In one embodiment, R¹⁰ is aryl.

[0218] In an exemplary embodiment, R¹⁰ means -phenyl.

[0219] In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAC10, hBR96, hS2C6, h1F6 and h2F2.

[0220] Examples of drug-antibody conjugates disclosed herein include those o

<img file="PL1725249T3_D0010.tif" />

wherein L is antibody, Val is valine and Cit is citrulline.

[0221] Drug coverage is represented by p, the average number of drug molecules per antibody in the molecule (e.g., of formula Ia, Ia 'and Ic). Drug coverage may be in the range of 1 to 20 [molecules] of drugs (D) per ligand (eg Ab or mAb). Compositions of formula Ia and formula Ia 'include collections of antibodies conjugated to a range of drugs, from 1 to 20. The average number of [molecules] of drug per antibody obtained by conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA and HPLC assays. The quantitative distribution of ligand-drug conjugates can also be determined relative to p. In some cases, separation, purification and characterization of homogeneous ligand-drug conjugates, in which p is a specific value from ligand-drug conjugates, with different drug loading, can be achieved by methods such as reverse phase HPLC or electrophoresis.

4.2.2 DRUG COMPOUNDS OF FORMULA (Ib) [0222] In a further aspect, the invention provides drug compounds of formula (Ib):

<img file="PL1725249T3_D0011.tif" />

or a pharmaceutically acceptable salt or solvate thereof as defined in claim 1.

[0223] In one embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ means -H. In an exemplary embodiment, each R³ and R⁴ is isopropyl, R⁵is -H, and R⁷ means sec-butyl.

[0224] In another embodiment, each R² and R⁶ is methyl and R⁹ means -H.

[0225] In yet another embodiment, each occurrence of R⁸ means -OCH3.

[0226] In an exemplary embodiment, each R³ and R⁴ is isopropyl, each R² and R⁶is methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ means -OCH3 and R⁹ means -H.

[0227] In one embodiment, R¹⁰ is aryl.

[0228] In an exemplary embodiment, R¹⁰ means -phenyl.

[0229] Illustrative compounds disclosed herein, each of which may be used as drug (D) moieties in the ADC, include compounds with the following structures:

<img file="PL1725249T3_D0012.tif" />

<img file="PL1725249T3_D0013.tif" />

HOOC ^^ Ν ^^, ΟΟΟΗ 7

<img file="PL1725249T3_D0014.tif" />

and

<img file="PL1725249T3_D0015.tif" />

and their pharmaceutically acceptable salts or solvates. COMPOUNDS WITH FORMULA (Ic) [0230] In another aspect, the invention provides o-drug antibody (ADC) conjugate compounds of formula Ic:

<img file="PL1725249T3_D0016.tif" />

containing the antibody covalently attached to one or more drug units (moieties). Antibody-drug conjugate compounds include pharmaceutically acceptable salts or solvates thereof.

[0231] Compounds of formula Ic are defined in which:

Ab is an antibody that binds to one or more receptor associated with the tumor associated antigen (1) - (35):

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20Ra (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22 ) Ephb2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763);

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

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

(28) CD79a (CD79A, CD79a, linked to immunoglobulin alpha, a B cell specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, causes transduction of a signal involved in B cell differentiation, Genbank accession number NP_001774 .1);

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

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

(31) P2X5 (P2X purinergic receptor, ligand-gated ion channel 5, extracellular ATP-gated ion channel, may participate in synaptic conduction and neurogenesis, deficiency may contribute to pathophysiology of spontaneous detrusor instability, Genbank accession number NP_002552.2);

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

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein from the leucine rich repeat family (LRR), regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, number accession Genbank NP_005573.1);

(34) FCRH1 (Fc receptor-like protein 1, putative receptor for an immunoglobulin Fc domain that contains Ig-like and ITAM-like C2 domains, may play a role in B cell differentiation, Genbank accession number NP_443170.1); and (35) IRTA22 (superfamily of immunoglobulin receptors associated with translocation 2, putative immunoreceptor with a potential role in B cell development and lymphoma formation; gene deregulation by translocation occurs in certain B cell tumors, Genbank accession number NP_112571.1).

A is a stretch unit, and is 0 or 1, each W is independently an amino acid unit, w is an integer in the range of 0 to 12,

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

-D is the drug moiety of the formula DF:

<img file="PL1725249T3_D0017.tif" />

in which the wavy line D_E and D_F indicates a site for covalent attachment to A, W or Y. [0232] In one embodiment, -Ww- is -Val-Cit-.

[0233] In another embodiment, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ means -H. In an exemplary embodiment, each R³ and R⁴ is isopropyl, R⁵is -H, and R⁷ means sec-butyl.

[0234] In yet another embodiment, each R² and R⁶ is methyl and R⁹ means -H.

[0235] In yet another embodiment, each occurrence of R⁸ means -OCH3.

[0236] In an exemplary embodiment, each R³ and R⁴ is isopropyl, each R² and R⁶is methyl, R⁵ is -H, R⁷ is sec-butyl, each occurrence of R⁸ means -OCH3 and R⁹ means -H.

[0237] In one embodiment, R¹⁰ is aryl.

[0238] In an exemplary embodiment, R¹⁰ means -phenyl.

[0239] Illustrative antibody-drug conjugates described herein include:

<img file="PL1725249T3_D0018.tif" />

<img file="PL1725249T3_D0019.tif" />

wherein Ab is an antibody that binds to one or more receptors of the tumor associated antigens (1) - (35); Val means valine; and Cit is citrulline.

[0240] Drug coverage is represented by p, the average number of [molecules] drug per antibody in the molecule of formula I. Drug coverage can be in the range of 1 to 20 [molecules] drug (D) per antibody (Ab or mAb). The ADC compositions of Formula I include collections of antibodies conjugated to a range of drugs [molecules], from 1 to 20. The average number of [drug] molecules per antibody in the preparation of ADC in conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA and HPLC assays. You can also specify the quantitative distribution of ADC in relation to p. In some cases, the separation, purification and characterization of homogeneous ADCs in which p is a specific value from an ADC with a different drug loading can be achieved by methods such as reverse phase HPLC or electrophoresis.

[0241] For some antibody drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, where attachment occurs through a cysteine ​​thiol, as in the exemplary embodiment above, the antibody may have only one or several cysteine ​​thiol groups, or may have only one or several sufficiently reactive thiol groups through which the linker may be attached.

[0242] Typically, less than the theoretical maximum of the drug moieties is conjugated to the antibody during the coupling reaction. The 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 can react with the amine reactive linker reagent. In general, antibodies do not contain many, if any, free and reactive cysteine ​​thiol groups that can be linked to a drug moiety. Most thiols of cysteine ​​residues in the antibodies of the compounds of the invention occur as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). In addition, the antibody should be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. Occlusion (drug / antibody ratio) in ADC can be controlled in several different ways, including: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the coupling reaction, and (iii) partial or limiting reducing conditions for modification of cysteine ​​thiols.

[0243] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent followed by a drug moiety reagent, then the resulting product is a mixture of ADC compounds with the distribution of one or more drug moieties attached to the antibody . The average number of [drug] molecules per antibody can be calculated from the mixture by means of a dual antibody ELISA, specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry, and separated by HPLC, e.g. hydrophobic interaction chromatography ("Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibodydrug conjugate", Hamblett, KJ, et al, abstract No. 624, American Association for Cancer Research; 2004 Annual Meeting, 27-31 March 2004, Proceedings of the AACR, volume 45, March 2004; "Controlling the Location of Drug Attachment in Antibody-Drug Conjugates", Alley, SC, et al, abstract No. 627, American Association for Cancer Research; 2004 Annual Meeting, March 27-31 March 2004, Proceedings of the AACR, volume 45, March 2004). Hence, homogeneous ADCs with a single occupancy value can be isolated from the mixture after coupling by electrophoresis or chromatography.

4.3 LINKER UNIT [0244] "Linker unit" (LU) means a bifunctional compound that can be used to link a drug unit and a ligand unit to form drug-linker-ligand conjugates, or one that is useful in forming immunoconjugates directed against associated antigens with cancer. Such immunoconjugates allow the selective delivery of toxic drugs to cancer cells. In one embodiment, the linker unit of the drug-linker compound and drug-linker-ligand conjugate has the formula:

-Aa-W_in-Yy what:

-A- is a stretch unit; a is 0 or 1;

each -W- is independently an amino acid unit;

w is independently an integer between 0 and 12;

-Y- is the distance unit, and y is 0, 1 or 2.

[0245] In drug-linker-ligand conjugates, the linker is capable of linking the drug moiety and ligand unit.

4.3.1 STRETCH UNIT [0246] The stretch unit (-A-), when present, is capable of linking a ligand unit with an amino acid unit (-W-). In this regard, ligand (L) has a functional group that can form a bond with the functional group of the tensile element. Useful functional groups that may be present on the ligand, either naturally or through chemical manipulation, include, but are not limited to, thiol (-SH), amino, hydroxy, carboxy, carbohydrate anomeric hydroxyl and carboxy. In one aspect, the ligand functional groups are thiol and amino. Thiol groups can be generated by reducing the intramolecular disulfide ligand bond. Alternatively, thiol groups can be generated by reacting the lysine amino group of a ligand moiety using 2-iminothiolane (Traut's reagent) or other thiol generating reagent.

[0247] In one embodiment, the tensile unit forms a bond with the sulfur atom of the ligand unit. The sulfur atom may be from the thiol group of the ligand. Representative stretch units according to this embodiment are shown in square brackets in formulas IIIa and IIIb, in which L-, -W-, -Y-, -D, wiy are as defined above, and R¹⁷ is selected from -C₁-C₁₀ alkylene-, -C₃-C₈ carbocyclo, -O- (C.₁-C₈ alkyl) -, -arylene-, -C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, -C1-C10 alkylene- (C3-C8 carbocyclo) -, - (C₃-C₈ carbocyclo) -C₁-C₁₀ alkylene-, -C₃-C₈ heterocyclo, -C₁-C₁₀ alkylene- (C₃-C₈ heterocyclo) -, - (C₃-C₈ heterocyclo) -C₁-C₁₀ alkylene-, - (CH₂CH₂ABOUT)_r- their₂CH₂ABOUT)_rCH2-; and r is an integer in the range of 1 to 10. It should be understood, from all exemplary embodiments of formula Ia, such as III-VI, that even when not explicitly indicated, from 1 to 20 drug moieties are attached to the ligand. (p = 1-20).

<img file="PL1725249T3_D0020.tif" />

<img file="PL1725249T3_D0021.tif" />

IN_in—Yy — D mb [0248] An illustrative stretch unit is one of formula IIIa, wherein R¹⁷ means - (CH2) 5-:

<img file="PL1725249T3_D0022.tif" />

[0249] Another illustrative tensile unit is one of formula IIIa, wherein R¹⁷means - (CH2CH2O) r-CH2-; ir is 2:

<img file="PL1725249T3_D0023.tif" />

[0250] Yet another illustrative stretch unit is one of formula IIIb, at

<img file="PL1725249T3_D0024.tif" />

[0251] In another embodiment, the tensile unit is connected to the ligand unit via a disulfide bond between the sulfur atom of the ligand unit and the sulfur atom of the stretch unit. A representative stretch unit according to this embodiment is shown in square brackets in formula IV, wherein R¹⁷, L-, -W-, -Y-, -D, wi are as defined above.

<img file="PL1725249T3_D0025.tif" />

[0252] In yet another embodiment, the reactive group of the tensile element comprises a reactive site that can form a bond with the primary or secondary amino group of the ligand. Examples of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative stretch units according to this embodiment are shown in square brackets in formulas Va and Vb, where R¹⁷, L-, -W-, -Y-, -D, wi are as defined above;

<img file="PL1725249T3_D0026.tif" />

[0253] In yet a further aspect, the reactive stretch group includes a reactive site that is reactive with a modified carbohydrate (CHO) group that may be present on the ligand. For example, the carbohydrate can be gently oxidized using a reagent such as sodium periodate, and the resulting (-CHO) oxidized carbohydrate unit can be condensed with a tensile element that contains functionality such as hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide such as those described by Kaneko, T. et al. (1991) Bioconjugate Chem 2: 133-41. Representative stretch units according to this embodiment are shown in square brackets in formulas VIa, VIb and VIc, where R¹⁷, L-, -W-, -Y-, -D, wi are as defined above.

<img file="PL1725249T3_D0027.tif" />

4.3.2 AMINO ACID UNIT [0254] The amino acid unit (-W-), when present, connects the stretch unit to the spacer unit, if the spacer unit is present, connects the stretch unit to the drug moiety, if the spacer unit is absent, and connects the ligand unit with the drug unit if the stretch unit and spacer unit are absent.

[0255] Ww- is a unit of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide. Each unit -W- independently has the formula noted below in square brackets, and means an integer between 0 and 12:

<img file="PL1725249T3_D0028.tif" />

with R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, phydroxybenzyl, -CH2OH, -CH (OH) CHs, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, CH2CH2CONH2, -CH2CH2COOH, - (CH2) 3NHC NH2, - (CH2ĄNH2, - (CH2ĄNHCOCH3, - (CH2ĄNHCHO, - (CH2) 4NHC (= NH) NH2, - (CH2ĄNH2, - (CH2ĄNHCOCH3, - (CH2ĄNHCHO, - (CH2ĄNHCONH2, - CH2ĄHH, CH2 CH2NH2, 2-pyridylmethyl-, 3-

<img file="PL1725249T3_D0029.tif" />

<img file="PL1725249T3_D0030.tif" />

[0256] The amino acid unit may be enzymatically cleaved by one or more enzymes, including a tumor associated protease, to release the drug unit (-D), which in one embodiment is protonated in vivo after release, to provide drug (D). Illustrative units of W._in are represented by the formulas (vii) - (ix):

<img file="PL1725249T3_D0031.tif" />

in which R²⁰ and R²¹ mean as follows:

R²⁰ R²¹ benzyl methyl isopropyl isopropyl benzyl isobutyl sec-butyl

<img file="PL1725249T3_D0032.tif" />

(CH2) 4NH2;

(CH2) 4NH2;

(CH2) 4NH2;

(CH2) 3NHCONH2 (CH2) 3NHCONH2 (CH2) 3NHCONH2 (CH2) 3NHCONH2 (CH2) 3NHCONH2 benzyl benzyl methyl; and (CH2) 3NHC (= NH) NH2;

<img file="PL1725249T3_D0033.tif" />

21 22 in which R, R and R are as follows:

R20 R21 R22 benzyl isopropyl benzyl (CH₂)₄NH₂;

benzyl (CH₂)₄NH₂; and

<img file="PL1725249T3_D0034.tif" />

in which R²⁰, R²¹, R²² and R²³ mean as follows:

H benzyl isobutyl H; and methyl isobutyl methyl isobutyl [0257] Exemplary amino acid units include, but are not limited to, those of formula (VII) wherein: R²⁰ is benzyl and R²¹ means - (CH2) 4NH2; R²⁰

twenty 21 [is] isopropyl and R²¹ means - (CH2) 4NH2; R²⁰ [means] isopropyl and R²¹ means (CH2) 3NHCONH2. Another exemplary amino acid unit is a unit of formula (VIII), wherein R²⁰ is benzyl, R²¹ is benzyl, and R²² means - (CH2) 4NH2.

[0258] Useful -W units can be designed and optimized_in- in terms of their selectivity for enzymatic cleavage by specific enzymes, for example a tumor-associated protease. In one embodiment, the -Ww- unit is one whose cleavage is catalyzed by cathepsin B, C and D, or plasmin protease.

[0259] In one embodiment, -W_in- means a dipeptide, tripeptide, tetrapeptide or pentapeptide.

[0260] When R¹⁹, R²⁰, R²¹, R²² or R²³ are different from hydrogen, the carbon atom to which R is attached¹⁹, R²⁰, R²¹, R²² or R²³ is chiral.

[0261] Each carbon atom to which R is attached¹⁹, R²⁰, R²¹, R²² or R²³ occurs independently in the (S) or (R) configuration.

[0262] In one aspect of the amino acid unit, the amino acid unit is valine-citrulline. In another aspect, the amino acid unit is phenylalanine lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinolinecarboxylate-lysine, cyclohexyl-alanine-lysine, isonipecotinic acid lysine, beta-alanine-lysine, glycine-serine-valine-glutamine and acid.

[0263] In certain embodiments, the amino acid unit may contain natural amino acids. In other embodiments, the amino acid unit may contain non-natural amino acids.

4.3.3 DISTANCE UNIT [0264] The spacer unit (-Y-), when present, connects the amino acid unit to the drug moiety when the amino acid unit is present. Alternatively, the spacer unit connects the stretch unit to the drug moiety when the amino acid unit is absent. The spacer unit also connects the drug moiety to the ligand unit when both the amino acid unit and the stretch unit are absent.

[0265] Distance units are of two general types: self-destructive and non-self-destructive. A non-self-destructive spacer unit means one in which part or all of the spacer unit remains attached to the drug moiety after cleavage, particularly enzymatic, of the amino acid unit from the drug-linker-ligand or drug-linker conjugate. Examples of a non-self-destructive spacer unit include, but are not limited to, a spacer unit (glycine-glycine) and a glycine spacer unit (both shown in Diagram 1) (below). When an exemplary compound comprising a glycine-glycine spacer unit or glycine spacer unit is subjected to enzymatic cleavage by means of a tumor cell-associated protease, cancer cell-associated protease or lymphocyte-associated protease, glycine-glycine drug grouping or glycine-drug group is cleaved from L-Aa -W w. In one embodiment, an independent hydrolysis reaction occurs within the target cell, with the glycine-drug moiety cleaved and the drug released.

[0266] In another embodiment, -Yy- is a p-aminobenzyl alcohol unit (PAB) (see Schemes 2 and 3) whose phenylene portion is substituted with Qm, wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; im is an integer in the range 0-4.

Scheme 1 fWjj — Gly — DJ

Ab-fA_and-IN_in-Gly-Gly-jD enzymatic cleavage

Gly-D hydrolysis and drug enzymatic cleavage

Gly-Gły-D hydrolysis drug [0267] In one embodiment, the non-self-destructive spacer unit (-Y-) is -Gly-Gly-. In another embodiment, the non-self-destructive spacer unit (Y-) means -Gly-.

[0268] In one embodiment, a drug-linker compound or drug-linkerligand conjugate is provided in which the spacer unit is absent (y = 0), or a pharmaceutically acceptable salt or solvate thereof.

[0269] Alternatively, an exemplary compound comprising a self-destructive spacer unit may release -D without having to perform a separate hydrolysis step. In this embodiment, -Y- is a PAB group that is connected to the -Ww- via the amino nitrogen of the PAB group, and connected directly to -D via a carbonate, carbamate, or ether group. Without being bound to any particular theory or mechanism, Scheme 2 illustrates the possible mechanism of drug release from the PAB group that is attached directly to -D via a carbamate or carbonate group according to Toki et al. (2002) J Org. Chem. 67: 1866-1872.

<img file="PL1725249T3_D0035.tif" />

<img file="PL1725249T3_D0036.tif" />

drug wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; m is an integer in the range of 0-4; ip ranges from 1 to about 20.

[0270] Without being bound to any particular theory or mechanism, Scheme 3 illustrates a possible mechanism for releasing a drug from the PAB group that is attached directly to -D via an ether or amine bond.

Diagram 3

<img file="PL1725249T3_D0037.tif" />

wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; m is an integer in the range of 0-4; ip ranges from 1 to about 20.

[0271] Other examples of self-destructive spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9: 2237) and ortho or para-aminobenzylacetals. Spacers that are cyclized after hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo ring systems [2.2.1] and bicyclo [2.2.2] (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990 , 55, 5867). Also, the elimination of amine-containing drugs that are substituted at the α-glycine position (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is an example of a self-destructive spacer useful in exemplary compounds.

[0272] In one embodiment, the spacer unit is a branched bis (hydroxymethyl) styrene unit (BHMS), a unit as shown in Scheme 4, which can be used to incorporate and release a number of drugs.

<img file="PL1725249T3_D0038.tif" />

Diagram 4

mr

<img file="PL1725249T3_D0039.tif" />

EH_and(O (C (O))) "- D CH₂(0 {C (O))) "- DI enzymatic cleavage drugs wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; m is an integer in the range of 0-4; n is 0 or 1; ip ranges from 1 to about 20.

[0273] In one embodiment, the -D moieties are the same. In yet another embodiment, the -D moieties are different.

[0274] In one aspect, the spacer units (-Yy-) are represented by formulas (X) - (XII):

<img file="PL1725249T3_D0040.tif" />

wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; and m is an integer in the range of 0-4;

and

<img file="PL1725249T3_D0041.tif" />

NHCH₂C (O) NHCH₂WHAT)

<img file="PL1725249T3_D0042.tif" />

[0275] Embodiments of the antibody-drug conjugate compounds of Formula Ia 'and Ic include:

and

<img file="PL1725249T3_D0043.tif" />

<img file="PL1725249T3_D0044.tif" />

each wiy is 0,

<img file="PL1725249T3_D0045.tif" />

and

<img file="PL1725249T3_D0046.tif" />

[0276] Drug (D) antibody conjugate (ADC) moieties are of the dolastatin / aurystatin type (US Patent Nos. 5635483; 5780588) which have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and exhibit anti-tumor (US Patent No. 5,663149) and anti-fungal (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965) [0277] D is a drug unit (moiety) having a nitrogen atom that can form a bond with a spacer unit when y = 1 or 2, with the C-terminal carboxyl group of the amino acid unit, when y = 0, with the carboxy group of the stretch unit when wiy = 0, and the carboxy group of the drug unit when a, w, iy = 0. It should be understood that the terms "drug unit" and "drug moiety" are synonymous and are used interchangeably in the description. The drug unit has a structure as defined in the claims.

[0278] Also described herein are drug units of the formula DE:

<img file="PL1725249T3_D0047.tif" />

and independently for each location: R² is selected from H and C₁-C₈ alkyl;

₃

R is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C.₁-C₈ alkylaryl, C.₁C₈ alkyl- (C₃-C₈ carbocycle), C₃-C₈ heterocycle and C.₁-C₈ alkyl- (C₃-C₈ a heterocycle);

R⁴ selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C.₁-C₈ alkylaryl, C.₁C₈ alkyl- (C₃-C₈ carbocycle), C₃-C₈ heterocycle and C.₁-C₈ alkyl- (C₃-C₈ a heterocycle); R⁵selected from H and methyl;

or R⁴ and R⁵ together form a carbocyclic ring and have the formula - (CR^andR^b) n-, where R^and and R^b are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle, and n are selected from 2, 3, 4, 5 and 6; R⁶ is selected from H and C1-C₈ alkyl;

R⁷ selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C.₁-C₈ alkylaryl, C.₁C₈ alkyl- (C₃-C₈ carbocycle), C₃-C₈ heterocycle and C.₁-C₈ alkyl- (C₃-C₈ a heterocycle);

each R⁸ is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O (C1-C8 alkyl); R⁹ is selected from H and C1-C₈ alkyl;

R¹⁸ is selected from -C (R⁸) 2-C (R⁸) 2-aryl, -C (R⁸) 2-C (R⁸) 2- (C₃-C₈ heterocycle) and C (R⁸) 2-C (R⁸) 2- (C3-C8 carbocycle); and n is an integer in the range of 0 to 6.

[0279] In one case, R³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵means -H. In the example case, eachR^{3 and} R⁴ is isopropyl, R⁵ is H, and R⁷means sec-butyl.

[0280] In a further case, each R² and R⁶ is methyl, and R⁹ means H.

[0281] In yet a further case, each occurrence of R⁸ means -OCH₃.

[0282] In an exemplary case, each R³ and R⁴ is isopropyl, each R² and R⁶ is methyl, R⁵ means H, R⁷ is sec-butyl, each occurrence of R⁸ means -OCH3 and R⁹ means H.

[0283] Illustrative drug units (-D) include drug units with the following structures:

<img file="PL1725249T3_D0048.tif" />

<img file="PL1725249T3_D0049.tif" />

and

<img file="PL1725249T3_D0050.tif" />

<img file="PL1725249T3_D0051.tif" />

and their pharmaceutically acceptable salts or solvates.

[0284] As described herein, hydrophilic groups such as, but not limited to, methylene glycol esters (TEG) as shown above can be attached to the drug unit at R¹¹. Without being bound by theory, hydrophilic groups help internalize and prevent drug units from sticking together.

4.5 LIGAND UNIT [0285] A ligand (L-) unit includes within its scope any ligand (L) units that bind to or reactive binds or complexes with a receptor, antigen or other susceptible moieties associated with a given target cell population. A ligand is a molecule that binds to, forms complexes with, or reacts with, a cell population moiety that is expected to be therapeutically or biologically modified in another way. In one aspect, the ligand unit works by delivering a drug unit to the specific target cell population with which the ligand unit reacts. Such ligands include, but are not limited to, high molecular weight proteins, such as, for example, full-length antibodies, antibody fragments, lower molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, transporting molecules. nutrients (such as, but not limited to, transferrin), or any other cell-binding molecule or substance.

[0286] The ligand unit may form a bond with a stretch unit, amino acid unit, distance unit, or drug unit. The ligand unit may form a bond with the linker unit via a ligand heteroatom. Heteroatoms that may be present on the ligand unit include sulfur (in one embodiment from the thiol group of the ligand), oxygen (in one embodiment from the carbonyl, carboxyl or hydroxyl group of the ligand) and nitrogen (in one embodiment from the primary or secondary amino group ligand). These heteroatoms may be present on the ligand in the natural state of the ligand, for example, a naturally-occurring antibody, or may be introduced into the ligand by chemical modification.

[0287] In one embodiment, the ligand has a thiol group and the ligand binds to the linker unit through a sulfur atom of the thiol group.

[0288] In yet another aspect, the ligand has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. The ligand unit binds to the linker unit through the sulfur atom of the thiol group. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl Sacetylthioacetate (SATA) and 2-Iminothiolate Hydrochloride (Traut's Reagent).

[0289] In another embodiment, the ligand may have one or more carbohydrate groups that can be chemically modified to have one or more thiol groups. A ligand unit binds to a linker unit, such as a stretch unit, through a sulfur atom of a thiol group.

[0290] In yet another embodiment, the ligand may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, e.g., Laguzza, et al., J. Med. Chem. 1989, 32 ( 3), 548-55). The corresponding aldehyde can form a bond with the reactive site on the tensile element. Reactive sites on a tensile element that can react with a carbonyl group on a ligand include, but are not limited to, hydrazine and hydroxylamine. Other protocols for protein modification, for attachment or binding to drug entities are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley &amp; Sons (2002), incorporated herein by reference.

[0291] Useful ligands of a non-immunoreactive protein, polypeptide or peptide include, but are not limited to, transferrin, epidermal growth factors ("EGF"), bombesin, gastrin, gastrin releasing peptide, platelet-derived growth factor, IL-2, IL-6 transforming growth factors ("TGF") such as TGF-α and TGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins and low density lipoprotein apoprotein.

[0292] 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 to produce polyclonal antibodies against the antigen of interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injecting the subject antigen or derivative thereof, including but not limited to rabbits, mice, rats and guinea pigs. Various adjuvants may be used to increase the immune system response, depending on the host species, and include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, polyols Pluronic, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol and potentially useful human adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0293] Useful monoclonal antibodies are homogeneous antibody populations for a particular antigenic determinant (e.g., cancer cell antigen, viral antigen, microbial antigen, protein, peptide, carbohydrate, chemical, nucleic acid or fragments thereof). A monoclonal antibody (mAb) for the subject antigen can be obtained using any technique known in the art that ensures the production of antibody molecules by means of continuously maintained cell line cultures. These include, without limitation, the hybridoma technique first described by Kohler and Milstein (1975, Nature 256, 495-497), the hybridoma technique of human B cells (Kozbor et al., 1983, Immunology Today 4: 72) and the hybridoma technique EBV (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be from any class of immunoglobulins including IgG, IgM, IgE, IgA and IgD, and any subclass thereof. Hybridomas producing mAbs for use in the invention can be cultured in vitro or in vivo.

[0294] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be obtained by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

[0295] The antibody may also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditionally, the production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two chains having different specificities (Milstein et al., 1983, Nature 305: 537-539). Due to the random selection of immunoglobulin light and heavy chains, these hybridomas (quadromes) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[0296] In another approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are conjugated to immunoglobulin constant domain sequences. The fusion preferably occurs with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge regions, CH2 and CH3. It is preferred that it has a first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. Nucleic acids with sequences encoding immunoglobulin heavy chain fusions and, if desired, an immunoglobulin light chain fusion are introduced into separate expression vectors, and are co-transfected into the appropriate host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments, when the uneven proportions of the three polypeptide chains used in the construction provide optimal yields. It is, however, possible to introduce coding sequences for two or all three polypeptide chains into one expression vector, where the expression of at least two polypeptide chains in equal proportions results in high yield or when the proportions have no particular meaning.

[0297] In an embodiment of this approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid heavy chain-light chain immunoglobulin pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains, as the presence of an immunoglobulin light chain in only half of the bispecific molecules provides an easy separation path (International Publication No. WO 94/04690, which is incorporated herein by reference in its entirety).

[0298] For further details on the generation of bispecific antibodies, see. for example, Suresh et al., Methods in Enzymology, 1986, 121: 210; Rodrigues et al., 1993, J. of Immunology 151: 6954-6961; Carter et al., 1992, Bio / Technology 10: 163-167; Carter et al., 1995, J. of Hematotherapy 4: 463-470; Merchant et al., 1998, Nature Biotechnology 16: 677-681. Using such techniques, bispecific antibodies can be obtained for use in treating or preventing a disease as defined herein.

[0299] Bifunctional antibodies are also described in European Patent Publication No. EPA 0105360. As disclosed in this reference, hybrid or bifunctional antibodies can be obtained biologically, i.e. by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-forming reagents , and they may contain whole antibodies or fragments thereof. Methods for making such hybrid antibodies are disclosed, for example, in International Publication No. WO 83/03679, and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.

[0300] An antibody may 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 the tumor cells or matrix. In this regard, "functionally active" means that the fragment, derivative or analog is capable of raising anti-anti-idiotype antibodies that recognize the same antigen as the antibody from which the fragment, derivative or analog is derived. Specifically, in an exemplary embodiment, the idiotype antigenicity of an immunoglobulin molecule may be enhanced by deleting framework and CDR sequences that are C-terminal relative to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind an antigen, synthetic peptides containing the CDR sequences in antigen binding assays can be used using any of the prior art binding assays (e.g., the BIAcore assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, fifth edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. of Immunology 125 (3): 961-969).

[0301] Other useful antibodies include antibody fragments, such as, but not limited to, F (ab ') 2 fragments that contain a variable region, a light chain constant region, and a heavy chain CH1 domain that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing disulfide bridges of F (ab ') 2 fragments. Other useful antibodies are antibody heavy chain and light chain dimers, or any minimal fragment thereof, such as Fvs or single chain antibodies (SCAs) (e.g., as described in US Patent No. 4946778; Bird, 1988, Science 242: 423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-54), or any other molecule with the same specificity as the antibody.

[0302] In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions that can be produced using standard recombinant DNA techniques, are useful antibodies. Chimeric antibody means a molecule in which different parts are from different animal species, such as those having a variable region derived from murine monoclonal immunoglobulins and human immunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from a non-human species having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, US Patent No. 5,585,089, which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be made using recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171496; European Patent Publication No. 173494; International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Publication No. 122,023; Berter et al., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al., 1987, Cancer Res. 47: 999-1005; Wood et al., 1985, Nature 314: 446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al., 1986, BioTechniques 4: 214; U.S. Patent No. 5,225,339; Jones et al., 1986, Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al., 1988, J. Immunol. 141: 4053-4060; each of which is incorporated by reference in its entirety.

[0303] Completely human antibodies are particularly desirable and can be produced using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. Transgenic mice are immunized in the normal manner with the selected antigen, e.g. all or part of the polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation, and then subject to class switching and somatic mutations. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see. e.g. U.S. Patent Nos. 5,625,126; 5633425; 5569825; 5661016; 5545806; each of which is incorporated by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

[0304] Completely human antibodies that recognize a selected epitope can be generated using a technique termed "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to direct selection of a completely human antibody that recognizes the same epitope. (Jespers et al. (1994) Biotechnology 12: 899-903). Human antibodies can also be made using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991); Quan, MP and Carter, P. 2002. The rise of monoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease, Jardieu, PM and Fick Jr., R. B, eds., Marcel Dekker , New York, NY, Chapter 20, pp. 427-469).

[0305] In other embodiments, the antibody is an antibody fusion protein, or a functionally active fragment thereof, for example, wherein the antibody is fused via a covalent bond (e.g., a peptide bond), N-terminus or C-terminus, with an amino acid sequence another protein (or part thereof, preferably at least 10, 20 or 50 amino acids part of the protein) that is not an antibody. Preferably, the antibody or fragment thereof is covalently linked to another protein at the N-terminus of the constant domain.

[0306] Antibodies include analogs and derivatives that are modified, i.e., by covalent attachment of any type of molecule, as long as such covalent attachment allows the antibody to retain its antigen binding immuno-specificity. For example, without limitation, antibody derivatives and analogs include those which have been further modified, e.g. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivative production by known protecting / blocking groups, proteolytic cleavage, binding to a cellular antibody unit or other protein, etc. Any of a variety of chemical modifications may be carried out by known techniques, including without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the analog or derivative may contain one or more non-natural amino acids.

[0307] Antibodies include antibodies having modifications (e.g., substitutions, deletions or additions) at amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications in amino acid residues identified as participating in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies that are immune to the cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, CA) or produced by any method known to a person skilled in the art, such as, e.g. chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding the cancer cell antigen-specific antibodies can be obtained, e.g., from a GenBank database or similar database, from literature publications, or by routine cloning and sequencing.

[0308] In a specific embodiment, known antibodies can be used to treat or prevent cancer. Antibodies that are immunospecific to a cancer cell antigen can be obtained commercially or produced by any method known to one of ordinary skill in the art, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a cancer cell antigen can be obtained, e.g. from the GenBank database or similar database, from literature publications, or by routine cloning and sequencing. Examples of antibodies available for cancer treatment include, but are not limited to, humanized anti-HER2 monoclonal antibody, HERCEPTIN® (Trastuzumab; Genentech) for the treatment of patients with metastatic breast cancer; RITUXAN® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a mouse antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an anti-EGFR chimeric IgG antibody for the treatment of cancers with positive epidermal growth factor such as head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I / H (Leukosite, MA) which is a humanized IgG antibody₁ 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 ID 10 (Protein Design Labs, Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment of non-granulomatous lymphoma; Oncolym (Techniclone, Inc., CA) which is a radiolabeled mouse anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA) which is a humanized anti-VEGF antibody for the treatment of lung cancer and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0309] Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies to the following antigens: CA125 (ovary), CA15-3 (cancer), CA199 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA 242 (large intestine), placental alkaline phosphatase (tumors), prostate-specific antigen (prostate), acid prostate phosphatase (prostate), epidermal growth factor (tumors), MAGE-1 (tumors), MAGE-2 (tumors), MAGE-3 (tumors) , MAGE -4 (tumors), anti-transferrin receptor (tumors), p97 (melanoma), MUC1-KLH (breast cancer), CEA (large intestine), gp100 (melanoma), MART1 (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 (cancer), CD38 (multiple myeloma), CD40 (lymphoma), mucin (cancer), P21 (cancer), MPG (melanoma) and Neu oncogen product (tumors). Some specific useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, SJ, Henderson, AJ, Hofstead, SJ, Casazza, AM, Firestone, RA, Hellstrom, I., Hellstrom, KE, "Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates" Science 1993, 261, 212-215), BR64 (Trail, PA, Willner, D, Knipe, J., Henderson, AJ, Lasch, SJ, Zoeckler, ME, Trailsmith, MD, Doyle, TW, King, HD, Casazza, AM, Braslawsky,

GR, Brown, JP, Hofstead, SJ, (Greenfield, RS, Firestone, RA, Mosure, K., Kadow, DF, Yang, MB, Hellstrom, KE, and Hellstrom, I. "Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates ”Cancer Research 1997, 57, 100-105, mAbs against CD40 antigen, such as mAb S2C6 (Francisco, JA, Donaldson, KL, Chace, D., Siegall, CB, and Wahl, AF "Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, 60, 3225-3231), mAbs against CD70 antigen such as mAb 1F6 and mAb 2F2, and mAbs against CD30 antigen such as AC10 (Bowen, MA, Olsen, KJ, Cheng, L., Avila, D., and Podack, ER "Functional effects of CD30 on a large granular lymphoma cell line YT" J. Immunol., 151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res. 62 (13): 3736-42). Many other internalized antibodies that bind to tumor-associated antigens can be used and have already been reviewed in the review (Franke, AE, Sievers, EL, and Scheinberg, DA, "Cell surface receptortargeted therapy of acute myeloid leukemia: a review "Cancer Biother Radiopharm. 2000,15, 459-76; Murray, JL," Monoclonal antibody treatment of solid tumors: a coming of age "Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0310] In certain embodiments, the antibody is other than Trastuzumab (full length, humanized anti-HER2 antibody (MW 145167)), HerceptinF (ab ')₂(derived from anti-HER2 [enzymatically cleaved (MW 100,000)), 4D5 (full length, mouse anti-HER2, from hybridoma), rhu4D5 (transiently expressed, full-length humanized antibody), rhuFab4D5 (recombinant humanized Fab (MW 47738) ), 4D5Fc8 (full length, mouse anti-HER2, with mutated FcRn binding domain), or Hg ("Hingeless" full-length humanized 4D5, with heavy chain cysteines mutated to serines. They were expressed in E. coli (hence no glycosylation)).

[0311] In another particular embodiment, known antibodies for treating or preventing an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies that are immunospecific to a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university researcher or company) or produced by any method known to a person skilled in the art, e.g. chemical synthesis or recombinant expression techniques. In another embodiment, useful immuno-specific antibodies for treating autoimmune diseases include, but are not limited to, an anti-nuclear antibody; anti-dsDNA; anti-ssDNA, anti-cardiolipin IgM antibody, IgG; anti-phospholipid IgM antibody, IgG; anti-SM antibody; anti-mitochondrial antibody; thyroid antibody; microsomal antibody; thyroglobulin antibody; anti-SCL-70; Anti-Jo; anti-U1RNP; Anti-La / SSB; anti-SSA; anti-SSB; anti-parietal cell antibody; Anti-Histones; anti-RNP; C-ANCA; P-ANCA; antycentromer; anti-fibrillin and anti-GBM antibody.

[0312] In certain embodiments, useful antibodies can bind to either the receptor or the receptor complex expressed on activated lymphocyte. The receptor or receptor complex may include a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable members of the immunoglobulin superfamily are CD2, CD3, CD4, CD8, CD 19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1 and ICOS. Non-limiting examples of suitable members of the TNF receptor superfamily are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNFR1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3. Non-limiting examples of suitable integrins are CD 11 a, CD 11 b, CD11c, CD 18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103 and CD104. Non-limiting examples of suitable lectins are type C, S and 1 lectins.

[0313] In one embodiment, the ligand binds to an activated lymphocyte that is associated with an autoimmune disease.

[0314] In another particular embodiment, useful immuno-specific antigen-specific microbial ligands are monoclonal antibodies. Antibodies can 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 gp120, HIV nef, RSV F glycoprotein, influenza neuraminidase, influenza hemagglutinin, HTLV tax, Herpes simplex glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen that are capable of to elicit an immune response. As used herein, the term "microbial antigen" includes, without limitation, any peptide, polypeptide, protein, saccharide, polysaccharide, or lipid of a microbial molecule (e.g. polypeptide of bacterial, fungal, pathogenic protozoa, or yeast (e.g. LPS and capsular polysaccharide 5/8) that are capable of eliciting an immune response.

[0315] Immuno-specific antibodies to the viral or microbial antigen can be obtained commercially, for example, from BD Biosciences (San Francisco, CA) Chemicon International, Inc. (Temecula, CA), or Vektor Laboratories, Inc. (Burlingame, CA) or produced by any method known to a person skilled in the art, such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific to a viral or microbial antigen can be obtained, e.g. from the GenBank database or similar database, from literature publications, or by routine cloning and sequencing.

[0316] In a particular embodiment, useful ligands are those that are useful for treating or preventing viral or microbial infections in accordance with the methods disclosed herein. Examples of available antibodies useful for treating viral or microbial infections include, but are not limited to, SYNAGIS (Medlmmune, Inc., MD) which is a humanized monoclonal antibody against respiratory syncytial virus (RSV) useful for treating patients with an RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody useful for treating HIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.

[0317] Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies to antigens from pathogenic bacterial strains (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium Clothridium perfumeium botulinum, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexumi, Shigella trepumum, Salmonella, Salmonella , Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma sstr., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia sstr.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania brazil

Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or worms (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosomajaponicum, Schistosoma mansoni, Schistosoma haematobium and hookworms).

[0318] Other antibodies useful in the invention for the treatment of viral diseases include, but are not limited to, antibodies to antigens of pathogenic viruses, including, by way of example and not limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza virus, measles, syncytial virus, syncytial , 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.

[0319] In attempting to discover effective cellular targets for the diagnosis and treatment of cancer, researchers sought to identify transmembrane or other tumor associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cell compared to one or more normal non-cancer cells. Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancer cells. Identification of such tumor associated cell surface antigens polypeptides has led to the ability to specifically target cancer cells to be destroyed in antibody-based therapies.

[0320] Antibodies that contain Ab in drug antibody (ADC) conjugates of formula Ic and that may be useful in the treatment of cancer include, but are not limited to, antibodies to tumor associated antigens (TAAs). Such tumor-associated antigens are known in the art, and can be obtained for use in generating the antibody using methods and information that are well known in the art. Examples of TAA include (1) - (35), but are not limited to TAA (1) - (35) listed below. For convenience, information relating to these antigens, all of which are known in the art, is detailed below and includes names, alternative names, Genbank accession numbers, and original references. Tumor-associated antigens that target antibodies include all amino acid sequence variants and isoforms having at least about 70%, 80%, 85%, 90%, or 95% sequence identity to the sequences identified in the corresponding specified sequences (SEQ ID NOS: 1-35) or sequences identified in the cited references. In some embodiments, TAAs having amino acid sequence variants exhibit essentially the same biological properties or characteristics as TAAs having a sequence found in corresponding sequences specified (SEQ ID NOS: 1-35). For example, TAA having a variant sequence is generally capable of binding specifically to an antibody that specifically binds to TAA with the corresponding specified sequence. The sequences and disclosures specifically cited herein are expressly incorporated by reference.

[0321] CANCER ANTIGENES (1) - (35):

(1) BMPR1B (bone morphogenetic protein receptor type IB, accession number

Genbank NM_001203, Ten Dijke, P., et al. Science 264 (5155): 101-104 (1994), Oncogene 14 (11): 1377-1382 (1997)); WO2004063362 (claim 2)

WO2003042661 (claim 12); US2003134790-A1 (pages 38-39); WO2002102235 (claim 13; page 296) WO2003055443 (pages 91-92); WO200299122 (example 2; pages 528-530); WO2003029421 (claim 6) WO2003024392 (claim 2; Fig 112); WO200298358 (claim 1; page 183); WO200254940 (page 100-101) WO200259377 (page 349-350); WO200230268 (claim 27; page 376); WO200148204 (example; Fig 4) NP_001194 bone morphogenetic protein receptor, type IB / pid = NP_001194,1 - Cross references: MIM: 603248; NP_001194.1; NM_001203_1

502 aa

MLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPBDSVNNICSTDGYCFTMIEED DSGLPWTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPLKNRDFVD GPIHHRALLISVTVCSLLLVLIILFCYFRYKRQETRPRYSIGLEQDETYI P PGESLRDLI EQSQSSGSGSGLPLLVQRTIAKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFFTTEEAS WFRETEIYQTVLMRHENILGFIAADIKGTGSWTQLYIiITDYHENGSLYDYLKSTTLDAXS MLKLAYSSVSGLCHLHTSIFSTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVKFISD TNEVDIPPNTRVGTKRYMPPEVLDBSLNRNHFQSYIMADMYSFGLILWEVARRCVSGGIV EEYQLPYHDLVPSDPSYEDMREIVCXKKLRPSFPNRWSSDECLRQMGKLMTECWAHNPAS RLTALRVKKTLAKMSESQDIKL (SEQ ID NO: 1) (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486); Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699): 288-291 (1998), Gaugitsch,

HW, et al. (1992) J. Biol. Chem. 267 (16): 11267-11273); WO2004048938 (example 2); WO2004032842 (example IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (example 2); WO200299074 (claim 19; pages 127-129); WO200286443 (claim 27; pages 222,393); WO2003003906 (claim 10; page 293); WO200264798 (Claim 33; pages 93-95); WO200014228 (claim 5; pages 133-136); US2003224454 (Fig 3); WO2003025138 (claim 12; page 150); NP_003477 solute carrier family 7 (cationic amino acid transporter, y + system), member 5 /pid=NP_003477.3 -Homo sapiens Links:

MIM: 600182; NP_003477.3; NM_015923; NM_003486_1

507 aa

MAGAGPICRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAIIV GTIIGSGIFVTPTGVLKEAGSPGLALWWAACGVFSIVGALCYAELGTTISKSGGDYAYM LEVYGSŁPAFLKLMIELLIIRPSSQYIVALVFATYLLKPLFPTCPVPEEAAKLVACECVL ΕΒΤΑνΝσΥβνΚΑΑΤΗνΟϋΑΡΑΑΑΚΙ, ΙΑΙΑΒΙΙΙ, ΙΧΪΡναίαΚΟννΒΝΙ.ϋΡΝΡΕΓΕαΤΚΕΕν GNIVLALYSGLFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPI VTLVYVLTNLAYFTTL STEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLFFVGSREGHLP SILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRH RKPELERPIKVNLALPVFFILACLFLIAVSFWKTPVECGIGFTIILSGLPVYFFGVWWKN KPKWLLOGIFSTTVLCQKLMQ WPQET {SEQ ID WO: 2) (3) STEAP1 (epithelial prostate antigen with six transmembrane domains, accession Genbank NM_012449 Cancer Res. 61 (15), 58575860 (2001), Hubert, RS, et al. ( 1999) Proc. Natl. Acad. Sci. USA. 96 (25): 1452314528); WO2004065577 (claim 6); WO2004027049 (Fig 1L); EP1394274 (example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (example 5); US2003185830 (example 5); US2003064397 (Fig 2); WO200289747 (example 5; pages 618-619); WO2003022995 (example 9; Fig 13A, example 53; page 173, example 2; Fig 2A); NP_036581 epithelial prostate antigen with six transmembrane domains [cross reference] MIM: 604415; NP_036581.1; NM_012449_1

339 aa

MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE LQHTQELFPQWHLPIKIAAIIASLTPLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM VSITLIALVYLPGVIAAIVQLHNGTKYKKFPHWIiDKWMLTRKQFGLLSFFFAVLHAIYSL SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS VSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNXWIDIKQFVWYTPPTFMIAVFLPIV VLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO 3) (4) 0772P (CA125, MUC16, Genbank accession number AF361486 J. Biol. Chem. 276 (29): 27371-27375 (2001)); WO2004045553 (claim 14); WO200292836 (claim 6; Fig 12); WO200283866 (claim 15; pages 116-121); US2003124140 (example 16); US2003091580 (claim 6); WO200206317 (claim 6; pages 400-408); Links: GI: 34501467; AAK74120.3; AF361486_1

6995 aa

PVTSLLTPGL VI1TDRMGISRE PGTSSTSWLSSTSBERLTTIiEDTTOTEAMOPSTHTATO NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSL T SGLRETS SS ER ISSATEG STVLS E VPSGATTE VSRTEVISSRGTSMSGPDOFT IS PDIS ΤEAITRLS TS ΡIMTES AE SAI71ETGS PGATS EGTLTLDTSTTTFWSGTHSTASPGP SHS EMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALL TLG PVKTTDMLR TSSE ΡΞ TS SPPNLSSTSAEILATSEVTKDREKIH PSSNT P WNVGTVI YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREIS TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIS TPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSR GPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKT TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYS SYPGLPEPSKVTSPWTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIH SATKPSTVPYKALTSATIEDSMTQVWSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTES TBMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPWLS HPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSE P ET SS PPNLS STS AE ILATTEVTTDTEKLEMTNVVT SGYTHESPSSVLADSVTTKAT S SM GI T ¥ FTGDTNVŁTSTPAFSDTSRI QTKSKLSLTPGLMETSISEET SATEKSTVLS S £ VPT GATTEVSRTEAISS SRTSIPGPAOSTM3 SDTSMETITRIST PLTRKESTDMAITPKTGPS GATSQGTFTLDSSSTASWPGTHSATTQRFPRSWTTPMSRGPEDVSWPSPLSVEKNSPPS SLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNI TSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPWTSSS IRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPE VSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTBSAEITITTQTGYSLATSQ VTLPLGTSMTFLSGTOSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLP LTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIP 2LTSEIMTDADVSI TST SMTSTSSTSSTA

FT ΞΝΡ A? S ETRRIPTEPTFSLTPGFRETSTS ΕΕΤΤΞIT ETSAVLFGVPTSATTEVSMTEI mssnrthipdsdqstmspdiitevitrlssssmmsestqmtittqksspgataqstltla TTTAPLARTHSTVP PRFLHSEWTTLMSRS penP SWKS SP FVEKTSSSSSLLSLPVTTSPS VSSTLPQSI PS SS FS VTSLLTPGHVKTTDTSTE PGTS LS PNLSGTS VEILAASBVTTDTE KIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFET TGFEAE FF SHLTSGLRKTNMSLDTS S YTPTNTPSS POSTHLLOS SKTDFTSS AKTSS PD WP PASGYTEIP VDIITP FNASPSI testgiTS fpesrftm SVTESTHHLSTDLLPSAETIS TGTVMPSŁSEAMTS FATTGVPRAISGSG SP FSRTESGFGDATLSTIAES L PS STP VP FS S ST FTTTDSSTIPALHEITSSSAT PYRVDTSLGTESSTTEGRLVMVSTLDTS SQPGRTS £ S PILDTRMTBSVEL GTVTSA YQ VPSLSTRLTRTDGIKĘHITKIPNEAAHRGTIRPVKGPQT STS PAS PKGLHTGGTKRHETTTTALKTTTT ALKTTS RATLTTSVYTPTLGTLTPLNASMG MASTIPTEMMITTP YVF PWPBTTSSLATSLGAETSTAL PRTT P SVFNRESRTTASLVSR SGAERS P VIQTLDVS SSEPDTTASWVIH PASTIPTVS KTTPNFFHS ELDTVSSTATSHGA D VS SA IPTTU S PSELDALTPL VTISGTDTSTTFPTLTKS PHETETRTTWLTHFABTS ST PRTIPN FSHR SDATPS IATSPGAETSSAIPIMTVS PGAEDLVTSQVTSSGTDRNMTI PT LTLS PGE PKTI ASLVTHPEAQTSSAl PTSTIS PAVSRLVTSMVTSLAAKTSTTNRALTNS PGEPATTVSLVTHSAQTSPTVP "TTSIFFHSKSDTTPSMTTSHaAESSSAVPTPTVSTEV PGWTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGW TSLVTSS RAYTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPE VPGMVTS LVAS SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGWTSLVTSSSGVNS TSIPTLILS PGELETTP SMATS HGAEAS SAVPTPTVS PG VSGWTPLVTSSRAVTSTTI p ILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTISS DE PETTTSLVT HS EAKMISAIPTLGVS PTVQGLVTS DVTS SGSETSAFSNLTVASSQPET IDSWAKPGTEAS Ξ In PTLT VSTGE FTNISL VTH P P AE SSSTLPRTTSRPSHSELDTM PS TVTS PEAESSSAISTTISFGI PG VLTSL VTSSGRD ϊ S ATFPTVPES PHESEATASWVTHP AVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPT1TVSPDVPDMVTSQVTSSGTD TS IΤIPTLTLS SGEPETTTSFITYSETHTSSAIPTLPVS PDASKMLTSLVI SSGTDSTTT FPTLTETPYEP STTAIQLI HP AETNTMVPRTT PKFSHSK SDTTLP7AITSPGPEAS SA VS TTT ISPDMSDL VTS LUPSSGTDTSTTF PTLS ETPYE PBTTATWLTHPAETS TTUSGTI PN fshrgsdtapsmvtspgvetrsgvpttti ppsipgwtsqvtssatdtstai ptltpspg EPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPD ATPUMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALL S THPRTETSKT FPASTVFPQVSETTASLTIRPGAETSTAL PTQTTSSLFTLLVTGTSRUD LSPTASPGVSAKTAPLSTHPGTttTSTMIPTSTLSLGLLETTGEJiATfiSSABTSTSTLTLT VS PAVSGLS SAS ITTDKPQTVTSWNTETS PS PS VTSVGP PEFSRTVTGTTMTLI EMPTP P KTSHGEGUS PTT and LR TTMVEATNLAT TGS S PWAKTTTT FNTLAGSŁFTPLTTPGMSTLA S ESVTSRTS YNHRS WISTTSSYNRRYWTPATSTPUTSTFS PG ISTS SIPSSTAAT VP FMV P FTLN FTI TNLQYEEDMRHPGSHKFNATBRfiLQGLL KP LFRNSSLE YLYSG C RLASLRPE KDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHR SSMPTTS TPGT SWDVGTSGTPS SSPS PTTAG PLLMPFTLNFTITNLOYEEDMRRTGSRK FNTME S F VLQGDLKPL KNTS VGPLY SG CRLTLLRPEKDGAATGVDAICTHRLDPKS PgDn REQL YWEL Ξ KLTNDIEELGP YTLDRNSL YVNGFTHQSS VSTTSTPGTSTVDLRTSGT PSS L SS PTIMAAG PLL VP VG FTLNFTITNLQ YGBDMGHPGSRKFNTTERVLQGLLGPIFKNTS PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYT LDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITN

ICTHRLDPKSPGVDREQLYWELSQLTNGIKELGP YTLDRHSLYWGFTHWIPVFTSSTPG TSTVDLGSGTPSSL PS PTS ATAGPLLWFTLNFT ITKLKYEEDMHCPGSRKFNTTERVLQ S LLGPMFKMTS VGPLYSGCRLTLLRSEKDGAATGVDA ICTHRWPKS PGVDRBQLYWELS QLTHGIKELGP ytldrnslyvngpthqts APNTSTPGTSTVOLG TSGTPSSLPS PTSAG LLVPFTLNFTITNLqYEECMHHPGSRXFNTrERVLQGLLGFMFKNT P, PE SVGLLYSGCRLTI.LR KHGAATGMDAI CSHRLDPKSPGLHREQLYWBLSQLTHGIKE LG? YTLLRNSLYYHGF THRSSVAPTSTPGTSTVDLGTaGTPSSL PSPTTAVPLL VPFTLWFTITNLQYGEDMRHPG SRKFHTTERVLQGLLG PLFKWS SVG PLYSGCRL1S LRS EKDGAATGVD Al CTHHLMPQSP GLDKEOLYMOLSęMTNGiKELGPYTLDRfISLYTNGPTURSSGLTTSTPWTSTyDLGTSGT P3P VPS PTT AGPLLVPFTLNFT Ϊ TNLQYEEDMHRPGSRKHNPTSPLGLSPGLSPGLKSPGLSPKL_JYWKLSQI_tTHlIITELGPY SWRDSL YVNGFTHQWS VPTTSTPGTSTVWATTGTPS S FPGHTE PGPLLIPPTFNFTIT NLH YEENMQH PGS RKFRTTERVLQGLLKPLFKNTS VGPL YSGCRLTLLRPEKQEAATG VD TI CTHR VDPIGPGLDRER LYMELSQLTWS ITELG PYTLDRDSLYVNGPNPWSSVPTTS TP GTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQ GLLKPLFKSTSVGPI »YSGCRI.TLLR PEKHGAATGVDAICTLRLDPTGPGLDRERLYWELS QLTNSVTELGP YTLDRDSLYWGFTHRSS YPTTSIPOTS AVHLETSGTPASLPGHTAPGP LLVPFTL WFTITKLQ YEEDMRH PGSRKFKTTERVLQGLL KPLFKSTS VG PLYSGCRLTLL R PK KRC .ATGWTI CTH ^ RT DPI .NPGI , DP. KQI.WKI.SX: .TRGI IELGPYLLDRGSLYVNGP THRNPVPItstpgtstvklgtsetpsslprpiVPGPLl, VPPTLNFTitnlqyeeamrHPG SRKFttTTERVLQGLLR EN FKNTSIGPLYSSCRLTL LRPEKDKAATRVDAICTHHPDPOS P glnreql ™ elsqlthgitelgpytldrdslyvdg fthwsp ipttstpgtsi wtlctsci PPSLPETTATGPLLVPFTLNFTITHLQYEENMGHPGSRKFWlTESVLCGl_LLKF¹I.FKSTSV GPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPY TLDRDSLYVHGFTQRS SVPTTSTPGTFTVQPETS ETP3 S LPG PTATGP VLLP PTLNFTII nlqyeedmhrpgsrkfnttervlqgllmplfkntsvsslysgcrltllrpekdgaatrvd AVCTHRPD PKSPGLDRERL YWKLSQLTHGITELGPYTLDRHS LYVNG FTHQS £ MTTTRTP DTSTHHLATSRTPASLSGPTTASPLLVLFTIHFTITNLRYEEHMHHPGSRKFłnTERVLQ GLLRPyFKWTSYGPLYSGCRLTLLRPKKDGAATfiYDAiCTYRPDPKSPGLDREOLYWELS QLTHSITELGPYTLDRDSLYVNGPTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPS AAS P LL YLFTLNFTITNLRYE RNMqiiPGSRKENTTERVLQGLLRSLEKS TS VGPL YSGCRLTLŁ RPEKDGTATGVDAICTHHPD PKS PRLDREQL TOEŁSQLTHNI TELGP YALDNDSLFYHGF THRSS VS TTSTPGT PTVYLCASKTPASIF3PSAASHLLIL FTLN PTI TNLfcYEEHWW PGS RKFNTTERVLCGLLRPLFKNTS VGPL YSGCRLTLLR PEKDGEATG TOAICTHRPDPTG PG LDREQLYLEL SOLTHS ITELGPYTLDRDSLYVMG FTHRS S VPTTS TG WSEE PFTLNFTI NKLR YMADMGOPGS LKFNITDHVMQHLLSPLFQRS SLGARTTGCRVIALR ΞVKHGAETRV DLLCT YLQPLSGPGLPIKQVFHELSQQTHGITRLGP YSLDKDS L YLHG YJTEPGPDE P PTT PKPATTFLPPLSEATTAMGYRLKTLTLNFTISWLQYSPDMGKG3ATPNSTEGVLQHLLRP L FQKS SMG PP YLGCQLIS L RPEKDGAATGVDTTCTYHPDPVGPGLDIQQL YWELSOLTHG VTQLGFYVWRDSLFIEJa YAPQNLSIRGBYQIWFHIVNWHLSETPDPTSSEYITLLRDIQD KVTTLYKGSQLHDTFRFCLVTHLTMDSVLVTVKALPSSEILDPSLVEQVFLDKTLNASFHW LGST YQL VDIHVTEMES S VYQPTS SSSTQH P YLNFTITNLP YSQDKAQ PGTTNYCRNKRII IEDALBQLFRHSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFS PLARRVDRVAIYEEFL RMTRUGTQLQNFTLDRSSVLVDGYS PNKNEPLTGNSDL PFWAYILIGLAGLLGLITCLIC

GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ (3EQ ID W0; 4) (5) MPF (MPF, MSLN, SMR, megakaryocyte growth enhancer, mesothelin, Genbank accession number NM_005823 Yamaguchi, N., et al. Biol. Chem. ), Proc. Natl. Acad. Sci. USA. 96 (20): 11531-11536 (1999), Proc. Natl. Acad. Sci. USA. 93 (1): 136-140 (1996), J. Biol. Chem. 270 (37): 21984-21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; page 287-288); WO2002101075 (claim 4; page 308-309); WO200271928 (page 320-321); WO9410312 (page 52-57); Cross-references: MIM: 601051; NP_005814.2; NM_005823_1

622 aa

malptarpllgscgtpalgsllflłfslgwvqpsrtlagetgqeaapldgvlanppniss LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL DLLLFLNPDAFSGPQACTRFFSRITKANVDLLiPRGAPERQRIiLPAALACWGVRGSLLSEA DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW SVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKARBIDESLIFYKKWBLEACVDAALLATQMDRVNAIPFTYEQIiDVljKHKLDELY PQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVK GRGQLDKDTLDTLTAFYPGYL CSLS peels SVP PSSIWAVRPQDLDTCDPRQLDVLYPKA RLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQ KLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMLLAGALLA

CSEQ ID NO; 5) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II, sodium-dependent phosphate transporter 3b, accession number Genbank NM_006424, J. Biol. Chem. 277 ( 22): 19665-19672 (2002), Genomics 62 (2): 281-284 (1999), Feild, JA, et al. (1999) Biochem. Biophys. Res. Commun. 258 (3): 578-582); WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17-19); WO200157188 ​​(claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; pages 139-140); Links: MIM: 604217; NP_006415,1; NM_006424_1

<img file="PL1725249T3_D0052.tif" />

MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIELLPSYSTATLI

DEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGRLILLLGFLYFFVCSLDILSS

AFQLVGGKMAGQFFSNSSIMSNPLLGLVIGVLVrVLVQSSSTSTSIWSMVSSSLLTVRA

AIPIIMGANIGTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHY

LEIITQL1VESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAXNKSLVK

AND WC KT FTNKTQINVTVPSTANCTSPSLCWTD3IQNWTMKNVTYKENIAKCQHIFVNFHLP

FOR VGTI LL ILSLLVLCGCLIMIVKILGS VLKGQVATVIKKTINTDFP FPFAWLTGYLAI

LVGAGMTFIVQSSSVFTSALTPLIGIGVITIERAYPLTLGSNIGTTTTAILAALASPGNA

LRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMAKGLGNISAKYRWFAVFYLIIFFFLI

PLTVFGLSLAGWRVLVGVGVPWFIIILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSL

KPWDAWSKFTGCFQMRCCYCCRVCCRACCLLCGCPKCCRCSKCCEDLEEAQEGQDVPVK

APETFDNITISREAQGEVPASDSKTECTAL (SEQ ID NO: 61 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878, Nagase T., et al. (2000) DNA Res. 7 (2): 143150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; page 50); WO200188133 (claim 1; pages 4143, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); accession numbers: Q9P283; EMBL; AB040878; BAA95969.1. Geneva; HGNC: 10737;

1093 aa

MVLAGPLAVSLLLPSLTLLVSHLS SS QDVS S BP SS EQQLCALS KHPTVAFEDLQ p WVSNF TYPGAHD FSQLALDPSGNQL X VGARN YLFRLSLAMV SLLQATEWAS SEDTRRS CQSKGKT EEECQWYVRVL IVAGRKVPMCGTNAFS PMCTSRO VG NL S RT portfolio NG7ARC PYDPRHWST AVI SS QG ELYAATTIDFSGRDP AIYRS LGSGP PLRTAQ YHS KWLNEPNTVAAYDIGLFAY FFLRENAVĘHDCGRTVYSRVAR VCKNDVGGR flle DTWTTFMKARLNCS RPGEV PFY yne LQS AF HL PEQDLIYG VFTTWNSIAAS AVCAFWLS AIS QAFWG PFRYQEN PRAAWLPI AW PI PN FQCGTLPETGPWENLTER £ LQDAQRL FLMSEAVQ PTT PE PCVTQDS VR FSHL WDL VQAKDTLYHVLYIGTESGTILKALS TASRSLHGC YLEEŁHVŁ PPGRREPLRS L RILHS AR ALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCOWDGKQQRCSTLEDSSKMSLWTQHI TAC P VRKVT RDGGFG PWS PWQP CE HLDGDNSGS CLCRARS CDSPRPRCGGLDCLGPAIHI AWCSKWGAWT P WSSWALCST SCGIGFQVRQRSCSWPAPRHGGR IC VGKS REER FCKENTP CP VPIF WAS WGS WSKCSSWCGGGMQSRRRACE WG WS CLGCGVEFKTCNPEGCPEVRRNTP WTP WL P VWVTQGGARQEQRFRFTC RA Plada PHGLQFGRRRTETRTCPADGSGSCETDALV EDLLRSGSTS PHTVS GGWAAWG PWSSCSRDC ELGFRVRKRTCTNPE PRNGGLPCVGDAAE YQDC NPQACPVRGAWS CWTSWS PCSASCGGGHYQRTRS CTS PAPS PG E DlCLGŁHTEEAIi CATQACPEGWS P WSE WSKCTDDGAOSRSRHCEEIiLPGS SACAGNSSQSRPCPYSEIP VIL PAS 3MEEATGCAGFNL IHLVATGIS C FLGSQYQSL

HLHYKGGGT PKNEKYTPMEFKTLNKNN LI PDDRAWFYPLQQTNVYTT T YYPS PLN KHSFR PEASPGQRCPPHS {SEQ ID HO; 7) (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKENCNA gen 2700050C12 cdna gen 2700050C12 US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (example 5); WO2003105758 (claim 12); US2003206918 (example 5); EP1347046 (claim 1); WO2003025148 (claim 20); Links: GI: 37182378; AAQ88991.1;

AY358628_1

141 aa

MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQIiNNDCSSPEFIVNCTVNVQDMCQKEVME

QSAGIMYRKSCASSAACLIASAGYQSFCSPGKLNSVCISCCNTPLCNGPRPKKRGSSASA

LRPGLRTTILFLKLALFSAHC (SEQ ID NO: 8) (9) ETBR (type B endothelin receptor, Genbank accession number AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy NA, et al. J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J.

Cardiovasc. Pharmacol. 20, S1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg RL, et al. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij JB, et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra RMW, et al. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger NP et al. Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Moth. Genet. 4, 2407-2409, 1995; Auricchio A., et al. Hum. Moth. Genet. 5: 351-354, 1996; Amiel J., et al. Hum. Moth. Genet. 5, 355-357, 1996; Hofstra RMW, et al. Nat. Genet. 12, 445-447, 1996; Svensson PJ, et al. Hum. Genet. 103, 145-148, 1998; Fuchs S., et al. Moth. Med. 7, 115124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (claim 1); WO2004048938 (example 2); WO2004040000 (claim 151); WO2003 087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (Fig 1); WO2003016494 (Fig 6); WO2003025138 (claim 12; page 144); WO200198351 (claim 1; pages 124-125); EP522868 (claim 8; Fig 2); WO200177172 (claim 1; pages 297-299); US2003109676; US6518404 (Fig 3); US5773223 (claim 1a; Col 31-34); WO2004001004;

442 aa

MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPTKTLWPKGSNA

SLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTWSCLVFVLGIIGNS

TLLRIIYKNKCMRNGPNILIASLALGDLLHIVIDIPINVYKLLAEDWPFGAEMCKLVPFI

QKASVGITVLSLCALSIDRYRAVASWSRlKGIGVPKWTAVEIVLIWWSWLAVPEAIGF

DIITMDYKGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM rjRKKSGMQIALNDHLKQRREVAKTVFCLVLVFA LCWLPLHLSRILKLTIjYNQNDPNRCEL

LSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSCLCCWCQS FEEKQSLEEKQSC

LKFKANDHGYDNFRSSNKYSSS (SEQ ID NO: 9) (10) MSG783 (RNF124, hypothetical FLJ20315 protein, Genbank accession number NM_017763); WO2003104275 (claim 1); WO2004046342 (example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; Fig 93);

WO200166689 (example 6); Links: Locus ID: 54894; NP_060233.2;

NM_017763_1

783 aa

MSGGHQLQLAALWPWLLMATLQAGFGRTGLVIAAAVESERSAEQKAIIRVIPLKMDPTGK

LNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDDDNLEPGFISIVKLESPRRAPRPCL

SLASKARMAGERGASAVLFDITEDRAAAEQLQQPLGLTWPWLIWGNDAEKLMEFVYKNQ

KAHVRIELKEPPAWPDYDVWILMTWGTIFVHLASVLRIRCRPRHSRPDPLQQRTAWAI

SQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVD

PWLHQHRTCPLCVFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAHYHLPAAYLLG

PSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPGEQQRLAGAQHPYAQGWGMSH

LQSTSQHPAACPVPLRRARPPDSSGSGESYCTBRSGYLADGPASDSSSGPCHGSSSDSW

NCTDISLQGVHGSSSTFCSSLSSDFDPLVYCSPKGDPQRVDMQPSVTSRPRSLDSWPTG

ETQVSSHVHYHRHRHHHYXKRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTG

SNSAAPSGRLSNPQCPRALPEPAPGPVDASSICPSTSSLFNLQKSSLSARHPQRKRRGGP

SEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLICGPPGLDKRLLPETPGPCYS

NSQPVWLCLTPRQPLEPHPPGEGPSEWSSDTAEGRFCPYPHCQVLSAQPGSEEELEELCE

QAV (SEQ ID KO: 10) (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, gene 1 associated with prostate cancer, protein 1 associated with prostate cancer, epithelial prostate antigen with six [ transmembrane domains 2, prostate protein with six transmembrane domains, Genbank accession number AF455138, Lab. Invest. 82 (11): 1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; Fig 1); WO200272596 (claim 13; pages 54-55); WO200172962 (claim 1; Fig 4B);

WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; Fig 10); WO200226822 (claim 23; Fig 2); WO200216429 (claim 12; Fig 10); Links: GI: 22655488; AAN04080.1; AF455138_1

490 aa

MESISMMGSPKSLSETVLPNGINGIKDARKVTVGVIGSGDFAKSLTIRLIRCGYHWIGS

RNPKFASEFFPHWDVTHHEDALTKTNIIFVAIHREHYTSLWDLRHLLVGKILIDVSNNM

RINQYPESNAEYLASLFPDSLIVXGFNWSAWALQLGPKDASRQVYICSNNIQARQQVIE

LARQLNFIPIDLGSLSSAREIENLPLRLFTLWRGPVWAISLATFFFLYSFVRDVIHPYA

RNQQSDFYKIPIEIVNKTL PIVAITLLSLVYLAGLLAAAYQLYYGTKYRRFPPWIiETWLQ

CRKQLGLLSFFFAMVHVAYSLOLPMRRSERYLFLNMAYQQVHANIENSWNEEBVWRIEMY

ISFGIMSLGLLSLLAVTSIPSVSNALNWREFSFIQSTLGYVALLISTFHVLIYGWKRAFE

EEYYRFYTPPNFVLALVLPSIVII&gt; GKIILFLPCISQKLKRIKXGWEKSQFLEEGIGGTIP

HVSPERVTVM (SEQ ID NO: 11) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor of a potential cationic channel, M subfamily, member 4, Genbank accession number NM_017636 Xu, XZ, et al. Proc. Natl. Acad. Sci. USA. 98 (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J. Biol. Chem. 278 (33): 30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; pages 100-103); WO200210382 (claim 1; Fig 9A); WO2003042661 (claim 12); WO200230268 (claim 27; page 391); US2003219806 (claim 4); WO200162794 (claim 14; Fig 1A-D); Links: MIM: 606936; NP 060106.2; N 0176361

1214 aa

MW PE KEQS WIPKIFKKKTCTTFIVDSTD PGGTLCQCG RPRTAHP AVAMEDAFGAAWTV «DS DAHTTEKPTDAYGELDF TGAORKH StTFLRLSDRTDPAAVYSJj VTRT BY FRAPNL WS VLGGSGGPVLQTWLQDLLRRGLVRAAQSTGAWIVTGGLHTGIGRHVGVAVRDHQHASTGG TKWAMGVAPWGWRNIWTLIKPKGS F PARYRWRGDPE VQ DG F PLD YS YN IA FLVDDGTH

GCL.GGENRFRLRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRIENATQAQLPCLL

VAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIRRFFPKGDLEVLQAQVERIMTRKEL

LTVYSSEDGSEEFETIVLKALVKACGSSEASAYLDELRLAVAWNRVDIAQSELFRGDIQW rsfhleaslmdallndrpefvrllishglslghfltpmrlaqlysaapsnslirnlldqa

SHSAGTKAPALKGGAAELRPPDVGHVLRMLLGKMCAPRYPSGOAWDPHPGQGFGESMYLL

SDKATSPLSU3AGLGQAPWSDIiLLWALLLNRAQMAMYFWEMGSNAVSSALGACLLLRVMA

RLEPDAEEAARRKDLAFKFEGMGVDLFGECYRSSEVRAARLLLRRCPLWGDATCLQIAMQ

ADARAFFAQDGVQSLLTQKWWGDMASTTPIWALVLAFFCPPLIYTRLITFRKSEEEPTRE

ELEFDMDSVINGEGPVGTADPAEKTPLGVPRQSGRPGCCGGRCGGRRCLRRWFHFWGAPV

TIFMGNWSYLLFLLLFSRVLLVDFQPAPPGSLELLLYFWAFTLLCEELRQGLSGGGGSL asggpgpghaslsqrlrlyladswnqcdlvaltcfllgvgcrltpglyhlgrtvlcidfm

VFTVRLLHIFTVNKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEGLLRPRDSDFP

SILRRVFYRPYLQIFGQIPQEDKDVALMEHSNCSSEPGFWAHPPGAQAGTCVSQYANWLV

VLLLVIFLLVANILLVNLLIAMFSYTFGKVQGNSDLYWKAQRYRLIREFHSRPALAPPFI

VISHLRLLLRQLCRRPRSPQPSSPALEHFRVYLSKEAERKLLTWESVHKENFLLARARDK

RESDSERLKRTSQKVDLALKQLGHIREYEQRIiKVLEREVQQCSRVI&gt; GWVAEALSRSAI.LP

PGGPPPPDLPGSKD (SEQ ID Nd! 12) (13) CRIPTO, (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma growth factor, accession number Genbank NP_003203 or NM_003212, Ciccodicola, A., Et al. EMBO J. 8 ( 7): 1987-1991 (1989), Am. J. Hum. Genet. 49 (3): 555-565 (1991)); US2003224411 (claim 1); WO2003083041 (example 1); WO2003034984 (claim 12); WO200288170 (claim 2; pages 52-53); WO2003024392 (claim 2; Fig 58); WO200216413 (claim 1; pages 94-95, 105); WO200222808 (claim 2; Fig 1); US5854399 (example 2; Col 17-18); US5792616 (Fig 2); Links: MIM: 187395; NP_003203.1; NM_003212_1

188 aa mdcrkmarfsysviwimaiskvpelglvaglGhqefarpsrgylafrddsiwpqeepair

PRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPH

DTWLPKKCSLCKCWHGQLRCFPQAFLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGI

CLSIQSYY {SEQ ID MO: 13} (14) CD21 (CR2 (complement receptor 2) or C3DR (receptor C3d / virus

Epstein-Barr) or Hs.73792, Genbank accession number M26004, Fujisaku et al. (1989) J. Biol. Chem. 264 (4): 2118-2125); Weis JJ, et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Natl. Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., et al. Moth. Immunol. 35, 1025-1031, 1998; Weis JJ, et al. Natl. Acad. Sci. USA. 83, 56395643, 1986; Sinha SK, et al. (1993) J. Immunol. 150,5311-5320; WO2004045520 (example 4); US2004005538 (example 1); WO2003062401 (claim 9); WO2004045520 (example 4); WO9102536 (Fig. 9.1-9.9); WO2004020595 (claim 1); accession numbers: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

1033 aa

MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGE

KSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKT

NFSMNGNKSVWCQANNMWGPTRLPTCVSVFPLECPALPMIHNGHHTSENVGSIAPGLSVT

ΥδαΕβΟΥΙΟινσΕΚΙΙΝσΒΞδσΚΜδΑνΡΡΤΟΕΕΑΡσΚβΕΟΡΡΡΝΟΚνΚΕΡΡΙΙ, κνσνΤΑΝΡ

FCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHIGNSLANVSYGSI

VTYTCDPDPEEGVNFILIGESTLRCT7DSQKTGTWSGPAPRCELSTSAVQCPHPQILRGR

MVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEPSAPVCEKECQAPPNIŁNGO

KEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLT

KPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWPMEIRLCKEITCPPPPVIYNGAHTG

SSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAV

QCSHVHIANGYKISGKEAPYFYNDTVTFKCYSG FTLKGS SQIRCKADNTWDPEIPVCEKE

TCQHVRQSLQELPAGSRVELVNTSCQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCH

PPPVIVNGKHTGMMAE2JFLYGNEVSYECDQGFYLLGEKKLQCRSDSKGHGSWSGPSPQCL

RSPPVTRCPNPEVKHGYKLNKTHSAYSHNDIVYVDCHPGFIMNGSRVIRCHTDNTWVPGV

PTCIKKAFIGCPPPPKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTW

SQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAWTLECEDGYMLEGSPQSQCQSDHQ

WNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRBRNYYTDTSQKEAFHLEA

REVYSVDPYNPAS {SEQ ID NO; 14) (15) CD79b (CD79B, CD79B, IGb (beta-linked immunoglobulin), B29, Genbank accession number NM_000626 or 311038674, Proc. Natl. Acad. Sci. USA. (2003)

100 (7): 4126-4131, Blood (2002) 100 (9): 3068-3076, Muller et al. (1992) Eur. J.

Immunol. 22 (6): 1621-1625); WO2004016225 (claim 2, Fig 140);

WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (example 2); US2002150573 (claim 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Fig 17A / B); WO200055351 (claim 11, pages 1145-1146); Links: MIM: 147245; NP_000617.1; NM_000626_1

229 aa marlalspvpshwmvalllllsaepvpaarsbdryrnpkgsacsriwqsprfiarkrgft VKMHCYMNSASGNVSWLWKOEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIY FCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLL LDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO: 15} (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank Accession No. NM_030764, Genome Res. 13 (10): 2265-2270 (2003), Immunogenetics 54 (2): 87-95 (2002), Blood 99 (8): 2662-2669 (2002), Proc. Natl. Acad. Sci. USA. 98 (17): 9772-9777 (2001), Xu, MJ, et al. (2001) Biochem. Biophys. Res. Commun. 280 (3): 768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; Fig 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25); Links: MIM: 606509; NP 110391.2; NM 0307641

508 aa

MLLWSLLVIFDAVTEQACSLTLVAPSSVFEGDSIVLKCQGEQNWKIQKMAYHKDNKELSV

FKKFSDFLIQSAVLSDSGNYFCSTKGQLFLWDKTSNIVKIKVQELFQRPVLTASSFQPIB

GGPVSLKCETRLSPQRLDVQLQFCFFRENQVLGSGWSSSPELQISAVWSEDTGSYWCKAE

TVTHRIRKQSLQSQIHVQRIΡISNVSLEIRAPGGQVTEGQKLILLCSVAGGTGNVTFSWY

REATGTSMGKKTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKWNIPVRIPVSRP vltlrspgaqaavgdllelhcealrgsppilyqfyhedvtlgnssapsggGasfnlslta

EHSGNYS CEANNGLGAQCSEAVPVSISGPDGYRRDLMTAGVLWGLFGVLGFTGVALLLYA

LFHKISGESSATNEPRGASRPNPQEFTYSSPTPDMEELQPVYVNVGSVDVDWYSQVWSM

QQPESSANIRTLLENKDSQVIYSSVKKS (SEQ ID NO: 16} (17) HER2 (ErbB2, Genbank accession number M11730, Coussens L., et al. Science (1985) 230 (4730): 1132-1139); Yamamoto T., et al. Nature 319 , 230-234, 1986; Semba

K., et al. Natl. Acad. Sci. USA. 82, 6497-6501, 1985; Swiercz JM, et al. J. Cell Biol. 165, 869-880, 2004; Kuhns JJ, et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al. Nature 421, 756-760, 2003; Ehsani A., et al. (1993) Genomics 15, 426-429; WO2004048938 (example 2); WO2004027049 (Fig 11); WO2004009622;

WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1);

US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29;

Fig 1A-B); WO2003025228 (claim 37; Fig 5C); WO200222636 (example 13;

pages 95-107); WO200212341 (claim 68; Fig 7); WO200213847 (pages 71-74);

WO200214503 (pages 114-117); WO200153463 (claim 2; pages 41-46); WO200141787 (page 15); WO200044899 (claim 52; Fig 7); WO200020579 (claim 3; Fig 2); US5869445 (claim 3; Col 31-38); WO9630514 (claim 2; pages 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (example 3; Fig 4); accession numbers: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1. 1255 aa

MELAALCRWGLLLAIiL&gt; PPGAASTQVCTGTDMKLRLPASPETHLDMLRHIiYQGCQWQGN1i

ELTYIiPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYAIAVLDNG

DPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTII, WKDIFHKNNQIA

ΏΤΕΙϋΤΝΕΞΡΑΟΗΡΩβΡΜΟΚαΕΡΟίΟΕΕβΕΟΟΟδΒΤΕΤνσΑΟΟΟΑΚΟΚΟΡΕΡΤϋΟΟΗΕΟσ

AAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP

YNYLSTDVGSCTLVCPLHNQBVTAEDGTQRCEKCSKPCARVCYGLGMBHLREVRAVTSAN

IQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITOYLYISAWPDSl4P

DLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTV

PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQEC

VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC

PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSI ISAWG

ILLWVLGWFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETEL

RKVKVLGSGAPGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSP

YVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVR

LVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT

HQSDVWS YGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM

IDSECRPRPRELVSEFSRMARDPQRFWIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDA

EEYLVPCQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEG

AGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYV

NQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGWKDVFAFGGAVENPEYŁTPQ

GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID NO: 17) (18) NCA (CEACAM6, Genbank accession number M18728); Barnett T., etal Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988 Strausberg RL, et al. Natl. Acad. Sci. USA. 99: 16899-16903, 2002; WO2004063709; EP 143 93 93 (claim 7) WO2004044178 (example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (example 2) WO200286443 (claim 27; page 427); WO200260317 (claim 2); accession numbers: P40199; Q14920; EMBL; M29541; AAA59915,1. EMBL; M18728;

100

344 aa

MOPPSAPPCRLiHVPWKEVLLTASLI «TFWNPPTTAKLTIESTPFNVAEGKEVLIjIiAKNIiPQ

NRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLŁiIQNVTQNDTGFY

TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWV

NGQSLPVSPRLQLSNGNMTLTLLSVKRHDAGSYECEIQNPASANRSDPVTLMVLYGPDVP

TISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQ

AHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI (SEQ ID NO: 18) (19) MDP (DPEP1, Genbank accession number BC017023, Proc. Natl. Acad. Sci. USA. 99 (26): 16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 (Fig 6-8); WO9946284 (Fig 9); Links: MIM: 179780; AAH17023.1; BC017023_1

411 aa

MWSGWWLWPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQLLDMFNNRLQDERANLTT

LAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVRRTLEQMDVVHRMCRMYPETFLYVT

SSAGIRQAFREGKVASLIGVEGGHSIDSSLGVLRALYQLGMRYI, TLTHSCNTPWADNWLV

DTGDSEPQSQGLSPFGQRWKELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYS

VCASRRNVPDDVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLDHIKEVAGARAVG

FGGDFDGVPRVPEGLEDVSKYPDLIAELLRRNWTEAEVKGALADNLLRVFEAVEQASNLT

QAPEEEPIPLDQLGaSCRTHYGYSSGASSLHRHWGLLLASLAPLVLCLSLL (SEQ ID NO: 19) (20) IL20Ra (IL20Ra, ZCYTOR7, Genbank accession number AF184971); Clark HF, 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; Dumoutier L., et al. J. Immunol. 167,3545-3549,2001; Parrish-Novak J., et al. J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry 42: 12617-12624; Sheikh F., et al. (2004) J. Immunol. 172, 2006-2010; EP1394274 (example 11); US2004005320 (example 5); WO2003029262 (pages 74-75); WO2003002717 (claim 2; page 63); WO200222153 (pages 45-47); US2002042366 (pages 20-21); WO200146261 (pages 57-59); WO200146232 (pages 63-65); WO9837193 (claim 1; pages 55-59); accession numbers: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

101

553 aa

MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGLPKPANITFLSINMKNVLQWTPPE glqgvkvtytvqyfiygqkkwlnksecrninrtycdlsaetsdyehqyyakvkaiwgtkc

SXWAESGRPYPFLETQIGPPEVAI.TTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLK

YNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTL kdqssefkakiifwyvlpisitvflfsvmgysiyryihvgkekhpanliliygnefdkrf

FVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNDPQPSGNLRPPQEEEBVKHL

GYASHLMEIFCDSEENTEGTSFTQQESLSRTIPPDKTVIEYEYDVRTTDICAGPEEQELS

LQEEVSTQGTLLESQAALAVLGPQTLQYSYTPQLQDLDPLAQBHTDSEEGPEEEPSTTLV

DWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQ

FMEEWGLYVQMEN (SEQ ID NO: 20) (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053) Gary SC, et al. Gene 256, 139-147, 2000; Clark HF, et al. Genome Res. 13, 2265-2270, 2003; Strausberg RL, et al. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; Fig 52); US2003119122 (claim 1; Fig 52); US2003119126 (claim 1); US2003119121 (claim 1; Fig 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; Fig 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1);

911 aa

MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVH YLRPPPSRRA.VLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDV SIALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGWFLYREGSARYAFSFSGAQEACA RIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGW DPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGABrATTGQLYAAWDGGLDH CSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPS AIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSST PEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEBKYEDSEEKEEEEEEEEVEDEALW AWPSELSSPGPEASLPTEPAAQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPT ETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRA PEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAWPASGDCVPSPCHNGGT CLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYG AHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLS GENCWMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVL RYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEED PEGRQGRLLGRWKAL

LIPPSSPMPGP fSBQ ID NOe21)

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

NM_004442) Chan, J. and Watt, VM, Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5): 897-905 (1995), Annu. Rev. Neurosci. 21: 309-345 (1998), Int. Rev. Cytol. 196: 177244 (2000)); WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9);

WO2003004529 (pages 128-132); WO200053216 (claim 1; page 42); Links: MIM: 600997; NP_004433.2; NM_004442_1

987 aa

MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIRTYQ

VCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSIPSVPGSCKETFNLYYYEADF

DSATKTFPNWMENPWVKVDTIAADESFSQVDLGGRVMKINTEVRSFGPVSRSGFYLAFQD

YGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLY ·

CNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGA

TNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLMLEWTPPRDSGGREDLVYNI

ICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAVNGVTDQSPF

S PQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKELSE

YNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEK

LPLIIGSSAAGLVFLIAWVIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDP

FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVCSGHLKLPGKREIFVAIKTLKSGY

TEKQRRDFLSEASIMGQFDHPNVIHLEGWTKSTPVMIITEFMENGSLDSFLRQNDGQFT

VIQLVGMLRGIAAGMKYLADMNYVHRDLAARNILVNSNLVCKVSDFGLSRFLEDDTSDPT

YTSALGGKIPIRWTAPEAIOYRKFTSASDVWSYGIVMWEVMSYGERPYHDMTNQDVINAI

EQDYRLPPPMDCPSALHQLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSG

INLPLLDRTIPDYTS FNTVDEWLEAIKMGQYKE SFANAGFTS FDWSQMMMEDILRVGVT

LAGHQKKILNSIQVMRAQMNQIQSVEV (SEQ ID NO.-22) (23) ASLG659 (B7h, Genbank accession number AX092328) US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (Fig 3); US2003165504 (claim 1); USA2003124140 (example 2); US2003065143 (Fig 60); WO2002102235 (claim 13; page 299); US2003091580 (example 2); WO200210187 (claim 6; Fig 10); WO200194641 (claim 12; Fig 7b); WO200202624 (claim 13; Fig 1A-1B); US2002034749 (claim 54; pages 45-46); WO200206317 (example 2; pages 320-321, claim 34; pages 321-322); WO200271928 (pp. 468-469); WO200202587 (example 1; Fig 1); WO200140269 (example 3; pages 190-192); WO200036107 (example 2; pages 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pp. 233-234,452-453); WO 0116318;

103

232 aa

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP DIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV QLTDAGTYKCYIITSKGKKNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTW WASQVDQGANFS EVSNTSFELNS ENVTMKWS VLYNVTINNTYSCMIENDIAKATGDIKV TESEIKRRSHLQLLNSKASLCVSS FFAISWALLPLS PYLMLK (SEQ ID NO: 23l (24) PSCA (precursor antigen stem cells, prostate cancer, GenBank Accession No. AJ297436) Reiter RE et al., Proc. Natl Acad. Sci. USA. 95, 17351740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res Commun. (2000) 275 (3): 783-788; WO2004022709; EP1394274 (example 11); US2004018553 (claim 17) WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO2003003906 (claim 10; page 288) WO200140309 (example 1; Fig 17); US2001055751 (example 1; Fig 1b); WO200032752 (claim 18; Fig 1) W09851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; Fig 1B); accession numbers: 043653; EMBL; AF043498; AAC39607.1.

123 aa

MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLT

VISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGP

GQL (SEQ ID NO: 24) (25) GEDA (Genbank accession number AY260763); AAP14954 HMGIC lipoma phase-like protein / pid = AAP 14954.1 -Homo sapiens species: Homo sapiens (human) WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (example 3, Claim 20); US2003194704 (claim 45); Links: GI: 30102449; AAP14954.1; AY2607631

236 aa

MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVRNSRAIGVLWAIFTICFAIVNVVCFIQPYW igdgvdtpqagyfglfhycigngfsreltcrgsftdfstlpsgafkaasffiglsmmlii

ACIICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKRMCGEKTDKYT

LGACSVRWAYIIAIIGILDALILSFLAFVLGNRQDSLMAEELKAENKVLLSQYSLE {SEQ ID NO: 25) (26) BAFF-R (B cell activating factor receptor, BLyS 3 receptor, BR3,

NP_443177.1); NP_443177 BAFF receptor / pid = NP_443177.1 -Homo sapiens Thompson, JS, et al. Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (example; pages 32-33); WO2003014294 (claim 35; Fig 6B); WO2003035846 (claim 70; pages 615-616);

104

WO200294852 (Col 136-137); WO200238766 (claim 3; page 133); WO200224909 (example 3; Fig 3); Links: MIM: 606269; NP_443177.1; NM_052945_1

184 aa

MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ

ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD

KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG

PEQQ (SEQ ID NO: 26) (27) CD22 (CD22-B B cell receptor isoform, Genbank accession number NP001762,1); Stamenkovic, I. and Seed, B., Nature 345 (6270), 74-77 (1990); US2003157113; US2003118592; WO2003062401 (claim 9); WO2003072036 (claim 1; Fig 1); WO200278524 (example 2); Links: MIM: 107266; NP_001762.1; NM_001771_1

847 aa

MHLLGP WLL YIAFSD £ 3KWVFEHPETLYAWEGACWIP GT YRALDGDLE £ F IL FH

NPEYNKNT'SKFEXjTRLYESTKDGECVPSBQKRVQFLGDKIiKNCTLSIl-iPVHLNDSG &lt;3U3I "R VS MES KTEKWERIHAN ERPFPPHIQLFPEIQESQEVTLTCLLNFS C YGYPIQLQWLLEG VPMRQAAVTSTS LTI KS VFTRS J3LKFS PQWSHHGKIVTCQLQDADGKFL £ NDTVQLNVKH TPKLEIKVTPSDAIVREGDSVTMTCEVSSSWPEYTTVSWLKDGTSLKKQNTFTLWLREVT KDQSGK YC CQVSNDVGPGRSBEVFLQVQYAPEPS T VQILHS PAVEGSGVEFLCMSLANPL PIWYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSC VAENILGTGQRGPGAELDVQYP PK

KVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYKWKPHGAWEEPSLGVLKIQNVGWDNT

TIACARCNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQ

FFWEKNGRLLGKESQLNFDS IS PEDAGS YSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM

SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPHHSQKLRLEPVKVQHSGAYWCQ

GTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQG

LQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAES

SEMQRPPRTCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENV

DYVILKH (SEQ ID NO: 27) (28) CD79a (CD79A, CD79a, bound to immunoglobulin alpha, a B cell specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, causes transduction of the signal involved cell differentiation B) PROTEIN SEQUENCE Full mpggpgv ... dvqlekp (1..226; 226 aa), pI: 4.84, MW: 25028 TM: 2 [P] Chromosome gene: 19q13.2, Genbank accession number NP_001774. 1; WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658

105 (claim 13, Fig 16); WO9207574 (Fig 1); US5644033; Ha et al. (1992) J. Immunol. 148 (5): 1526-1531; Mueller et al. (1992) Eur. J. Biochem. 22: 1621-1625; Hashimoto et al. (1994) Immunogenetics 40 (4): 287-295; Preud'homme et al. (1992) Clin. Exp. Immunol. 90 (1): 141-146; Yu et al. (1992) J. Immunol. 148 (2) 633-637; Sakaguchi et al. (1988) EMBO J. 7 (11): 3457-3464;

226 aa

MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASI, MVSLGEDAHFQCPHNSSN

NANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCG

TYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLPCAWPGTLLLFRKRWQNEKLGLDAGD

EYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 28) (29) CXCR5 (Burkitt's 1 lymphoma receptor, a G-protein coupled receptor that is activated by the chemokine CXCL13, is involved in the migration of lymphocytes and may be involved in the defense of HIV infection and may be involved in the development of AIDS, lymphoma, myeloma and leukemia) PROTEIN SEQUENCE Full mnypltl ... atslttf (1..372; 372 aa), pI: 8.54 MW: 41959 TM: 7 [P] Gene on chromosome: 11q23.3, Genbank accession number NP_001707.1; WO2004040000; WO2004015426; US2003105292 (example 2); US6555339 (example 2); WO200261087 (Fig 1); WO200157188 ​​(claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (example 1, pages 152153, example 2, pages 254-256); WO9928468 (claim 1, page 38); US5440021 (example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, Fig 5); Dobner et al. (1992) Eur. J. Immunol. 22: 2795-2799; Barella et al. (1995) Biochem. J. 309: 773-779;

372 aa

MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSL

IFLLGVIGNVLVLVrLERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTP

LCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLL

ALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVG

WHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSI&gt;

PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSL

SESENATSLTTF (SEQ ID NO: 29) (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen) which binds peptides and presents them to CD4 + T lymphocytes) PROTEIN SEQUENCE Full mgsgwvp ... vllpqsc (1..273; 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene on chromosome: 6p21.3, accession number Genbank NP_002111,1; Tonnelle et al. (1985) EMBO J. 4 (11): 2839-2847; Jonsson et al. (1989) Immunogenetics 29 (6): 411-413; Beck et al. (1992) J. Mol. Biol. 228: 433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99: 16899-16903; Servenius et al. (1987) J. Biol. Chem. 262: 8759-8766; Beck et al. (1996) J. Mol. Biol. 255: 1-13; Naruse et al. (2002) Tissue Antigens 59: 512-519;

106

WO9958658 (claim 13, Fig 15); US6153408 (Col 35-38); US5976551 (col 168170); US6011146 (col 145-146); Kasahara et al. (1989) Immunogenetics 30 (1): 66-68; Larhammar et al. (1985) J. Biol. Chem. 260 (26): 14111-14119;

273 aa

MGSGWVP WWALLVHLTRLDSSMTQGTDS PED F VIQAKADC Y FTtTGTEKVQ F WRFIFHL EEYVR FDS DVGMFVALTKLGQ PDAEQ WKSRLDIiLERSRQAVDG VCRHHYRLGAPFTVGRK VQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGP IRiIGDWT FQTWMLFMTPELGH VYTCLVDHSSLLS PVSVEWRAQ £ E YS WRKMLSGIAAFLLGLI PLL VGIVIQLRAQKGYVRTQMS GNEV SRAVL LPQSC (31) P2X5 (purinergic receptor P2X ion channel ligand gated 5, an ion channel gated extracellular ATP, may participate in synaptic conduction and neurogenesis, deficiency may contribute to the pathophysiology of detrusor instability) PROTEIN SEQUENCE Full mgqagck ... lephrst (1..422; 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene on chromosome: 17p13.3 , Genbank accession number NP_002552.2; Le et al. (1997) FEBS Lett. 418 (1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10: 165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);

422 aa mgqagckglclslfdyktekyviaknkkvgllyrllqasilaylwwvflikkgyqdvdt slqsavitkvkgvaftntsdlgqriwdvadyvipaqgenvffwtnlivtpnqrqnvcae

NEGIPDGACSKDSDCHAGEAVTAGNGVKTGRCLRRENLARGTCEIFAWCPLETSSRPEEP

FLKEAEDFTIFIKNHIRFPKFNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRW

AGSDFQDIALEGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARY

YRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKKYEEVRGLEDS

SQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKRGSSSQKGNGSVCPQLLEPHR

ST {SEQ ID NO: 31) (32) CD72 (B72 CD72 differentiation antigen, Lyb-2) PROTEIN SEQUENCE Full maeaity ... tafrfpd (1..359; 359 aa), pI: 8.66, MW: 40225 TM: 1 [P] Gen on chromosome: 9p13.3, accession number Genbank NP_001773.1; WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105106); Von Hoegen et al. (1990) J. Immunol. 144 (12): 4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99: 16899-16903;

107

359 aa

MAEAITYADLRFVKAPLKKSISSRLGQDPGADDDGEITYENVQ VPAVLGVPS SLAS S VLG

DKAAVKSEQPTASWRAVTSPAVGRILPCRTTCLRYLLLGLLLTCLLLGVTAICLGVRYLQ

VSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQA

AEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMH

QKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTG

LSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICEMTAFRFPD (SEQ ID NO; 321 (33) LY64 (lymphocytic antigen 64 (RP105), a type I membrane protein from the family of repeats rich in leucine and disease-dependent, activates B-cell activity) with systemic lupus erythematosus) PROTEIN SEQUENCE Full mafdvsc ... rwkyqhi (1..661; 661 aa), pI: 6.20, MW: 74147 TM: 1 [P] Gene on chromosome: 5q12, accession number Genbank NP_005573,1 ; US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38 (3): 299-304; Miura et al. (1998) Blood 92: 2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26);

661 aa

MAFDVSCFFWWLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEFLEF SFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLSTLVLTGNPLIFMAETS LNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARNLKVLDF QNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIBLGAFDSTVFQSLNFGGTPNLSVIFN GLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDISSTTFQCFTQL QELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCQ ISAANF PS LTHLYIRGNVKKLH LGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQTLNLSHNEPLGLQSQAFKECP QLELLDLAFTRLHINAPQSPFQtmHFLQVLNLTYCFLDTSNQHLLAGLPVLRHLNLKGNH FQDGTITKTNLLQTVGSLEVLILSSCGLLSIDQQAFHSLGKMSHVDLSHNSLTCDSIDSL SHLKGIYLNLAANSINIISPRLLPILSQQSTINLSHNPLDCTCSNIHFLTWYKENLHKLE GSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLIVFLLLLAILLFFAVKYLLRWKYQH (SEQ ID NO: 33) (34) FCRH1 (like protein 1 Fc receptor, a putative receptor for the immunoglobulin Fc domain that contains C2 domain of a type similar to the Ig and ITAM, may play a role in the differentiation of B cells) protein sequences Full mlprlll ... vdye-dam (1..429; 429 aa), pI: 5.28, MW: 46925 TM: 1 [P] Gene on chromosome: 1q21-1q22, accession number Genbank NP_ 443170.1; WO2003077836; WO200138490 (claim 6, Fig 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci. USA 98 (17): 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7);

108

429 aa

ML PRLLLLICAPLCEPAELFLI AS PS HPTEG SPVTLTCKMP FLQS SDAQFQFCFF RDTRA LG PG W SS S PKLQIAAMWKEDTGS YWCE AQTMASKVLR SR RS QINVH RVPVADVSLBTQPeGLSLSLSLY

YYCVAENGYGPSPSGLVSITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILY

WFYHEDITLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAQRSEAVTLNFTVPTGA

RSNHLTSGVTEGLLSTLGPATVALLFCYGLKRKIGRRSARDPLRSLPSPLPQEFTYLNSP tpgqlqpiyenvnwsgdevyslayynqpeqbsvaaetlgthmedkvsldiysrlrkani

TDTOYEDAM (SEQ ID NO: 34) (35) IRTA22 (superfamily of immunoglobulin receptors associated with translocation 2, putative immunoreceptor with a potential role in B cell development and lymphoma; gene deregulation by translocation occurs in certain B cell tumors) PROTEIN SEQUENCE Full mllwvil. ..assaphr (1..977; 977 aa), pI: 6.88 MW: 106468 TM: 1 [P] Chromosome gene: 1q21, Genbank accession number NP_112571.1; WO2003024392 (claim 2, Fig 97); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277 (1): 124-127; WO2003077836; WO200138490 (claim 3, Fig 18B-1-18B-2);

977 aa

MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFYSPQKTKWYHR ylgkeilretpdnilevqesgeyrcqaqgsplsspvhldfssaslilqaplsvfegdsw LRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHI PHACbKDNGAYRCTGYKESCCPVSSNT VKIQVQEPFTRPVLRASSFQPISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWS LSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFE GTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNG LGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAXVTLHCEAQRGSLPILYQFHHEDAA LERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSITVPVSHPVLTLSSA EALTFEGATVTLHCEVQRGS PQILYQPYHEDMPLWSSSTP SVGRVS FS FSLTEGHSGNYY CTADNGFGPQRSEWSLFVTVPVSRPILTLRVPRAQAWGDLLELHCEAPRGSPPILYWF YHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVSRPI LT FRAPRAQA WGDLLELHCEALRGSS PI LYWFYHEDVTLGKISAPSGGGAS FNLSLT TE HSGIYSCEADNGPEAQRSEMVTLKVAVPV3RPVLTLRAPGTHAAVGDLLELHCEALRGSP LILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGL TANRSGPFATGVAGGLLSIAGIAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYH NVPAWEELQPVYTNANPRGENWYSEVRIIQEXKKHAVASDPRHLRNKGSPXIYSEVKVA STPVSGSLFLASSAPHR (SEQ ID NO: 35) [0322] See. also: W004 / 045516 (03 Jun 2004); WO03 / 000113 (Jan 3, 2003);

WO02 / 016429 (Feb 28, 2002); WO02 / 16581 (Feb 28, 2002); WO03 / 024392 (March 27, 2003);

WO04 / 016225 (Feb 26, 2004); WO01 / 40309 (07 June 2001) and provisional American

109 Patent Application No. 60/520842 "COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN", filed November 17. 2003; all of which are attached to the description by reference in their entirety.

[0323] In an embodiment, the ligand-linker-drug conjugate has formula IIIa wherein the ligand is an Ab antibody, including one that binds at least one of the CD30, CD40, CD70 antigen, Lewis Y antigen, w = 0, y = 0 and D have the formula Ib. Exemplary conjugates of formula IIIa include those wherein R¹⁷ means - (CH2) 5-. Also included are conjugates of formula IIIa in which D has the structure of compound 2 in Example 3 and their esters. Also included are conjugates of formula IIIa containing about 3 to about 8, in one aspect about 3 to about 5 drug D moieties, i.e. conjugates of formula Ia, wherein p is a value in the range of 3-8, e.g. about 3- 5. Conjugates containing combinations of structural features noted in this paragraph are also considered to fall within the scope of the compounds of the invention.

[0324] In another embodiment, the ligand-linker-drug conjugate has formula IIIa, wherein the ligand is an Ab antibody that binds to one of the CD30, CD40, CD70 antigens, Lewis Y antigen, w = 1, y = 0, and D has formula Ib. Included are conjugates of formula IIIa in which R¹⁷means - (CH2) 5-. Also included are conjugates of formula IIIa in which W is -Val-Cit-, and / or in which D has the structure of compound 2 in Example 3 and their esters. Also included are conjugates of formula IIIa containing about 3 to about 8, preferably about 3 to about 5 drug D moieties, i.e. conjugates of formula Ia, wherein p is a value in the range 3-8, preferably about 3-5. Examples are also conjugates containing combinations of structural features noted in this paragraph.

[0325] In an embodiment, the ligand-linker-drug conjugate has formula IIIa, wherein the ligand is an Ab antibody that binds to one of the CD30, CD40, CD70 antigens, Lewis Y antigen, w = 1, y = 1 and D has the formula Ib . Included are conjugates of formula IIIa in which R¹⁷ means - (CH2) 5-. Also included are conjugates of formula IIIa in which: W is -ValCit-; Y has the formula X; D has the structure of compound 2 in Example 3 and their esters; p is about 3 to about 8, preferably about 3 to about 5 drug groups D. Drug conjugates containing combinations of structural features noted in this paragraph are also contemplated to be within the scope of the compounds of the invention.

[0326] A further embodiment is an antibody drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody that binds to one of the tumor associated antigens (1) - (35) noted above ("TAA compound" ).

[0327] A further embodiment is a TAA compound or a pharmaceutically acceptable salt or solvate thereof, which exist in isolated and purified form.

[0328] A further embodiment is a method for killing or inhibiting the proliferation of a cancer cell or cancer cell, comprising administering to a patient, for example a human with hyperproliferative disorder, an amount of a TAA compound or a pharmaceutically acceptable salt or solvate thereof, said amount being

110 effective in killing or inhibiting the reproduction of a cancer cell or cancer cell.

[0329] A further embodiment is a method for treating cancer comprising administering to a patient, for example a human with hyperproliferative disorder, an amount of a TAA compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective in treating cancer alone or with an effective amount of additional anti-cancer agent.

[0330] A further embodiment is a method for treating an autoimmune disease comprising administering to a patient, for example a human with hyperproliferative disorder, an amount of a TAA compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective in treating an autoimmune disease.

[0331] Antibodies suitable for use in the invention may be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

4.5.1 PRODUCTION OF RECOMBINANT ANTIBODIES [0332] The antibodies of the invention may be produced using any method known in the art to be useful in the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression.

[0333] The recombinant expression of antibodies, or fragments, derivatives or analogs thereof, requires a nucleic acid construct that encodes the antibody. If the nucleotide sequence of the antibody is known, the nucleic acid encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g. as described in Kutmeier et al., 1994, BioTechniques 17: 242), which requires the synthesis of overlapping oligonucleotides containing parts of the antibody coding sequence, hybridization and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides, e.g. by PCR.

[0334] Alternatively, the nucleic acid molecule encoding the antibody may be generated from a suitable source. If the clone containing the nucleic acid encoding the specific antibody is not available, but the antibody sequence is known, the 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 immunoglobulin) through, for example. PCR amplification using synthetic primers capable of hybridizing to the 3 'and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the sequence of a particular gene.

[0335] 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), specific antigen specific antibodies can be generated

111 by any method known in the art, for example, by immunizing a patient, or a suitable animal model, such as a rabbit or mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g. as described in Kohler and Milstein (1975, Nature 256: 495-497) or as described in 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 a portion of the antibody Fab 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 a specific antigen or by assay screening of antibody libraries (see, e.g., Clackson et al., 1991, Nature 352: 624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937).

[0336] Once the nucleic acid sequence encoding at least the antibody variable domain has been obtained, it can be inserted 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 US Patent No. 5122464). Vectors are available that contain complete light or heavy chains that allow expression of the complete antibody molecule. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletions necessary for the substitution (or deletion) of cysteine ​​residues of one or more variable regions involved in intra-chain disulfide bonds on an amino acid residue that does not contain a thiol group. Such modifications can be carried out by any method known in the art to introduce specific mutations or deletions in the nucleotide sequence, for example, without limitation, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551).

[0337] In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608 ; Takeda et al., 1985, Nature 314: 452-454) by splicing genes from a mouse antibody molecule with appropriate antigen specificity together with genes from a human antibody molecule with appropriate biological activity. Chimeric antibody means a molecule in which different parts are from different animal species, such as those having a variable region derived from mouse monoclonal antibodies and human immunoglobulin constant regions, e.g., humanized antibodies.

[0338] Alternatively, the techniques described for the production of single chain antibodies can be adapted (US Patent No. 44,694,778; 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) for the production of single chain antibodies. Single chain antibodies are formed by linking fragments of the heavy and light chain Fv region through an amino acid bridge, resulting in a single chain antibody. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., 1988, Science 242: 1038-1041).

112 [0339] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to, F (ab ') fragments₂which can be produced by pepsin digestion of antibody molecules and Fab fragments that can be produced by reducing disulfide bridges of F (ab ') 2 fragments.

[0340] Once the nucleic acid sequence encoding the antibody has been obtained, a vector for producing the antibody can be generated using 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 antibody coding sequences and appropriate transcription and translation 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, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY).

[0341] An expression vector containing the nucleotide sequence of the antibody, or the nucleotide sequence of the antibody can be transferred to the host cell by conventional techniques (e.g., electroporation, liposome transfection and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce an antibody. In certain embodiments, the expression of the antibody is regulated by a constitutive, inducible or tissue-specific promoter.

[0342] Host cells used to express recombinant antibodies may be bacterial cells, such as Escherichia coli, or, preferably, eukaryotic cells, in particular for expressing whole recombinant immunoglobulin molecules. In particular, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector such as the main intermediate promoter element of the early gene from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45: 101 ; Cockett et al., 1990, BioTechnology 8: 2).

[0343] A variety of host-vector expression systems can be used to express antibody immunoglobulins. Such expression host systems represent the tools by which the antibody coding sequences can be obtained and then purified, but also represent cells that can, after transformation or transfection with the appropriate nucleotide coding sequences, express the antibody molecule in situ. This includes, but is not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant DNA bacteriophage, DNA plasmid or cosmid expression DNA vector containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g. baculovirus) containing coding sequences

113 immunoglobulins; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, and tobacco mosaic virus (TMV)) or transformed recombinant expression plasmid vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the mammalian cell genome (e.g. metallothionein promoter) or from mammalian viruses (e.g. adenovirus late promoter; 7.5K vaccinia virus promoter).

[0344] In bacterial systems, a number of expression vectors can preferably be selected depending on the intended use of the expressed antibody molecule. For example, when a large amount of such a protein is to be produced, vectors that direct the expression of high levels of fusion protein products that are easily purified can be desirable. Such vectors include, but are not limited to, an E. coli vector for the expression of pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody coding sequence can be ligated individually to the vector in reading frame with the lacZ coding region such that a fusion protein is produced; pIN vectors (Inouye &amp; Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke &amp; Schuster, 1989, J. Biol. Chem. 24: 5503-5509); and similar. PGEX vectors can also be used to express foreign polypeptides as fusion proteins with S-glutathione transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads, followed by elution in the presence of free glutathione. PGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0345] In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or an analogous virus from Drosophila Melanogaster is used as a vector for expressing foreign genes. The virus multiplies in Spodopterafrugiperda cells. The antibody coding sequence can be cloned individually into viral regions that are not essential (e.g., the polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

[0346] A variety of virus-based expression systems can be used in mammalian host cells. In cases where the adenovirus is used as an expression vector, the sequence encoding the subject antibody can be ligated into an adenovirus transcription / translation control complex, e.g., a late promoter and triple leader sequence. This chimeric gene can then be introduced into the adenovirus genome by in vitro or in vivo recombination. The introduction of a viral genome region that is not essential (e.g., E1 or E3 region) results in a recombinant virus that is viable and capable of expressing immunoglobulin molecules in the infected host. (e.g., see Logan &amp; Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). Specific initiation signals may also be required for effective translation of the introduced antibody coding sequences. These signals

114 include ATG initiation codon and contiguous sequences. In addition, 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 translation control signals and initiation codons can make a range of initiation sites, both natural and synthetic. Expression efficiency can be enhanced by the addition of appropriate transcription enhancers, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153: 51-544).

[0347] In addition, a host cell strain is selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a specific, desired manner. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for protein function. Different host cells have characteristic and defined mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure correct modification and processing of the foreign expressed protein. To this end, eukaryotic host cells that have cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRŁ7030 and Hs578Bst.

[0348] For long-term, high-yield production of recombinant proteins, constant expression is preferred. For example, cell lines that stably express an antibody can be engineered. Rather than using expression vectors that contain viral origin 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. After the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in enriched media and then converted to selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to produce foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines that express antibodies. Such engineered cell lines may be particularly useful in screening and evaluating tumor antigens that interact directly or indirectly with the antibody.

[0349] A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase genes (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska &amp; Szybalski, 1992, Proc. Natl. Acad Sci. USA 48: 202) and adenine phosphoribosyl transferase (Łowy et al., 1980, Cell 22: 817 1980) in tk-, hgprt- or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis for selection for the following genes: DHFR, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 357; O'Hare et al., 1981, Proc Nat. Acad. Sci. USA 78: 1527); gpt, which confers acid resistance

115 mycophenol (Mulligan &amp; Berg, 1981, Proc. Natl. Acad. Sci. 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): 155-215) and hygroma, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art for recombinant DNA technology that 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 Prolocols in Human Genetics, John Wiley &amp; Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

[0350] Antibody expression levels 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 the marker in the antibody expression vector system is capable of amplification, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. As the amplified region is associated with the nucleotide sequence of the antibody, antibody production will also increase (Grouse et al., 1983, Mol. Cell. Biol. 3: 257).

[0351] The host cell may be co-transfected with two expression vectors, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector that encodes both heavy and light chain polypeptides can be used. In such situations, the light chain is preferably placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad. Sd. USA 77: 2197). The heavy and light chain coding sequences may contain cDNA or genomic DNA.

[0352] After expressing the antibody molecules by recombination, they can be purified by any method known in the art for purifying antibodies, for example, by chromatography (e.g. ion exchange, affinity, especially by affinity for a specific antigen after Protein A, and size exclusion chromatography), centrifugation, differential solubility or any other standard protein purification technique.

[0353] In yet another exemplary embodiment, the antibody is a monoclonal antibody.

[0354] In any case, the hybrid antibodies have dual specificity, preferably with one or more binding sites specific for the selected hapten or one or more binding sites specific for the target antigen, for example,

116 an antigen associated with a cancer, autoimmune disease, infectious organism, or other condition.

4.5.2 PRODUCTION OF ANTIBODIES [0355] Antibody production will be illustrated with respect to anti-CD30 antibodies, but it will be apparent to those skilled in the art that antibodies against other members of the TNF receptor family can be produced and modified in a similar manner. The use of CD30 for the production of antibodies is only exemplary and not intended to be limiting.

[0356] The CD30 antigen for use in the production of antibodies may exist, e.g., in the form of a soluble CD30 extracellular domain or portion thereof containing the desired epitope. Alternatively, cells expressing CD30 on their cell surface can be used for generating antibodies (e.g., Ł540 (Hodgkin lymphoma cell line with T cell phenotype) and Ł428 (Hodgkin lymphoma cell line with B cell phenotype)). Other forms of CD30 useful for generating antibodies will be apparent to those skilled in the art.

[0357] In another exemplary embodiment, the ErbB2 antigen for use in the production of antibodies may be, e.g., in the form of a soluble ErbB2 extracellular domain or portion thereof containing the desired epitope. Alternatively, cells expressing ErbB2 on their cell surface (e.g., NIH-3T3 cells transformed for overexpression of ErbB2; or a cancer cell line such as SK-BR-3 cells, can be used to generate antibodies; see Stancovski et al. Proc. Natl. Acad. Sci. USA 88: 8691-8695 (1991)). Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art.

(i) Polyclonal antibodies [0358] Polyclonal antibodies are preferably obtained in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen and adjuvant in question. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in species to be immunized e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g. maleimidobenzoylsulfosuccinimide ester (for coupling via cysteine ​​residues), N-hydroxysuccinimide (for lysine coupling), glutaraldehyde, succinic anhydride, SOCl₂ or R¹N = C = NR, where R and R¹ are different alkyl groups.

[0359] Animals are immunized against the antigen, immunogenic or derivative conjugates by combining, for example, 100 Lig or 5 Lig of the protein or conjugate (e.g. for rabbit or mouse, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally, in many places. One month later, the animals receive a booster dose of 1/5 or 1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant, by intradermal injection, in many places. Seven to 14 days later, the animals are bled and the serum is determined for antibody titers. The animals receive booster doses until the titer reaches value

117 plateau. Preferably, the animal receives a booster dose of a conjugate of the same antigen, but conjugated to another protein and / or via a different cross-linking reagent. Conjugates can also be prepared in recombinant cell cultures as fusion proteins. In addition, aggregation agents, such as alum, are appropriately used to enhance the immune response.

(ii) Monoclonal antibodies [0360] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Hence, the "monoclonal" modifier indicates the nature of the antibody, which is not a mixture of separate antibodies.

[0361] For example, monoclonal antibodies can be obtained using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be obtained by recombinant DNA methods (US Patent No. 4,816,567).

[0362] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to stimulate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then coupled to myeloma cells using a suitable coupling agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0363] The hybridoma cells thus obtained are inoculated and cultured in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of unconjugated myeloma stem cells. For example, if myeloma stem cells are lacking the guanine hydroxyanthine phosphinibosyltransferase enzyme (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin and thymidine (HAT medium), which prevent HG cell growth .

[0364] Preferred myeloma cells are those that efficiently undergo conjugation, maintain stable, high level production of antibodies by selected antibody producing cells, and are sensitive to a medium such as HAT medium. Of these, preferred myeloma cell lines are mouse myeloma lines, such as those derived from the 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- cells 653 available from the American Clean Culture Collection, Rockville, Maryland USA. Human myeloma and murine-human heterozygous cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker , Inc., New York, 1987)).

118 [0365] Culture medium in which hybridoma cells were cultured was assayed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined in vitro, using assays such as immunoprecipitation, a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis from Munson et al., Anal. Biochem., 107: 220 (1980).

[0366] After identification of hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, clones can be cloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be cultured in vivo as ascites in an animal.

[0367] Monoclonal antibodies secreted by subclones are appropriately separated from the culture medium, ascites fluid or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0368] DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of binding specifically to genes encoding mouse heavy and light chains). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be inserted into expression vectors, which are then transfected into host cells, such as E cells. coli, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that otherwise do not produce antibody protein to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

[0369] In a further embodiment, the monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552 (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 human antibodies (nM range) by chain shuffling (Marks et al., Bio / Technology 10: 779-783 (1992)) as well as combinatorial infections and in vivo recombination as strategies for constructing very high phage libraries (Waterhouse et al., Nucl Acids Res., 21: 2265-2266 (1993)). Hence, these techniques are practical alternatives to traditional hybridoma techniques for monoclonal antibodies, for the isolation of monoclonal antibodies.

119 [0370] DNA can also be modified, for example, by substitution of a coding sequence for the heavy and light chain constant domains in place of homologous mouse sequences (US Patent No. 4,816,567; and Morrison et al., (1984) Proc Natl Acad Sci USA 81: 6851) or by covalently linking to an immunoglobulin coding sequence, with all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0371] Typically, such non-immunoglobulin polypeptides are substituted for antibody constant domains, or are substituted for variable domains of one antibody antigen-joining site, to form a chimeric divalent antibody having one antigen-specific antigen-connecting site and another antigen-joining site with specificity for another antigen.

(iii) Humanized antibodies [0372] A humanized antibody may have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues that are typically taken from the "imported" variable domain. Humanization can generally be carried out as follows, according to Winter et al. (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 substitution hypervariable region sequences to the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than the intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and potentially some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0373] The choice of human variable domains, both light and heavy, for use in the production of humanized antibodies is important in reducing antigenicity. According to the so-called "best-fit" method, the rodent antibody variable domain sequence is screened against an entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol. , 196: 901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies from a particular subgroup of light or heavy chains. The same framework can be used in several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993) ).

[0374] In another embodiment, the antibodies can be humanized with retention of high affinity for the antigen and other favorable biological properties.

Humanized antibodies can be obtained by analyzing native sequences and various conceptual humanized products, using models

120 three-dimensional native and humanized sequences. Three-dimensional immunoglobulin models are widely available and are known to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected potential immunoglobulin sequences. Inspection of these presentations enables analysis of the likely role of residues in the functioning of potential immunoglobulin sequences, i.e. analysis of residues that affect the ability of a potential immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and imported sequences to achieve the desired antibody characteristic, such as increased affinity for the target antigen (s). In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0375] Various forms of humanized antibodies are contemplated. For example, a humanized antibody may be an antibody fragment such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0376] The examples describe the production of an exemplary humanized antiErbB2 antibody. For example, a humanized antibody may contain non-human hypervariable region residues incorporated into the human heavy variable domain and may additionally contain framework (FR) substitution at positions selected from the group consisting of 69H, 71H and 73H using the variable domain ring system numbering as given in Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody comprises FR substitutions at two or all positions 69H, 71H and 73H. Another example describes the preparation of purified Trastuzumab antibody from a HERCEPTIN® formulation.

(iv) Human antibodies [0377] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain (JH) region linking gene in chimeric mice and mouse germline mutants leads to complete inhibition of endogenous antibody production. The transfer of a human germline immunoglobulin gene matrix in such murine germline mutants will result in the production of human antibodies upon antigen challenge. See. e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and US Patent Nos. 5,591,669, 5,589,369 and 5,545,807.

[0378] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and antibody fragments in

121 in vitro, from the immunoglobulin variable (V) gene repertoire from non-immunized donors. According to this technique, the V domain genes of the antibody are cloned in reading frame with the gene of the major or minor filamentous bacteriophage coat protein, such as M13 or fd, and presented as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody may also result in selection of the gene encoding the antibody exhibiting those properties. Hence, the phage imitates some of the properties of the B cell. Phage display can be done in a number of formats; for a review of this subject, see e.g., Johnson, Kevin

S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed, and antibodies to a diverse array of antigens (including proprietary antigens) can be isolated essentially using the techniques described below by Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See. also US Patent Nos. 5565332 and 5573905. As discussed above, human antibodies can also be generated using in vitro activated B cells (see U.S. Patent Nos. 5567610 and 5229275). Human anti-CD30 antibodies are described in US Patent Application No. 10 / 338,366.

(v) Antibody fragments [0379] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal pf Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') fragments₂ (Carter et al., Bio / Technology 10: 163-167 (1992)). According to the next approach, fragments of F (_andb ')₂ can be isolated directly from a recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. 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 US Patent No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in US Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

(vi) Bispecific antibodies [0380] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the CD30 protein. Alternatively, the anti-CD30 arm may join

122 with the arm that binds to Fc receptors for IgG (Fc? R) such as Fc? RI (CD64), Fc? RII (CD32) and Fc? RIII (CD 16) to focus cell defense mechanisms on a CD30 expressing cell. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express CD30.

[0381] Traditionally, the production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two chains having different specificities (Milstein et al., Nature, 305: 537-539 (1983)). Due to the random selection of immunoglobulin light and heavy chains, these hybridomas (quadromes) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by means of affinity chromatography steps, is rather inconvenient and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). In another approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are conjugated to immunoglobulin constant domain sequences. The fusion preferably occurs with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge regions, CH2 and CH3. It is preferred that it has a first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding immunoglobulin heavy chain fusions and, if desired, immunoglobulin light chain fusion are introduced into separate expression vectors, and are co-transfected into the appropriate host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments, when the uneven proportions of the three polypeptide chains used in the construction provide optimal performance. It is, however, possible to introduce coding sequences for two or all three polypeptide chains into one expression vector, where the expression of at least two polypeptide chains in equal proportions results in high yield or when the proportions are not of particular importance.

[0382] In one embodiment of this approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid heavy chain-light chain immunoglobulin pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains, as the presence of the immunoglobulin light chain in only half of the bispecific molecules provides an easy separation path. This approach is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies, see for example Surcsh et al., Methods in Enzymology, 121: 210 (1986).

[0383] According to another approach described in US Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from

123 recombinant cell culture. The preferred interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" are created of identical or similar size for larger side chains on the interface of the second antibody molecule by replacing the side chains of large amino acids with smaller ones (eg, alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products, such as homodimers.

[0384] Techniques for generating bispecific antibodies from antibody fragments have also been reported in the literature. For example, bispecific antibodies can be obtained using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The Fab 'fragment generated is then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back to Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0385] 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) describes the production of a fully humanized F (ab ') 2 bispecific antibody molecule. Each Fab 'fragment was separately secreted from E. coli and subjected to direct chemical coupling in vitro to form a bispecific antibody.

[0386] Various techniques have also been described for the production and isolation of bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were combined with the Fab 'portions of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to form monomers, followed by re-oxidation to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described in Hollinger et al., Proc. Natl. Acad Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for producing bispecific antibody fragments. The fragments contain the variable domain of the heavy chain (V_H) linked to the light chain variable domain (V_L) by a linker that is too short to allow pairing between two domains on the same chain. Accordingly, the V domains_H and V_L from one fragment they are forced to pair with complementary V domains_L and V_H another fragment, with simultaneous

124 creating two antigen binding sites. Another strategy for producing bispecific antibody fragments by using single chain Fv dimers (sFv) has also been described. See. Gruber et al., J. Immunol., 152: 5368 (1994).

[0387] antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be obtained. Tutt et al. J. Immunol. 147: 60 (1991).

(vii) Other amino acid sequence modifications [0388] Modifications of the amino acid sequence of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody are obtained by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions relative to, and / or insertions for and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution is made to obtain the final construct, provided that the final construct has the desired characteristics. Changing amino acids can also change post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

[0389] A useful method for identifying specific residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells Science, 244: 1081-1085 (1989). In the present specification, a residue or group of target residues is identified (e.g. charged residues (such as arg, asp, his, lys and glu) and replaced with a neutral or negative charge (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. These locations of amino acids showing functional sensitivity to substitutions are then improved by introducing further or other variants in or substitution site substitutions. Hence, while the site for introducing amino acid sequence variations is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the functioning of a mutation at a given site, alanine scanning or random mutagenesis of the target codon or region is performed, and screened expressed antibody variants for desired activity.

[0390] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions that vary in length from one residue to polypeptides containing one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody conjugated to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include N- or C-terminal fusions of the antibody to an enzyme (e.g., in ADEPT) or polypeptide that increase the serum half-life of the antibody.

125 [0391] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by another residue. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR changes are also being considered.

[0392] Significant modifications of the biological properties of the antibody can be made by selecting substitutions that differ significantly in their effects on altering (a) the structure of the polypeptide backbone in the substitution area, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) side chain mass. 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, cheese, thr;

(3) acidic: asp, glu;

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

(5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0393] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0394] A particularly preferred type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variant (variants) selected for further development will have improved biological properties relative to the parent antibody from which it was generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each of these sites. The variants of the antibody thus generated are presented in a monovalent manner from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify potential hypervariable region sites for modification, mutagenesis may be performed by alanine scanning to identify hypervariable region residues significantly contributing to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and adjacent residues are candidates for substitution according to the technique described herein. After generating such variants, the variants panel is screened as

126 described herein, and can be selected for further development of antibodies with superior properties in one or more relevant assays.

[0395] It may be desirable to modify the antibody of the invention with respect to effector function, e.g., to increase antigen-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, a cysteine ​​residue may be introduced in the F region_c, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-dependent cell killing, and antibody-dependent cellular cytotoxicity (ADCC). See. Caron et al. J. Exp Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with increased anti-tumor activity may also be obtained using heterodifunctional cross linkers as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody that has double F regions can be engineered_c and thus may exhibit extensive complement lysis and ADCC capacity. See. Stevenson et al. AntiCancer Drug Design 3: 219-230 (1989).

[0396] To increase the serum half-life of the antibody, a rescue receptor binding epitope (in particular an antibody fragment) can be incorporated into the antibody as described in, e.g., US Patent No. 5739277. As used herein, the term "rescue receptor binding epitope" refers to an epitope of the Fc region of a molecule IgG (e.g., IgG₁, IgG₂, IgG₃ or IgG4), which is responsible for extending the serum half-life of the IgG molecule in vivo. (viii) Glycosylation variants [0397] Antibodies in the ADCs of the invention can be glycosylated at conserved positions in their constant regions (Jefferis and Lund, (1997) Chem. Immunol. 65: 111-128; Wright and Morrison, (1997) TibTECH 15 : 26-32). Oligosaccharide immunoglobulin side chains affect protein function (Boyd et al., (1996) Mol. Immunol. 32: 1311-1318; Wittwe and Howard, (1990) Biochem. 29: 4175-4180), and intramolecular interactions between glycoprotein parts, which may affect the conformation and the three-dimensional surface of the glycoprotein presented (Hefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7: 409-416). Oligosaccharides can also be used to direct a given glycoprotein to certain molecules based on the recognition of specific structures. For example, in galactosylated IgG it has been reported that the oligosaccharide moiety "pops" from the inter-CH2 space, and the N-terminal acetylglucosamine residues become available, resulting in binding of mannose binding protein (Malhotra et al., (1995) Nature Med. 1: 237-243). Removal of oligosaccharide glycopeptidase from CAMPATH-1H (recombinant humanized murine IgG1 monoclonal antibody that recognizes human lymphocyte CDw52 antigen) obtained in Chinese hamster ovary (CHO) cells resulted in a complete reduction of complement dependent lysis (CMCŁ) (Boyd et

127 al., (1996) Mol. Immunol. 32: 1311-1318), while the selective removal of sialic acid residues using neuraminidase did not result in a lack of DMCL. Antibody glycosylation has also been reported to affect antibody dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of 3 (1,4) -N-acetylglucosaminyltransferase III (GnTIII), glycosyltransferase catalyzing the formation of spacer GlcNAc, have been reported to have improved ADCC activity (Umana et al. (1999) Mature Biotech . 17: 176-180).

[0398] Glycosylation of an antibody typically occurs via N-glycosidic or O-glycosidic binding. N-glycosidic binding refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, in which X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Hence, the presence of any of these peptide triseventions in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition of one of the sugars - N-ethylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0399] Glycosylated antibody variants are variants in which the glycosylation pattern of the antibody has been changed. 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 glycosylation composition (glycosylation pattern), glycosylation range, etc.

[0400] 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 peptide trisection described above (for N-linked glycosylation sites). Changes can also be made by the addition or substitution of one or more serine or threonine residues to the antibody output sequence (for glycosylation sites with an Glycosidic linkage). Similarly, removal of glycosylation sites can be accomplished through amino acid changes within the native antibody glycosylation sites.

[0401] The amino acid sequence is usually changed by altering the basic 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-directed (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of the previously obtained variant or non-variant version of the antibody.

[0402] Glycosylation (including glycosylation pattern) of antibodies can also be changed without changing the amino acid sequence or the basic nucleotide sequence.

Glycosylation is highly dependent on the host cell used to express the antibody. Since the type of cell used to express the recombinant glycoprotein, e.g. antibody, as a potential therapeutic agent rarely means a native cell, it can be

128 expect significant variations in the glycosylation pattern of the antibodies. See. e.g. Hse et al., (1997) J. Biol. Chem. 272: 9062-9070. In addition to host cell selection, factors that affect glycosylation during recombinant antibody production include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed for changing the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (US Patent Nos. 5047335; 5510261; 5278299). Glycosylation, or specific types of glycosylation, can be enzymatically removed from glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered, e.g. make it defective in the treatment of certain types of polysaccharides. These and similar techniques are well known in the art.

[0403] The glycosylation structure of an antibody can be easily analyzed by conventional carbohydrate analysis techniques, including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, sequential enzymatic digestion, and HPAEC-PAD, which uses high-anion exchange chromatography pH for oligosaccharide separation based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, but are not limited to, enzymatic treatment (commonly performed using peptide-N-glycosidase F / endo-3-galactosidase), elimination using a crude alkaline environment, for release mainly O-linked glycosidic structures, and chemical methods using anhydrous hydrazine to release both N-glycosidic oligosaccharides and O-glycosidic linkages.

4.5.2a SCREENING FOR ANTIBODY-drug drug (ADC) conjugates [0404] Transgenic animals and cell lines are particularly useful in screening for antibody drug conjugates (ADCs) that have the potential for prophylactic or therapeutic treatment of diseases or disorders associated with protein overexpression including Lewis Y, CD30, CD40 and CD70. Transgenic animals and cell lines are particularly useful in screening for antibody drug conjugates (ADCs) that have the potential for prophylactic or therapeutic treatment of HER2 overexpression or disorders (US6632979). Screening for useful ADC may involve the administration of potential ADC at a series of doses to a transgenic animal, and assay, at various time points, of the effect of ADC on the disease or disorder being assessed. Alternatively, or in addition, the drug may be administered prior to or concurrently with exposure to the disease inducing agent, as appropriate. Potential ADC can mean screening serially and individually, or in parallel, in media format or in a high-throughput screening format. The rate at which ADC can be screened for usefulness for the preventive or therapeutic treatment of diseases or disorders is limited only by

129 synthesis rate or screening methodology, including data detection / measurement / analysis.

[0405] One embodiment is a screening method comprising (a) transplanting cells from a stable non-human animal kidney cancer cell line, (b) administering a potential ADC drug to a non-human animal, and (c) determining the candidate candidate's ability to inhibiting the formation of tumors from a transplanted cell line.

[0406] Another embodiment is a screening method comprising (a) contacting cells from a stable Hodgkin disease cell line with a potential ADC drug and (b) assessing the ability of the potential ADC to block CD40 ligand activation.

[0407] Another embodiment is a screening method comprising (a) contacting cells from a stable Hodgkin disease cell line with a potential ADC drug and (b) assessing the ability of the potential ADC to induce cell death. In one embodiment, the ability of potential ADC to induce apoptosis was evaluated.

[0408] One embodiment is a screening method comprising (a) transplanting cells from a stable cancer cell line to a non-human animal, (b) administering a potential ADC drug to a non-human animal, and (c) determining the ability of the candidate candidate to inhibit tumor formation from a transplanted cell line. The invention also relates to a method of screening potential ADCs for treating a disease or disorder characterized by HER2 overexpression, comprising (a) contacting cells from a stable breast cancer cell line with a potential drug, and (b) assessing the potential of potential ADC to inhibit the growth of a stable cell line .

[0409] Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with a potential ADC drug and (b) assessing the ability of the potential ADC to block HER2 ligand activation. In one embodiment, the ability of a potential ADC to block hegulin binding is assessed. In another embodiment, the ability of potential ADC to block ligand-stimulated tyrosine phosphorylation was evaluated.

[0410] Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with a potential ADC drug and (b) assessing the ability of the potential ADC to induce cell death. In one embodiment, the ability of potential ADC to induce apoptosis was evaluated.

[0411] A further embodiment is a screening method comprising (a) administering a potential ADC drug to a non-human transgenic mammal that overexpresses native human HER2 protein or a fragment thereof in its mammary gland cells, said transgenic mammal having a sequence stably integrated into the genome nucleic acid encoding native human HER2 protein or a protein thereof

130 a fragment having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences that direct its expression to the mammary gland, and the development of a breast tumor that does not respond or responds poorly to anti-HER2 antibody treatment; or a non-human mammal bearing a tumor transplanted from said non-human transgenic mammal; and (b) assessing the effect of potential ADC on the target disease or disorder. Non-limitingly, the disease or disorder may be HER2 overexpressing cancer, such as cancer of the breast, ovary, stomach, endometrium, salivary gland, lungs, kidney, colon, thyroid, pancreas and bladder cancer. The cancer is preferably a breast cancer that expresses HER2 at least about 500,000 copies per cell, more preferably at least about 2,000,000 copies per cell. Potential ADC drugs can, for example, be evaluated for their ability to induce cell death and / or apoptosis, using assay methods well known in the art and described below.

[0412] In one embodiment, potential ADCs are screened by administration to a transgenic animal in a dose range, and assessing the animal's physiological response to compounds over time. Administration can be oral, or by appropriate injection, depending on the chemical nature of the compound being tested. In some cases, it may be appropriate to administer the compound in combination with co-factors that could increase the effectiveness of the compound. if cell lines from the transgenic animals in question are used, for screening for compounds useful in the treatment of various disorders, the test compounds are added to the cell culture medium at the appropriate time, and the cell response to the compound is assessed over time, using appropriate biochemical and / or histological determinations. In some cases, it may be appropriate to administer the subject compound to the culture medium in combination with co-factors that could increase the efficacy of the compound.

[0413] Hence, assays for identifying ADCs that specifically target and bind to a target protein whose presence correlates with abnormal cell function, and with the pathogenesis of cell proliferation and / or differentiation that are causally related to the formation are provided herein cancer.

[0414] To identify ADC that blocks the activation of an ErbB receptor ligand (e.g., ErbB2), the compound's ability to block ErbB ligand binding to cells expressing the ErbB receptor (ErbB2) can be determined (e.g., by coupling to another ErbB receptor with which the subject the ErbB receptor forms the ErbB hetero-oligomer). For example, cells isolated from a transgenic HER2 overexpressing animal and transfected to express another ErbB receptor (with which HER2 forms a hetero-oligomer) can be incubated, i.e. cultured with 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 can then be evaluated.

131 [0415] For example, inhibition of hegulin binding (HRG) to breast cancer cell lines overexpressing HER2 and derived from a non-human transgenic mammal (e.g., mouse) as described herein, using potential ADC, can be performed using monolayer cultures ice, in 24-well plate format. Anti-ErbB2 monoclonal antibodies can be added to each well and incubated for 30 minutes. You can then add tagged ones¹²⁵I rHRG31 177-224 (25,000 cpm), and continue incubation for 4 to 16 hours. Dose-response curves can be obtained, and the IC50 value (cytotoxic activity) of the subject compound calculated.

[0416] Alternatively or additionally; the ADC ability to block ErbB ligand-stimulated tyrosine phosphorylation of the ErbB receptor present in the ErbB heterooligomer can be determined. For example, cell lines derived from transgenic animals as described herein with tested ADC can be incubated and then assayed for ErbB ligand dependent tyrosine phosphorylation activity using a phosphotyrosine monoclonal antibody (which is optionally conjugated to a detectable label). A kinase receptor activation assay, described in US Patent No. 5766863, is also available for determining ErbB receptor activation and blocking this activity by the compound.

[0417] In one embodiment, ADCs may inhibit HRG stimulation of p180 tyrosine phosphorylation in MCF7 cells essentially as described below. For example, a cell line derived from a HER2 transgenic animal may be applied to 24 well plates, and the compound may be added to each well and incubated for 30 minutes at room temperature; then rHR3p can be added₁₁₇7.₂₄₄ to each well, to a final concentration of 0.2 nM, and continue incubation for approximately 8 minutes. The media can be carefully aspirated from each well and the reactions quenched by the addition of 100 g SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 g) can be subjected to gel electrophoresis with a 4-12% gradient (Novex) and then electrophoretically transferred to the polyvinylidene difluoride membrane. Antiphosphotyrosine immunoblots (1gg / ml each) can be developed and the intensity of the main reactive band at M_r 180,000 using reflectance densitometry. An alternative method to assess inhibition of receptor phosphorylation is the KIRA assay (kinase receptor activation) according to Sadick et al. (1998) Jour. of Pharm. and Biomed. Anal. Some well-established anti-HER2 monoclonal antibodies that are known to inhibit HRG stimulation of p180 tyrosine phosphorylation as a positive control can be used in this assay. A dose response curve for inhibition of HRG stimulation of p180 tyrosine phosphorylation as determined by reflectance densitometry can be obtained and the IC50 of the subject compound calculated.

[0418] It is also possible to evaluate the inhibitory effect on growth, for the ADC tested, against 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, cells can be treated with the test compound at various concentrations for 4 days and

132 stain with crystal violet or Alamar Blue redox dye. Incubation with the compound may show an inhibitory effect on growth in this cell line, similar to that of 2C4 monoclonal antibody against MDA-MB-175 cells (Schaefer et al., Supra). In a further embodiment, exogenous HRG will not significantly reverse this inhibition.

[0419] To identify growth inhibitory compounds that specifically target the subject antigen, compounds that inhibit the growth of cancer cells overexpressing the subject antigen from transgenic animals can be screened using assay described in US Patent No. 5,677171. According to this assay, cancer cells that overexpress the subject antigen are cultured in a 1: 1 mixture of F12 medium and DMEM supplemented with 10% fetal bovine serum, glutamine and penicillin / streptomycin. Cells are seeded at 20,000 cells per 35 mm cell culture dish (2 ml / 35 mm dish) and the test compound is added at various concentrations. After six days, the number of cells, compared to untreated cells, is determined using an electronic COUŁTER ™ cell counting device. Those compounds that inhibit cell growth by about 20-100% or about 50-100% can be selected as growth inhibiting compounds.

[0420] To select for compounds that induce cell death, loss of membrane integrity can be assessed as indicated by, e.g., PI uptake, trypan blue or 7AAD uptake, relative to control. The PI uptake assay uses cells isolated from the tissue of the tumor of the transgenic animal in question. According to this assay, cells are cultured in Eagle Dulbecco's modified medium (D-MEM): Ham's F-12 (50:50) with the addition of 10% heat inactivated FBS (Hyclone) and 2 mM L-glutamine. Hence, the assay is performed in the absence of complement and immune effector cells. Cells are seeded at a density of 3 x 10⁶ on a weighing pan, on 100 x 20 mm dishes and allowed to stick overnight. The medium is then removed and replaced with fresh medium without additives or medium containing various concentrations of the compound. Cells are incubated for a period of 3 days. After each treatment, the monolayers are washed with PBS and detached by trypsinization. The cells are then centrifuged at 1200 rpm for 5 minutes at 4 ° C, the pellet resuspended in 3 ml cold Ca binding buffer²+ (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and separates into 12 x 75 mm tubes with a 35 mm mesh filter (1 ml per tube, 3 tubes per treatment group) to remove cell aggregates. PI (10 Lig / ml) is then added to the tubes. Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ CellQuest software (Becton Dickinson). Those compounds that induce statistically significant levels of cell death as determined by PI uptake can be selected as compounds inducing cell death.

[0421] To select compounds that induce apoptosis, an annexin binding assay is performed using cells derived from the tumor tissue of the transgenic animal in question. Cells are cultured and seeded in dishes as discussed in the previous paragraph. The medium is then removed and replaced with fresh without medium

133 additives or medium containing 10 Lig / mL antibody conjugate drug (ADC). After three days of incubation, the monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged, resuspended in Ca binding buffer²+ and aliquot into tubes as discussed above for the determination of cell death. Labeled annexin (e.g. annexin V-5 FITC) (1 Lig / ml) was then added to the tubes. Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ CellQuest software (Becton Dickinson). Those compounds that induce statistically significant levels of annexin binding relative to control are selected as compounds that induce apoptosis.

4.5.3 IN VITRO CELL EXPRESSION INDICATIONS [0422] In general, the cytotoxic or cytostatic activity of the antibody drug conjugate (ADC) is measured by: displaying mammalian cells having an ADC antibody protein receptor in cell culture medium; culturing the cells for a period of from about 6 hours to about 5 days; and measuring cell viability. In vitro cellular assays were used to measure viability (multiplication), cytotoxicity and induction of apoptosis (caspase activation) of the ADC according to the invention.

[0423] In vitro potency of antibody drug conjugates was measured by a cell proliferation assay (Example 18, Figures 7-10). Luminescence assay for CellTiter-Glo® cell viability is a commercially available (Promega Corp., Madison, WI) homogeneous assay method, based on recombinant expression of Coleoptera luceferase (US patents No. 5583024; 5674713 and 5700670). This cell proliferation assay determines the number of viable cells in culture, based on the amount of ATP present, the indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160: 81-88, US Patent No. 6602677). The CellTiter-Glo® assay was conducted in 96 well format, which predisposes it to automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6: 398-404). The homogeneous assay procedure requires the addition of a single reagent (CellTiter-Glo® Reagent) directly to cells grown in serum-supplemented medium. Cell washing, medium removal and numerous pipetting steps are not required. The system detects as few as 15 cells / well in a 384-well format, 10 minutes after adding the reagent and mixing. Cells can be treated continuously with ADC, or they can be treated and separated from ADC. In general, superficially treated cells, i.e. for 3 hours, showed the same potency effects as cells treated in continuous mode.

[0424] The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter-Glo® assay generates a luminescent "glow" signal, produced by a luciferase reaction, which has a half-life generally greater than five hours, depending on the type of cell and medium used. Viable cells are reflected in relative luminescence units (RLUs). The substrate, beetle luciferin, is

134 oxidatively decarboxylated by recombinant firefly luciferase with simultaneous conversion of ATP to AMP and photon generation. The extended half-life eliminates the need for reagent injectors and provides flexibility for the continuous or batch processing of multiple plates. This cell proliferation assay can be used with various multi-well formats, e.g., 96 or 384 well format. Data can be recorded using a luminometer or CCD camera image processing device. The luminescence output signal is presented as relative light units (RLU), measured in time.

luciferase _Λ

ATP + luciferin + O₂-&gt; - oxylciferin + AMP + PPi + CO₂ + light

<img file="PL1725249T3_D0053.tif" />

[0425] The anti-proliferative effect of antibody drug conjugates was measured by cell proliferation in the above in vitro cell killing assay against four different breast cancer cell lines (Figures 7-10). IC50 values ​​were determined for SK-BR-3 and BT-474, which are known to overexpress HER2 receptor protein. Table 2a shows potency measurements (IC50) for exemplary antibody drug conjugates in a cell proliferation assay against SK-BR-3 cells. Table 2b shows the power measurements (IC50) for exemplary antibody drug conjugates in a cell proliferation assay against BT-474 cells.

[0426] Drug antibody conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF / Ab; Trastuzumab-MC- (N-Me) vc-PAB-MMAF, 3.9 MMAF / Ab; Trastuzumab-MC-MMAF, 4.1 MMAF / Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE / Ab; Trastuzumab-MC-vc-PABMMAE, 3.3 MMAE / Ab; and Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF / Ab did not inhibit the multiplication of MCF-7 cells (Figure 9).

[0427] Drug antibody conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE / Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE / Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF / Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF / Ab; Trastuzumab-MC- (NMe) vc-PAB-MMAF, 3.9 MMAF / Ab; and Trastuzumab-MC-MMAF, 4.1 MMAF / Ab did not inhibit the proliferation of MDA-MB-468 cells (Figure 10).

[0428] MCF-7 and MDA-MB-468 cells did not overexpress HER2 receptor protein. consequently, anti-HER2 drug antibody conjugates of the invention showed selectivity in inhibiting cells that express HER2.

Table 2a SK-BR-3 cells

Drug Antibody Conjugate

H = Trastuzumab, combined through cysteine ​​[cys], except in the reported cases

H-MC-MMAF, 4.1 MMAF / Ab

0,008

IC50 (gg

ADC / ml)

135

<td>Drug Antibody Conjugate H = Trastuzumab, combined through cysteine ​​[cys], except as noted</td><td>IC50 (gg ADC / ml)</td>

<td>H-MC-MMAF, 4.8 MMAF / Ab</td><td> 0,002</td>

<td>H-MC-vc-PAB-MMAE, (reference example)</td><td> 0,007</td>

<td>H-MC-vc-PAB-MMAE (reference example)</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 (reference example)</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 (reference example)</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 (reference example)</td><td> 0,0025</td>

<td>H-MC-MMAE, 8.8 MMAE / Ab (reference example)</td><td> 0,018</td>

<td>H-MC-MMAE, 4.6 MMAE / Ab (reference example)</td><td> 0,05</td>

<td>H-MC- (L) val- (L) cit-PAB-MMAE, 8.7 MMAE / Ab (reference example)</td><td> 0,0003</td>

<td>H-MC- (D) val- (D) cit-PAB-MMAE, 8.2 MMAE / Ab (reference example)</td><td> 0,02</td>

<td>H-MC- (D) val- (L) cit-PAB-MMAE, 8.4 MMAE / Ab (reference example)</td><td> 0,0015</td>

<td>H-MC- (D) val- (L) cit-PAB-MMAE, 3.2 MMAE / Ab (reference example)</td><td> 0,003</td>

<td>H-Trastuzumab</td><td> 0,083</td>

<td>H-vc-MMAE, connected via lysine [lys] (reference example)</td><td> 0,002</td>

<td>H-phe-lys-MMAE, connected through lysine [lys]</td><td> 0,0015</td>

136

<td>Drug Antibody Conjugate H = Trastuzumab, combined through cysteine ​​[cys], except as noted</td><td>IC50 (gg ADC / ml)</td>

<td>(reference example)</td><td></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>

<td>4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF / Ab</td><td> 0,02</td>

<td>Anti-TF Fab-MC-vc-PAB-MMAE * (reference example)</td><td> -</td>

Table 2b of the BT474 cell

<td>Drug Antibody Conjugate H = Trastuzumab, linked through cysteine ​​[cys]</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, 4.1 MMAE / Ab (reference example)</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 (reference example)</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 (reference example)</td><td> 0,0025</td>

<td>H-MC-MMAE, 8.8 MMAE / Ab (reference example)</td><td> 0,04</td>

<td>H-MC-MMAE, 4.6 MMAE / Ab (reference example)</td><td> 0,07</td>

<td>4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF / Ab</td><td> 0,008</td>

<td>Hg-MC-vc-PAB-MMAF, 4.1 MMAF / Ab</td><td> 0,01</td>

137

<td>Drug Antibody Conjugate H = Trastuzumab, linked through cysteine ​​[cys]</td><td>IC50 (gg ADC / ml)</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 * (reference example)</td><td> -</td>

<td colspan="2">H = Trastuzumab 7C2 = mouse anti-HER2 antibody that binds to an epitope other than Trastuzumab. Fc8 = mutant that does not bind to FcRn Hg = "Hingeless [hingeless]" full length humanized 4D5, with hinge heavy chain cysteines mutated to serines. Expressed in E. coli (hence no glycosylation). Anti-TF Fab = anti-tissue factor antibody fragment * activity against MDA-MB-468 cells</td>

[0429] As surprisingly and unexpectedly found, the ADC results for the in vitro cell proliferation activity of Tables 2a and 2b generally showed that ADC with low average drug moieties per antibody showed efficacy, e.g. IC₅₀ &lt;0.1 gg ADC / ml. The results suggest that, at least for Trastuzumab ADC, the optimal ratio of drug moieties per antibody may be lower than 8, and may be about 2 to about 5.

4.5.4 IN VIVO Plasma CLIRENS AND STABILITY [0430] Plasma clearance pharmacokinetics and ADC stability were studied in Cynomolgus rats and monkeys. Plasma concentration was measured over time. Table 2c shows the pharmacokinetic data for antibody drug conjugates and other dosed samples in rats. Rats are a non-specific model for ErbB receptor antibodies because rats are not known to express HER2 receptor protein.

Table 2c Pharmacokinetics in rats

<td colspan="6">H = Trastuzumab combined by cysteine ​​[cys] except in the reported cases 2 mg / kg dose, except as noted</td>

<td>Sample dose mg / kg</td><td>AUCinf day * gg / ml</td><td>CL ml / day / kg</td><td>Cmax gg / ml</td><td>T1 / 2 Term. days</td><td>% conjugates.</td>

<td>H-MC-vc-PAB-</td><td> 78,6</td><td> 26,3</td><td> 39,5</td><td> 5,80</td><td> 40,6</td>

138

<td colspan="6">H = Trastuzumab combined by cysteine ​​[cys] except in the reported cases</td>

<td colspan="4">2 mg / kg dose, except as noted</td><td></td><td></td>

<td>Sample dose mg / kg</td><td>AUCinf day *</td><td>CŁ ml /</td><td>Cmax Lig / ml</td><td>T1 / 2 Term.</td><td>% conjugates.</td>

<td></td><td>ng / ml</td><td>day / kg</td><td></td><td>days</td><td></td>

<td>MMAE (total Ab) H-MC-vc-PAB-MMAE (Conjugate).</td><td> 31,1</td><td> 64,4</td><td> 33,2</td><td> 3,00</td><td></td>

<td>(example reference)</td><td></td><td></td><td></td><td></td><td></td>

<td>H-MC-vc-PABMMAF (total Ab) H-MC-vc-PAB-</td><td> 170</td><td> 12,0</td><td> 47,9</td><td> 8,4</td><td> 50,0</td>

<td>MMAF (conjugate)</td><td> 83,9</td><td> 24,0</td><td> 44,7</td><td> 4,01</td><td></td>

<td>H-MC-MMAE</td><td> 279</td><td> 18,9</td><td> 79,6</td><td> 7,65</td><td> 33</td>

<td>(total Ab) H-MCMMAE (conjugate) 5 mg / kg (reference example)</td><td> 90,6</td><td> 62,9</td><td> 62,9</td><td> 4,46</td><td></td>

<td>H-MC-MMAF</td><td> 299</td><td> 6,74</td><td> 49,1</td><td> 11,6</td><td> 37</td>

<td>(total Ab) H-MCMMAF (conjugate)</td><td> 110</td><td> 18,26</td><td> 50,2</td><td> 4,54</td><td></td>

<td>H-MC-vc-MMAF</td><td> 306</td><td> 6,6</td><td> 78,7</td><td> 11,9</td><td> 19,6</td>

<td>wo / PAB, (total Ab) H-MC-vc-MMAF, wo / PAB, (conjugate)</td><td> 59,9</td><td> 33,4</td><td> 82,8</td><td> 2,1</td><td></td>

<td>H-Me-vc-PAB-MMAF</td><td> 186</td><td> 10,8</td><td> 46,9</td><td> 8,3</td><td> 45,3</td>

<td>(total Ab)</td><td></td><td></td><td></td><td></td><td></td>

<td>H-Me-vc-PAB-MMAF</td><td> 84,0</td><td> 23,8</td><td> 49,6</td><td> 4,3</td><td></td>

<td>(Conjugate).</td><td></td><td></td><td></td><td></td><td></td>

<td>H-Me-vc-PAB-</td><td> 135</td><td> 15,0</td><td> 44,9</td><td> 11,2</td><td> 23,8</td>

<td>MMAE (total Ab) H-Me-vc-PABMMAE (conjugate) (reference example)</td><td> 31,9</td><td> 63,8</td><td> 45,2</td><td> 3,0</td><td></td>

139

<td colspan="6">H = Trastuzumab combined by cysteine ​​[cys] except in the reported cases 2 mg / kg dose, except as noted</td>

<td>Sample dose mg / kg</td><td>AUCinf day * gg / ml &lt;</td><td>&gt; CL ml / day / kg</td><td>Cmax gg / ml</td><td>T1 / 2 Term. days</td><td>% conjugates.</td>

<td>H-MC-vc-MMAF, wo / PAB, (total Ab) H-MC-vc-MMAF, wo / PAB, (conjugate)</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-</td><td> 107</td><td> 19,2</td><td> 30,6</td><td> 9,6</td><td> 38,1</td>

<td>PAB-MMAE (total Ab) H-MC (D) val- (L) cit-PABMMAE (conjugate) (reference example)</td><td> 40</td><td> 50,4</td><td> 33,7</td><td> 3,98</td><td></td>

<td>H-MC- (Me) -vc-PAB-</td><td> 135,1</td><td> 15,0</td><td> 44,9</td><td> 11,2</td><td> 23,8</td>

<td>MMAE, total Ab H-MC- (Me) -vc-PABMMAE, conjugate. (reference example)</td><td> 31,9</td><td> 63,8</td><td> 45,2</td><td> 2,96</td><td></td>

<td>H-MC- (D) Val (D) cit-</td><td> 88,2</td><td> 22,8</td><td> 33,8</td><td> 10,5</td><td> 38,3</td>

<td>PAB-MMAE, total Ab H-MC (D) val- (D) cit-PABMMAE, conjugate. (reference example)</td><td> 33,6</td><td> 59,8</td><td> 36,0</td><td> 4,43</td><td></td>

<td>H-MC-vc-PABMMAE, total Ab H-MC-vc-PABMMAE, conjugate. H connected to MC via lysine [lys] (example reference)</td><td> 78,6 31,1</td><td> 26.3 64.4</td><td> 39,5 33,2</td><td> 5,8 3,00</td><td> 40,6</td>

<td>MMAF 200 mg / kg</td><td> 0,99</td><td> 204</td><td> 280</td><td> 0,224</td><td>_-</td>

<td>MMAE 206 mg / kg</td><td> 3,71</td><td> 62,6</td><td> 649</td><td> 0,743</td><td></td>

140

<td colspan="6">H = Trastuzumab combined by cysteine ​​[cys] except in the reported cases</td>

<td colspan="4">2 mg / kg dose, except as noted</td><td></td><td></td>

<td>Sample dose mg / kg</td><td>AUCinf day *</td><td>CL ml /</td><td>Cmax pg / ml</td><td>T1 / 2 Term.</td><td>% conjugates.</td>

<td></td><td>Pg / ml</td><td>day / kg</td><td></td><td>days</td><td></td>

<td>example) (reference example)</td><td></td><td></td><td></td><td></td><td></td>

<td>HER F (ab ') 2-MC-vc-</td><td> 9,3</td><td> 217</td><td> 34,4</td><td> 0,35</td><td> 95</td>

<td>MMAE, total Ab HER F (ab ') 2-MC-vcMMAE, conjugate. (reference example)</td><td> 8,8</td><td> 227</td><td> 36,9</td><td> 0,29</td><td></td>

<td>4D5-H-Fab-MC-VC-</td><td> 43,8</td><td> 46,2 68,1</td><td> 38,5 34,1</td><td> 1,49 1,12</td><td> 68</td>

<td>MMAF, total Ab 4D5-H-Fab-MC-vcMMAF, Koniug.</td><td> 29,9</td><td></td><td></td><td></td><td></td>

<td>4D5-H-Fab-MC-vcMMAE.</td><td> 71,5</td><td> 70,3</td><td> 108</td><td> 1,18</td><td> 59</td>

<td>total Ab</td><td> 42,2</td><td> 118,9</td><td> 114</td><td> 0,74</td><td></td>

<td>4D5-H-Fab-MC-vcMMAE, conjugate. (reference example)</td><td></td><td></td><td></td><td></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-</td><td> 170</td><td> 12,03</td><td> 47,9</td><td> 8,44</td><td> 49,5</td>

<td>MMAF, total Ab H-MC-vc-PABMMAF, conjugate.</td><td> 83,9</td><td> 23,96</td><td> 44,7</td><td> 4,01</td><td></td>

<td>H-MC-vc-PAB-</td><td> 211</td><td> 9,8</td><td> 39,8</td><td> 8,53</td><td> 34,3</td>

<td>MMAF-DMAEA, total Ab H-MCvc-PAB-MMAFDMAEA, conjugate.</td><td> 71,5</td><td> 28,2</td><td> 38,8</td><td> 3,64</td><td></td>

<td>H-MC-vc-PAB-</td><td> 209</td><td> 9,75</td><td> 53,2</td><td> 8,32</td><td> 29,7</td>

<td>MMAF-TEG, total Ab H-MCvc-PAB-MMAF-TEG,</td><td> 63,4</td><td> 31,8</td><td> 34,9</td><td> 4,36</td><td></td>

141

<td colspan="6">H = Trastuzumab combined by cysteine ​​[cys] except in the reported cases 2 mg / kg dose, except as noted</td>

<td>Sample dose mg / kg</td><td>AUCinf day * gg / ml</td><td>CL ml / day / kg</td><td>Cmax gg / ml</td><td>T1 / 2 Term. days</td><td>% conjugates.</td>

<td>conjugate.</td><td></td><td></td><td></td><td></td><td></td>

[0431] AUC inf is the area under the plasma concentration-time curve from time to dose to infinity, and is a measure of total exposure to the measured unit (drug, ADC). CL is defined as the volume of plasma released from the measured unit per unit of time and is expressed by normalizing against body weight. The term T1 / 2 means the half-life of the drug in the body, measured during its elimination phase. The concept of% conjugates. is the relative amount of ADC compared to total antibody eluted, in separate immunoaffinity ELISA ("Analytical Methods for Biotechnology Products", Ferraiolo et al, pp. 85-98 in Pharmacokinetics of Drugs (1994) PG Welling and LP Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110, Springer-Verlag. Calculation of% conjugates. it's just AUCinf with ADC + AUCinf total Ab, and it's an indicator of linker stability, although other factors and mechanisms may be important.

[0432] Figure 11 shows a plot of plasma concentration clearance study after administration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF antibody conjugates of Sprague-Dawley rats. Total antibody and ADC concentrations were measured over time.

[0433] Figure 12 shows a graph of a two-step plasma concentration clearance study with ADC administered at different doses and total antibody and ADC concentrations measured over time.

IN VIVO EFFECTIVENESS [0434] The in vivo efficacy of the ADC according to the invention was measured using a mouse transgenic model from an ecanthate expressing HER2 at a high level. Allograft was propagated from a Fo5 mmtv transgenic mouse that does not respond or responds poorly to HERCEPTIN® therapy. Subjects were treated once with ADC and monitored for a period of time

3-6 weeks to measure time to tumor doubling, cell killing logarithm and tumor shrinkage. Control experiments on dose response and experiments on the administration of multiple doses were conducted.

[0435] Tumors easily develop in transgenic mice that express the mutated activated form of neu, a HER2 rat homolog, but HER2 that is overexpressed in breast cancers is not mutated and tumor formation is much weaker in transgenic mice that overexpresses unmutated HER2 (Webster et al. (1994) Semin. Cancer Biol. 5: 69-76).

[0436] To improve tumor formation for unmutated HER2, transgenic mice were obtained using a HER2 cDNA plasmid in which ATG was removed above to prevent translation initiation from those ATG codons upstream which

142 otherwise they would reduce the frequency of translation initiation from the authentic HER2 initiation codon below (for example, see Child et al. (1999) J. Biol. Chem. 274: 2433524341). In addition, a chimeric intron was added at the 5 'end, which should also increase expression levels as previously reported (Neuberger and Williams (1988) Nucleic Acids Res. 16: 6713; Buchman and Berg (1988) Mol. Cell. Biol. 8: 4395 ; Brinster et al. (1988) Proc. Natl. Acad. Sci. USA 85: 836). The chimeric intron was from the Promega vector, pCI-neo mammalian expression vector (pz 890-1022). The 3 'end of the cDNA is flanked by human growth hormone exons 4 and 5 and polyadenylation sequences. In addition, 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 used the AIN 76A diet to increase susceptibility to tumor development (Rao et al. (1997) Breast Cancer Res. And Treatment 45: 149-158).

Table 2d Tumor measurements in the allograft mouse model - MMTVHER2 Fo5 breast cancer, Nude mice

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>excipient</td><td></td><td></td><td></td><td></td><td> 2-5</td><td> 0</td>

<td>H-MC-vc-PAB-MMAE 8.7 MMAE / Ab (Ref. Example)</td><td>1250 gg / m2²</td><td> 5/5</td><td> 4/7</td><td> 0/7</td><td> 18</td><td> 1,5</td>

<td>H-MC-vc-PAB-MMAF 3.8 MMAF / Ab</td><td>555 gg / sq m²</td><td> 2/5</td><td> 2/7</td><td> 5/7</td><td> 69</td><td> 6,6</td>

<td>H-MC (Me) -vcPAB-MMAF</td><td></td><td></td><td></td><td></td><td> &gt;50</td><td> 6,4</td>

<td>H-MC-MMAF 4.8 MMAF / Ab</td><td>9.2 mg / kg Ab 550 gg / m² in days 0, 7, 14 and 21</td><td> 7/7</td><td> 6/7</td><td> 0/7</td><td> 63</td><td> 9</td>

143

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>H-MC-MMAF 4.8 MMAF / Ab</td><td>14 mg / kg Ab 840 gg / m² on days 0, 7, 14 and 21</td><td> 5/5</td><td> 5/7</td><td> 2/7</td><td> &gt;63</td><td></td>

<td>H-MC-vc-PAB-MMAF 5.9 MMAF / Ab</td><td>3.5 mg / kg Ab 300 gg / m² on days 0, 21 and 42</td><td> 5/6</td><td> 1 /7</td><td> 3/7</td><td> &gt;36</td><td></td>

<td>H-MC-vc-PAB-MMAF 5.9 MMAF / Ab</td><td>4.9 mg / kg Ab 425 gg / m2 on days 0, 21 and 42</td><td> 4/7</td><td> 2/7</td><td> 5/7</td><td> &gt;90</td><td></td>

<td>H-MC-vc-PAB-MMAF 5.9 MMAF / Ab</td><td>6.4 mg / kg Ab 550 gg / m2 on days 0, 21 and 42</td><td> 3/6</td><td> 1 /7</td><td> 6/7</td><td> &gt;90</td><td></td>

<td>H- (L) val (L) cit-MMAE 8.7 MMAE / Ab (ref. Example)</td><td>10 mg / kg</td><td> 7/7</td><td> 1 /7</td><td> 0/7</td><td> 15,2</td><td> 1,1</td>

<td>H-MC-MMAE 4.6 MMAE / Ab (ref. example)</td><td>10 mg / kg</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 4</td><td> 0,1</td>

<td>H- (D) val (D) cit-MMAE 4.2 MMAE / Ab (ref. Example)</td><td>10 mg / kg</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 3</td><td></td>

144

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>H- (D) val (L) cit-MMAE 3.2 MMAE / Ab Example reference</td><td>13 mg / kg</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 9</td><td> 0,6</td>

<td>H-MC (Me) -vcMMAE 3.0 MMAE / Ab Example reference</td><td>13 mg / kg</td><td> 7/7</td><td> 3/7</td><td> 0/7</td><td> 17</td><td> 1,2</td>

<td>H- (L) val (D) cit-MMAE 3.5 MMAE / Ab Example reference</td><td>12 mg / kg</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0,2</td>

<td>H-vc-MMAE 8.7 MMAE / Ab Example reference</td><td>10 mg / kg</td><td> 7/7</td><td></td><td></td><td> 17</td><td></td>

<td>H-cys-vcMMAF 3.8 MMAF / Ab</td><td>1 mg / kg</td><td> 7/7</td><td></td><td></td><td> 3</td><td></td>

<td>H-cys-vcMMAF 3.8 MMAF / Ab</td><td>3 mg / kg</td><td> 7/7</td><td></td><td></td><td> &gt;17</td><td></td>

<td>H-cys-vcMMAF 3.8 MMAF / Ab</td><td>10 mg / kg</td><td> 4/7</td><td> 4/7</td><td> 3/7</td><td> &gt;17</td><td></td>

145

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except in cases noted b connected by cysteine ​​[cys] except in the reported cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>H-MC-vcMMAF-TEG 4 MMAF / Ab</td><td>10 mg / kg</td><td> 3/6</td><td> 1 /7</td><td> 6/7</td><td> 81</td><td> 7,8</td>

<td>H-MC-vcMMAF-TEG 4 MMAF / Ab</td><td>10 mg / kg q3wk x 3</td><td> 0/5</td><td> 0/7</td><td> 7/7</td><td> 81</td><td> 7,9</td>

<td>H-vc-MMAF (series 1)</td><td>10 mg / kg</td><td> 4/6</td><td> 2/8</td><td> 5/8</td><td></td><td></td>

<td>H-vc-MMAF (series 2)</td><td>10 mg / kg</td><td> 7/8</td><td> 1 /8</td><td> 1 /8</td><td></td><td></td>

<td>H-MC-MMAF</td><td>10 mg / kg 550 gg / m²</td><td> 8/8</td><td> 1 /8</td><td> 0/8</td><td> 18</td><td></td>

<td>H- (Me) -vcMMAF</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 Example reference</td><td>3.7 mg / kg on days 0, 7, 14, 21, 28</td><td> 6/6</td><td> 0/7</td><td> 1 /7</td><td> 17</td><td> 2,3</td>

<td>H-vc-MMAE 7.5 MMAE / Ab Example reference</td><td>7.5 mg / kg on days 0, 7, 14, 21, 28</td><td> 5/7</td><td> 3/7</td><td> 3/7</td><td> 69</td><td> 10</td>

<td>anti-IL8-vcMMAE</td><td>7.5 mg / kg on days</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0,5</td>

146

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>7.5 MMAE / Ab Example reference</td><td> 0, 7, 14, 21, 28</td><td></td><td></td><td></td><td></td><td></td>

<td>anti-IL8-vcMMAE 7.5 MMAE / Ab Example reference</td><td>3.7 mg / kg on days 0, 7, 14, 21, 28</td><td> 6/6</td><td> 0/7</td><td> 0/7</td><td> 3</td><td> 0,2</td>

<td>H-fk-MMAE 7.5 MMAE / A Reference example b</td><td>7.5 mg / kg on days 0, 7, 14, 21, 28</td><td> 7/7</td><td> 1 /7</td><td> 0/7</td><td> 31</td><td> 4,4</td>

<td>H-fk-MMAE 7.5 MMAE / Ab Example reference</td><td>3.7 mg / kg on days 0, 7, 14, 21, 28</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 8,3</td><td> 0,9</td>

<td>anti-IL8-fkMMAE 7.5 MMAE / Ab Example reference</td><td>7.5 mg / kg on days 0, 7, 14, 21, 28</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 6</td><td> 0,5</td>

<td>anti-IL8-fkMMAE 7.5 MMAE / Ab Example reference</td><td>3.7 mg / kg on days 0, 7, 14, 21, 28</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 3</td><td> 0,1</td>

147

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>trastuzumab</td><td>7.5 mg / kg on days 0, 7, 14, 21, 28</td><td> 7/7</td><td> 0/7</td><td> 0/7</td><td> 5</td><td> 0,4</td>

<td>H-vc-MMAE 8.7 MMAE / Ab Example reference</td><td>10 mg / kg 1250 gg / m2</td><td> 6/6</td><td> 3/6</td><td> 0/6</td><td> 15</td><td> 1,3</td>

<td>H-vc-MMAE Example reference</td><td>10 mg / kg 1250 gg / m2 on days 0, 7 and 14</td><td> 7/7</td><td> 5/7</td><td></td><td> &gt;19</td><td></td>

<td>H-vc-MMAE Example reference</td><td>3 mg / kg on days 0, 7 and 14</td><td> 7/7</td><td></td><td></td><td> 8</td><td></td>

<td>H-vc-MMAE Example reference</td><td>1 mg / kg on days 0, 7 and 14</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 on days 0, 7 and 14</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 on days 0, 7 and 14</td><td> 7/7</td><td></td><td></td><td> 6</td><td></td>

<td>H-vc-MMAF</td><td>1 mg / kg on days 0, 7 and 14</td><td> 8/8</td><td></td><td></td><td> 4</td><td></td>

148

<td colspan="7">single dose on day 1 (T = 0) except as noted</td>

<td>H = Trastuzuma</td><td colspan="6">b connected by cysteine ​​[cys] except in the reported cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>trastuzumab</td><td>10 mg / kg on days 0 and 7</td><td> 8/8</td><td></td><td></td><td> 3</td><td></td>

<td>Hg-MC-vc-PAB-MMAF 4.1 MMAF / Ab</td><td>10 mg / kg on day 0</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 on day 0</td><td> 7/7</td><td> 6/8</td><td> 0/8</td><td> 25</td><td> 2,1</td>

<td>7C2-MC-vc-PAB-MMAF</td><td>10 mg / kg on day 0</td><td> 5/6</td><td> 6/8</td><td> 1 /8</td><td> 41</td><td> 3,7</td>

<td>4 MMAF / Ab</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>H-MC-vc-PAB-MMAF 5.9 MMAF / Ab</td><td>10 mg / kg on day 0</td><td> 3/8</td><td> 3/8</td><td> 5/8</td><td> 62</td><td> 5,7</td>

<td>2H9-MC-vc-PAB-MMAE Example reference</td><td></td><td> 9/9</td><td></td><td></td><td>&gt; 14 days</td><td></td>

<td>2H9-MC-vc-PAB-MMAF</td><td></td><td> 9/9</td><td></td><td></td><td>&gt; 14 days</td><td></td>

<td>11D10-vc-PAB-MMAE Example reference</td><td></td><td> 9/9</td><td></td><td></td><td>&gt; 1 4 days</td><td></td>

149

<td>single dose H = Trastuzuma</td><td colspan="6">ka on day 1 (T = 0) except for recorded cases b connected by cysteine ​​[cys] except for recorded cases</td>

<td>Antibody drug samples</td><td>Dose</td><td>ti</td><td>PR</td><td>CR</td><td>Time to double [volume] tumor (days)</td><td>Average log of cell killing</td>

<td>11D10-vc-PAB-MMAF</td><td></td><td> 9/9</td><td></td><td></td><td>11 days</td><td></td>

<td colspan="7">7C2 = mouse anti-HER2 antibody that binds to an epitope other than Trastuzumab. Fc8 = mutant that does not bind to FcRn Hg = "Hingeless [hingeless]" full length humanized 4D5, with hinge heavy chain cysteines mutated to serines. Expressed in E. coli (hence no glycosylation). 2H9 = Anti-EphB2R 11D10 = Anti-0772P</td>

[0437] The term Ti means the number of animals in the cancer test group for T = 0 + total animals in the group. The concept of PR means the number of animals achieving partial tumor remission + animals with cancer for T = 0 in the group. The term CR means the number of animals achieving complete tumor remission + cancer animals for T = 0 in the group. The concept of Log from cell killing means time in days to double the tumor volume - time in days for doubling the control tumor volume, divided by 3.32 X time to double the tumor volume in the control animals (which were dosed). The calculation of Log from cell killing takes into account the delay in tumor growth resulting from the treatment and the time to double the tumor volume in the control group. ADC anti-tumor activity is classified, with reference

<td colspan="3">to the log value of killing cells as:</td>

<td> ++++</td><td> &gt; 3,4</td><td>(highly active)</td>

<td> +++</td><td> = 2,5-3,4</td><td></td>

<td> ++</td><td> = 1,7-2,4</td><td></td>

<td> +</td><td> = 1,0-1,6</td><td></td>

<td>inactive</td><td> = 0</td><td></td>

[0438] Figure 13 shows the mean tumor volume change over time in nude MMTV-HER2 Fo5 nude mice with breast tumor allografts that were dosed on day 0: vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 gg / m²) and

Trastuzumab-MC-vc-PAB-MMAF (555 gg / m2²). (H = Trastuzumab). The growth of tumors was

150 delayed by ADC treatment compared to control (vehicle) height level. Figure 14 shows the mean tumor volume change over time in nude MMTV-HER2 Fo5 mice with breast cancer allografts that were dosed on Day 0 at 10 mg / kg (660 pg / m²) Trastuzumab-MC-MMAE and 1250 qg / m² Trastuzumab-MC-vc-PAB-MMAE. Figure 15 shows the mean change in tumor volume over time in nude MMTV-HER2 Fo5 Nude mice transplanted with breast cancer allografts dosed with 650 μg / m² Trastuzumab-MC-MMAF. Table 2d and Figures 13-15 show that ADC has strong anti-tumor activity in allograft HER2 positive tumor (Fo5) that originated primarily in MMTV-HER2 transgenic mouse. The antibody alone (e.g., Trastuzumab) has no significant anti-tumor activity in this model (Erickson et al. US Patent No. 6632979). As illustrated in Figures 13-15, tumor growth was delayed by ADC treatment compared to control (vehicle) growth level.

[0439] As stated surprisingly and unexpectedly, the ADC results for anti-tumor activity in vivo from Table 2d generally demonstrated that ADC with low average number of drug moieties per antibody showed efficacy, e.g. time to double [tumor volume] &gt; 15 days and average log to kill cell&gt; 1.0. Figure 16 shows that for the drug antibody conjugate, Trastuzumab-MC-vc-PAB-MMAF, the mean tumor volume decreased and did not progress when the MMAF: Trastuzumab ratio was 2 and 4, while the tumor progressed for the ratio 5 , 9 and 6, but at a slower rate than the excipient (buffer). The rate of tumor progression in this mouse xenograft model was approximately the same, i.e. 3 days, for vehicle and Trastuzumab. The results suggest that, at least for Trastuzumab ADC, the optimal ratio of drug moieties per antibody may be less than about 8, and may be about 2 to about 4.

4.5.5 TOXICITY IN RODIES [0440] Antibody conjugates drug and ADC-minus - control, "excipient" were evaluated in a rat acute toxicity model. ADC toxicity was studied by treatment of male and female Sprague-Dawley ADC rats and subsequent inspection and analysis of effects on various organs. Gross observations included changes in body weight and signs of change and bleeding. Clinical parameters of pathology (serum chemistry and hematology), histopathology and autopsy were performed on animals after dosing.

[0441] It has been considered that the weight loss, or weight change relative to animals that were dosed with vehicle only, in animals after ADC dosing is a gross indicator and overall systemic or local toxicity. Figures 17-19 show the effect of different ADC and control (vehicle) after dosing on rat body weight.

[0442] Hepatotoxicity was measured by elevated liver enzymes, increased mitotic and apoptotic data, and hepatocyte necrosis.

Toxicity to the liver and lymphatic system has been observed by reducing the number of leukocytes, mainly granulocytes (neutrophils) and / or platelets, and organ involvement

151 lymphoid, i.e. atrophy or apoptotic activity. Toxicity has also been reported by gastrointestinal changes, such as increased mitotic and apoptotic data, and degenerative enteritis.

[0443] Enzymes indicative of liver damage that have been tested include:

AST (aspartate aminotransferase)

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

- liver: plasma ratio of 7,000: 1

- T1 / 2: 17 hours

ALT (alanine aminotransferase)

- Location: cytoplasmic; liver, kidney, heart, skeletal muscles

- liver: plasma ratio 3000: 1

- T1 / 2: 42 hours; daily fluctuations in GGT (g-glutamyl transferase)

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

- poor liver damage indicator; commonly elevated in biliary disorders [0444] The toxicity profiles of Trastuzumab-MC-val-cit-MMAF, TrastuzumabMC (Me) -val-cit-PAB-MMAF, Trastuzumab-MC-MMAF and Trastuzumab-MC-val-cit-PABMMAF were studied in female Sprague-Dawley rats (example 19). The humanized Trastuzumab antibody does not noticeably bind to rat tissues, and any toxicity would be considered non-specific. MMAF variants for dose levels 840 and 2105 ug / m²compared to Trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug / m².

[0445] Animals in groups 1, 2, 3, 4, 6 and 7 (vehicle, 9.94 and 24.90 mg / kg, respectively, Trastuzumab-MC-val-cit-MMAF, 10.69 mg / kg Trastuzumab-MC (Me) -val-cit-PAB-MMAF and 10.17 and 25.50 mg / kg Trastuzumab-MC-MMAF) gained weight during the study. Animals in groups 5 and 8 (26.78 mg / kg Trastuzumab-MC (Me) -val-cit-PABMMAF and 21.85 mg / kg Trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight over time. research. On study day 5, the change in body weights of the animals in groups 2, 6 and 7 was not significantly different from the animals in group 1. The change in body weights of the animals in groups 3, 4, 5 and 8 was statistically different from the animals in group 1 ( example 19).

[0446] Trastuzumab-MC-MMAF-treated rats (groups 6 and 7) were indistinguishable from control animals treated with vehicle at both dose levels; i.e. this conjugate showed an overriding safety profile in this model. TrastuzumabMC-val-cit-MMAF treated rats (without self-destructive PAB; groups 2 and 3) showed dose-dependent changes typical for MMAF conjugates; the range of lesions was smaller compared to the full-length MC-val-cit-PAB-MMAF conjugate (group 8). counts

152 platelets on day 5 reached approximately 30% of baseline in group 3 animals (high dose Trastuzumab-MC-val-cit-MMAF) compared to 15% in group 8 animals (high dose Trastuzumab-MC-val-cit -PAB-MMAF). Elevations in AST and ALT liver enzymes, bilirubin, and thrombocytopenia were most pronounced in animals treated with Trastuzumab-MC (Me) -val-cit-PAB-MMAF (groups 4 and 5) in a dose-dependent manner; Five animals of group 5 (high dose group) had ALT levels on day 5 of approximately 10x baseline, and platelets decreased by approximately 90% during necropsy.

[0447] Female Sprague-Dawley rats were also dosed at high levels (Example 19, high dose study: groups 2, 3, 4) Trastuzumab-MC-MMAF, and vehicle control (group 1). Signs of mild toxicity, including dose-dependent increases in liver enzymes ALT, AST and GGT, have been observed. On day 5, the animals in the highest dose group showed a 2-fold increase in ALT and a 5-fold increase in AST; GGT also increased (6U / L). Enzyme levels showed a trend toward normalization on day 12. Mild granulocytosis was observed in all three dose groups, on day 5, platelet levels remained essentially unchanged in all animals. Morphological changes were mild; animals treated at a dose of 4210 gg / m2² (group 2) presented normal histology of the liver, spleen, thymus, intestines and bone marrow. Mildly increased apoptotic and mitotic activity was observed in the thymus and liver, respectively, in animals treated at a dose of 5500 gg / m²(group 3). The bone marrow had normal cells but showed evidence of granulocyte proliferation, which is consistent with the absolute granulocytosis observed in peripheral blood counts in these animals. The animals in the highest dose group - group 4 showed the same qualitatively; mitotic activity in the liver appeared to be somewhat increased compared to animals in Group 3. Also, extramedullary hematopoiesis in the spleen and liver was observed.

[0448] EphB2R is a type 1 TM tyrosine kinase receptor with close homology between mouse and human, and is overexpressed in colorectal cancer cells. 2H9 is an antibody against EphB2R. The naked antibody has no effect on tumor union, but 2H9-val-cit-MMAE killed EphB2R expressing cells and showed efficacy in a mouse xenograft model using CXF1103 - human colon cancers (Mao et al (2004) Cancer Res. 64: 781-788). 2H9 and 7C2 are both murine anti-HER2 IgG1 antibodies. The toxicity profiles of 2H9-MC-val-cit-PABMMAF (3.7 MMAF / Ab), 7C2-MC-val-cit-PAB-MMAF (4 MMAF / Ab) and Trastuzumab-MCval-cit-PAB-MMAF (5.9 MMAF / ab). Differences in the structure of each immunoconjugate or part of the drug from the immunoconjugate may affect the pharmacokinetics and ultimately the safety profile. The humanized Trastuzumab antibody does not noticeably bind to rat tissues, and any toxicity would be considered non-specific.

TOXICITY / SAFETY IN CYNOMOLGUS MONKEYS [0449] As with the toxicity / safety study in rats, Cynomolgus monkeys were treated with ADC, followed by enzyme measurements

153 hepatic and inspection and analysis of the effects on various organs. Gross observations included changes in body weight and signs of change and bleeding. Clinical parameters of pathology (serum chemistry and hematology), histopathology and autopsy were performed on animals after dosing (Example 19).

[0450] The antibody drug conjugate, H-MC-vc-PAB-MMAE (H = Trastuzumab linked via cysteine) did not show any evidence of liver toxicity at any of the dose levels tested. There was a reduction in peripheral blood granulocyte counts after a single dose of 1,100 mg / m2 with complete recovery 14 days after dosing. Drug antibody H-MC-vc-PAB-MMAF conjugate showed elevation of liver enzymes for dose levels 550 (transient) and 880 mg / m2, no evidence of granulocytopenia, and dose-dependent, transient decrease (platelets 2 and 3) blood.

4.6 SYNTHESIS OF COMPOUNDS ACCORDING TO THE INVENTION [0451] Exemplary compounds and exemplary conjugates can be prepared using the synthetic procedures outlined below in Schemes 5-16. As described in more detail below, exemplary compounds or exemplary conjugates may conveniently be prepared using a linker having a reactive site for binding to the drug and ligand. In one aspect, the linker has a reactive site that has an electrophilic group that is reactive with the nucleophilic group present on the ligand, such as, but not limited to, an antibody. Useful nucleophilic groups on an antibody include, but are not limited to, thiol, hydroxyl and amino groups. The heteroatom of the nucleophilic group of the antibody is reactive with the electrophilic group on the linker and forms a covalent bond with the 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.

[0452] In another embodiment, the linker has a reactive site that has a nucleophilic group that is reactive with the electrophilic group present on the antibody. Useful electrophilic groups on an antibody include, but are not limited to, carbonyl groups of aldehydes and ketones. The heteroatom nucleophilic group of the linker may react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Useful nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and aryl hydrazide. The electrophilic group on the antibody provides a convenient place for attachment of the linker.

[0453] Carboxylic acid functional groups and chloroformate functional groups are also useful linker reactive sites because they can react with secondary amine groups of the drug to form an amide bond. 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 amine group of a drug, such as, without limitation, N-methylvaline to form a bond

154 carbamate. Typically, peptide-based drugs can be obtained by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be obtained, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, "The Peptydes", vol. 1, pp. 76-136, 1965, Academic Press) which is well known in peptide chemistry.

[0454] The synthesis of an illustrative tensile element having an electrophilic maleimide group is illustrated below in Schemes 8-9. General synthetic methods useful for linker synthesis are described in Scheme 10. Diagram 11 shows the construction of a linker unit having a val-cit group, an electrophilic maleimide group and a PAB self-destructive spacer group. Scheme 12 outlines the synthesis of a linker having a phe-lys group, an electrophilic maleimide group with and without PAB - a self-destructive spacer group. Scheme 13 presents an overview for the synthesis of a drug-linker compound, while Scheme 14 presents an alternative pathway for obtaining a drug-linker compound. Scheme 15 outlines the synthesis of the branched linker containing the 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, e.g., but not limited to, 2 or 4 drug per antibody moieties.

[0455] As described in more detail below, exemplary conjugates are conveniently prepared using a linker having two or more reactive sites for binding to the drug and to the ligand. In one aspect, the linker has a reactive site that has an electrophilic group that is reactive with the nucleophilic group present on the ligand, such as an antibody. Useful nucleophilic groups on an antibody include, but are not limited to, thiol, hydroxyl and amino groups. The heteroatom of the nucleophilic group of the antibody is reactive with the electrophilic group on the linker and forms a covalent bond with the 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.

[0456] In another embodiment, the linker has a reactive site that has a nucleophilic group that is reactive with the electrophilic group present on the ligand, such as an antibody. Useful electrophilic groups on an antibody include, but are not limited to, carbonyl groups of aldehydes and ketones. The heteroatom nucleophilic group of the linker may react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Useful nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and aryl hydrazide. The electrophilic group on the antibody provides a convenient place for attachment of the linker.

4.6.1 DRUG GROUPING SYNTHESIS [0457] Typically, peptide-based drugs can be obtained by forming a peptide bond between two or more amino acids and / or peptide fragments.

Such peptide bonds can be obtained, for example, according to a phase synthesis method

155 liquid (see E. Schroder and K. Lubke, "The Peptydes", vol. 1, pp. 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry.

[0458] Aurystatin / dolastatin drug moieties can be prepared according to the general methods of: US Patent No. 5635483; U.S. Patent No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15: 859-863.

[0459] In one embodiment, the drug is obtained by combining an approximately stoichiometric equivalent of dipeptide and tripeptide, preferably in a one-pot reaction, under appropriate condensation conditions. This approach is illustrated in diagrams 5-7 below.

[0460] Scheme 5 illustrates the synthesis of the N-terminal F unit of a tripeptide, which is a useful intermediate for the synthesis of drug compounds of formula Ib.

Diagram 5

PG O HN

Ry

JL + PyBrop, OIEA 9? C^{oh Η}ίΎΎ VH₄ Fk Fis Rfi o

R + R

AND

When PG = Z:

Re Ra Ó

B

Oh Ry

PG

When ru = Z:

1- ^, 10% Pd / C, EtOH &lt;R_S Re R_e ABOUT

Rj nN^oh

Rj&gt; O BB

Rj p | OR,^this'&lt;A-ⁿXS-Vyy

Ri! About Rą R₅ R₆ R_B ABOUT ⁿⁿ6 ng

D

HCl Dioxane

DERC, Et₃N yAy ^

Rg, O Rg Rg R3 O

OR₇ [0461] As illustrated in Scheme 5, protected amino acid A (wherein PG is an amine protecting group, R⁴ selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, -O- (C1-C8 alkyl), -aryl, alkylaryl, alkyl- (C3-C8 carbocycle), C3-C8 heterocycle, alkyl- (C3-C8 heterocycle) in which R⁵ selected from H and methyl; or R⁴ and R⁵ combine, have a pattern - (CR^andR^b) n- in which R^and and R^b are independently selected from hydrogen, C1-C8 alkyl and C3-C8 carbocycle and are selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached) is coupled to the t-butyl ester B ( with R⁶ is selected from -H and -C1-C₈ alkyl; and R⁷ selected from hydrogen, C.₁-C₈ alkyl, C₃C₈ carbocycle, -O- (C₁-C₈ alkyl), -aryl, alkylaryl, alkyl- (C₃-C₈ carbocycle), C₃-C₈heterocycle and alkyl- (C3-C8 heterocycle)) under appropriate coupling conditions, e.g.

156 presence of PyBrop and diisopropylethylamine, or using DCC (see for example, Miyazaki, K. et al. Chem. Pharm. Bull. 1995, 43 (10), 1706-1718).

[0462] Suitable PG protecting groups, and suitable synthetic methods for protecting an amino group with a protecting group are well known in the art. See. e.g. Greene, TW and Wuts, PGM, Protective Groups in Organic Synthesis, Second Edition, 1991, John Wiley &amp; Sons. Exemplary protected amino acids A are PG-Ile and, especially, PG-Val, while other suitable protected amino acids include, but are not limited to: PG-cyclohexylglycine, PG-cyclohexylalanine, PGaminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PG -phenylalanine, PGphenylglycine and PG-tert-butylglycine. Z represents an exemplary protective group. Fmoc means another example protecting group. An exemplary B-t-butyl ester is dolaisoleuine t-butyl ester.

[0463] Dipeptide C may be purified, e.g. by chromatography, and subsequently deprotected, e.g. using H₂ and 10% Pd-C in ethanol when PG is benzyloxycarbonyl, or using diethylamine to remove the Fmoc protecting group. The resulting amine D easily forms a peptide bond with the amino acid BB (where R¹selected from -H, -C1-C8 alkyl and -C3-C8 carbocycle; and R² selected from -H and -C1C8 alkyl; or R¹ and R² combine, have a pattern - (CR^andR^b) n- in which R^and and R^b are independently selected from -H, -C1-C8 alkyl and -C3-C8 carbocycle and are selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached; and R³ selected from hydrogen, C.₁-C₈ alkyl, C₃-C₈ carbocycle, -O- (C₁-C₈ alkyl), -aryl, alkylaryl, alkyl- (C₃-C₈carbocycle), C3-C8 heterocycle and alkyl- (C3-C8 heterocycle)). N, N-dialkyl amino acids are exemplary amino acids for BB, such as commercially available N, N-dimethylvaline. Other N, N-dialkyl amino acids can be obtained by reductive bisalkylation using known procedures (see, e.g., Bowman, RE, Stroud, HH J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two exemplary BB amino acids useful for the synthesis of N-monoalkyl derivatives. Amine D and amino acid BB react to provide tripeptide E, using DEPC coupling reagent with triethylamine as a base. The C-terminal protecting group E is then deprotected using HCl to provide the tripeptide compound of formula F.

[0464] The illustrative methodology for DEPC coupling and the PyBrop coupling methodology shown in Scheme 5 are presented below in general procedure A and general procedure B respectively. An illustrative methodology for removing the protecting group of Z-protected amine by catalytic hydrogenation is presented below in general procedure C.

[0465] General procedure A: Peptide synthesis using DEPC. N-protected or N, N-substituted amino acid or D-peptide (1.0 eq) and BB amine (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 added followed by DEPC (1.1 eq.). The resulting solution is stirred, preferably under argon, for a maximum of 12 hours with simultaneous monitoring by HPLC or TLC. The solvent is removed under vacuum at temperature

157 room temperature and the crude product is purified using, for example, HPLC or flash column chromatography (silica gel column). The relevant fractions are combined and concentrated in vacuo to afford tripeptide E, which is dried in vacuo overnight.

[0466] General procedure B: Peptide synthesis using PyBrop. 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 between 0.5 and 1.0 mM, then diisopropylethylamine (1) , 5 equivalents). Fmoc- or Protected Amino Acid (1.1 eq.) Is added to the resulting mixture as a solid in one portion, followed by PyBrop (1.2 eq.). The reaction is monitored by TLC or HPLC followed by a work-up procedure similar to that described in general procedure A.

[0467] General procedure C: Removal of Z by catalytic hydrogenation. The protected amino acid or C-peptide is diluted with ethanol to obtain a solution between 0.5 and 1.0 mM in a suitable vessel, such as a thick-walled round-bottomed flask. 10% palladium on carbon (5-10% w / w) is added and the reaction mixture placed under an atmosphere of hydrogen. The progress of the reaction is monitored by HPLC and is generally completed within 1-2 hours. The reaction mixture is filtered through a pre-washed celite pad and the celite is washed again after filtration with a polar organic solvent such as methanol. The eluent solution is concentrated in vacuo to a residue which is diluted with an organic solvent, preferably toluene. The organic solvent is then removed in vacuo to afford the amine C, which is deprotected.

[0468] Scheme 6 shows a method useful for producing a C-terminal dipeptide of formula K and a method for coupling a dipeptide of formula K with a tripeptide of formula F, for producing drug compounds of formula Ib.

158

Diagram 6

<img file="PL1725249T3_D0054.tif" />

[0469] Dipeptide K can easily be obtained by condensing the modified amino acid Boc-dolaproin G (see, for example, Pettit, GR, et al. Synthesis, 1996, 719725), with an amine of formula H using condensation agents well known in the art. peptide chemistry, such as, for example, DEPC in the presence of triethylamine as shown in Scheme 5.

[0470] The dipeptide of formula K can then be coupled to the tripeptide of formula F using general procedure D to obtain Fmoc protected drug compounds of formula L, which can then be deprotected using general procedure E to provide drug compounds of formula (Ib ).

[0471] General procedure D: drug synthesis. A mixture of dipeptide K (1.0 equiv.) And tripeptide F (1 equiv.) Is diluted with an aprotic organic solvent such as dichloromethane to form a 0.1 M solution, then a strong acid such as trifluoroacetic acid (1/2 is added) v / v) and the resulting mixture is stirred under nitrogen for two hours at 0 ° C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is dried twice azeotropically, preferably with toluene. The resulting residue is drained

159 under high vacuum for 12 hours and then diluted with an aprotic organic solvent such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq) is then added followed by PyBrop (1.2 eq) or DEPC (1.2 eq) depending on the chemical functionality of the residue. The reaction mixture is monitored by TLC or HPLC, and then after completion of the reaction, a work-up procedure similar or identical to that described in general procedure A is carried out.

[0472] General procedure E: Removal of Fmoc using diethylamine. Fmoc-protected drug L is diluted with an aprotic organic solvent such as dichloromethane and diethylamine (1/2 v / v) is added to the resulting solution. The progress of the reaction is monitored by TLC or HPLC, and is typically complete within 2 hours. The reaction mixture is concentrated in vacuo and the resulting residue is dried azeotropically, preferably using toluene, and then dried under high vacuum to obtain drug Ib having a deprotected amino group.

[0473] Scheme 7 shows a method useful for preparing MMAF derivatives

160

<img file="PL1725249T3_D0055.tif" />

When PG = Fmoc: WhcnPG = Fmoc: DEA / CHiCij

<img file="PL1725249T3_D0056.tif" />

<img file="PL1725249T3_D0057.tif" />

HCl / dioxane

<img file="PL1725249T3_D0058.tif" />

[0474] Dipeptide O can easily be obtained by condensing the modified Boc-dolaproin G amino acid (see, for example, Pettit, GR, et al. Synthesis, 1996, 719725), with a protected amino acid of formula M using condensation agents well known in peptide chemistry, such as, for example, DEPC in the presence of triethylamine as shown in Schemes 5 and 6.

[0475] The dipeptide of formula O can then be coupled to the tripeptide of formula F using general procedure D to obtain Fmoc protected MMAF compounds of formula P, which can then be deprotected using general procedure E to provide drug compounds of formula MMAF.

[0476] Hence, the above methods are useful for making medicaments that can be used in the invention.

161

4.6.2 DRUG-LINKER SYNTHESIS [0477] To obtain the drug-linker compound of the invention, the drug reacts with a reactive site on the linker. In general, the linker may have the structure:

Reactive site 2 —A 'Wyy —- Yy - ^ - fl ^ reactive site 1 ~ where both the spacer unit (-Y-) and the stretch unit (-A-) are present. Alternatively, the linker may have the structure:

<img file="PL1725249T3_D0059.tif" />

when the spacer unit (-Y-) is not present. [0478] The linker may also have the structure:

Reactive site 2

ΑΛ / γγ —-- Reactive site 1 when both the stretch unit (-A-) and the spacer unit (-Y-) are absent. [0479] The linker may also have the structure:

<img file="PL1725249T3_D0060.tif" />

when both the amino acid unit (W) and the spacer unit (-Y-) are absent.

[0480] In general, a suitable linker has an amino acid unit attached to the optional stretch unit and the optional spacer unit. Reactive site 1 is present at the end of the spacer and reactive site 2 is present at the end of the tensile element. If the spacer unit is not present, then reactive site 1 is present at the C-terminus of the amino acid unit.

[0481] In an exemplary embodiment, the reactive site No. 1 is reactive with the nitrogen of the drug, and reactive site No. 2 is reactive with the thiol group on the ligand. Reactive sites 1 and 2 may be reactive with different functional groups.

[0482] In one aspect of the invention, reactive site No. 1 is

<img file="PL1725249T3_D0061.tif" />

[0483] In another aspect of the invention, reactive site 1 is

<img file="PL1725249T3_D0062.tif" />

[0484] In yet a further aspect of the invention, reactive site 1 is a pnitrophenyl carbonate of formula

162

<img file="PL1725249T3_D0063.tif" />

[0485] In one aspect of the invention, Reactive Site # 2 is a thiol accepting group. Suitable thiol accepting groups include haloacetamide groups of the formula

<img file="PL1725249T3_D0064.tif" />

wherein X is a leaving group, preferably O-mesyl, O-tosyl, -Cl, -Br, or -I; or a maleimide group of the formula

<img file="PL1725249T3_D0065.tif" />

[0486] Useful linkers can be obtained from commercial sources such as Molecular Biosciences Inc. (Boulder, CO), or prepared as outlined in schemes 8-10 below.

163

<img file="PL1725249T3_D0066.tif" />

wherein X is -CH₂- or -CH₂OCH₂-; and n is an integer in the range of 0-10 when X is -CH₂-; or 1-10 when X is -CH₂OCH₂-.

[0487] The method shown in Scheme 9 combines maleimide with glycol under Mitsunobu conditions to form a stretch element of polyethylene glycol maleimide (see, for example, Walker, MAJ Org. Chem. 1995, 60, 5352-5) followed by the introduction of a site group reactive p-nitrophenyl carbonate.

164

<img file="PL1725249T3_D0067.tif" />

wherein E is -CH₂- or -CH₂OCH₂-; and e is an integer in the range 0-8;

[0488] Alternatively, PEG-maleimide and PEG-haloacetamide stretch members may be prepared as described in Frisch, et al., Bioconjugate Chem. 1996, 7, 180-186. Scheme 10 illustrates the general synthesis of an illustrative linker unit containing a maleimide stretch group and an optional self-destructive paminobenzyl ether spacer.

Diagram 10

Fmoc h₂n

1. NaHCOa, DME / HaO

2. diethylamine, CH₂CI₂

3. compound R, DMF o

R^L= Benzyl; R²= (GH2) 4NHMtr (U) R.¹= Isopropyl; R²= (CH2)₃NHCONH₂ (V)

Mtr

1. NaHCO₃, DME / H3O

1.

2.

3.

R '

Fmoc

R²

OH

S (m = 0) diethylamine, CHCl 3

R, DIEA, CH₂CI₂ bis {4mtrofenyloiwęglan

DIEA, GH₂CI₂

EEDQ wherein Q is -C1-C8 alkyl, -O- (C1-C8 alkyl), -halo, -nitro or-cyano; m is an integer in the range of 0-4; and n is an integer in the range of 0-10.

165 [0489] Useful tensile elements can be introduced into the linker using commercially available intermediates from Molecular Biosciences (Boulder, CO) described below, using known organic synthesis techniques.

The tensile elements of formula (IIIa) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid unit as shown in Schemes 11 and 12:

<img file="PL1725249T3_D0068.tif" />

wherein n is an integer in the range of 1-10 and T is -H or -SO₃On;

<img file="PL1725249T3_D0069.tif" />

wherein n is an integer in the range of 0-3;

and

<img file="PL1725249T3_D0070.tif" />

[0490] The stretch units of formula (IIIb) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid unit:

166

<img file="PL1725249T3_D0071.tif" />

wherein X is -Br or -I; and

<img file="PL1725249T3_D0072.tif" />

[0491] The stretch units of formula (IV) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid unit:

<img file="PL1725249T3_D0073.tif" />

and

<img file="PL1725249T3_D0074.tif" />

[0492] The stretch units of formula (Va) can be introduced into the linker by reacting the following intermediates with the N-terminus of the amino acid unit:

167

<img file="PL1725249T3_D0075.tif" />

and

<img file="PL1725249T3_D0076.tif" />

[0493] Other useful extensible elements can be synthesized according to known procedures. Aminooxy stretch elements of the formula shown below can be obtained by treatment with N-Boc-hydroxylamine alkyl halides according to the procedures described in Jones, DS et al., Tetrahedron Letters, 2000, 41 (10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23 (11), 4441-4447.

<img file="PL1725249T3_D0077.tif" />

with -R¹⁷- is selected from -C₁-C₁₀ alkylene-, -C₃-C₈ carbocyclo, -O- (C.₁-C₈ alkyl), -arylene-, -C₁-C₁₀ alkylenearyl-, -arylene-C₁-C₁₀ alkylene-, -C₁-C₁₀ alkylene- (C₃-C₈carbocyclo) -, - (C3-C8 carbocyclo) -C1-C10 alkylene-, -C3-C8 heterocyclo, -C1-C10 alkylene (C3-C8 heterocyclo) -, - (C3-C8 heterocyclo) -C1-C10 alkylene -, - (CH2CH2O) r-, - (CH2CH2O) rCH2-; and r is an integer in the range 1-10;

Isothiocyanate extenders of the formula shown below can be obtained from isothiocyanate carboxylic acid chlorides as described in Angew. Chem., 1975, 87 (14): 517.

<img file="PL1725249T3_D0078.tif" />

with -R¹⁷- means as described in the description.

[0494] Scheme 11 shows a method for preparing a val-cit linker dipeptide having a maleimide tensile element and optionally a p-aminobenzyl self-destructive spacer.

168

<img file="PL1725249T3_D0079.tif" />

wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; and m is an integer in the range of 0-4.

[0495] Scheme 12 illustrates the synthesis of the phe-lys (Mtr) dipeptide linker unit having a maleimide stretch unit and a p-aminobenzyl self-destructive spacer unit. The starting material AD (lys (Mtr)) is commercially available (Bachem, Torrance, CA) or can be obtained according to Dubowchik, et al. Tetrahedron Letters (1997) 38: 5257-60.

169

<img file="PL1725249T3_D0080.tif" />

wherein Q is -C₁-C₈ alkyl, -O- (C₁-C₈ alkyl), -halo, -nitro or -cyano; and m is an integer in the range of 0-4.

[0496] As shown in Scheme 13, the linker can be reacted with the amino group of the drug compound of formula (Ib) to form a drug-linker compound that contains an amide or carbamate group linking the drug unit with the linker unit. When reactive site No. 1 is a carboxylic acid group, as in the AJ linker, the coupling reaction can be carried out using HATU or PyBrop and the corresponding amine base, resulting in the formation of a drug-linker AK compound containing an amide bond between the drug unit and the linker unit. When reactive site # 1 is carbonate, as in the AL linker, the linker can be coupled to the drug using HOBt in a DMF / pyridine mixture for

170 providing a drug-linker AM compound containing a carbamate linkage between the drug unit and the linker unit.

[0497] Alternatively, when Reactive Site # 1 is a good leaving group, as in the AN linker, the linker may be coupled to the drug's amino group through a nucleophilic substitution process to provide a drug-linker compound having an amine linkage (AO) between the drug unit and unit linker.

[0498] Illustrative methods useful for linking a drug to a ligand to form a drug-linker compound are depicted in Scheme 13 and outlined in general GH procedures.

<img file="PL1725249T3_D0081.tif" />

[0499] General procedure G: Amide formation using HATU. Łek (Ib) (1.0 eq) and

The N-protected linker containing the carboxylic acid reactive site (1.0 eq) was diluted with a suitable organic solvent such as dichloromethane and the resulting solution was treated with HATU (1.5 eq) and an organic base, preferably pyridine (1.5 eq) . The reaction mixture was allowed to stir under an inert atmosphere, preferably argon, for 6 hours, during which time the reaction mixture was monitored using HPŁC. The reaction mixture was concentrated and the resulting residue was purified by HPLC to give the amide of formula AK.

[0500] Procedure H: Carbamate formation using HOBt. A mixture of AL linker having a reactive site of p-nitrophenyl carbonate (1.1 eq) and drug (Ib) (1.0 eq) was diluted with an aprotic organic solvent, such as DMF, to provide a solution having a concentration of 50-100 mM, and the resulting the solution was treated with HOBt (2.0 eq) and placed under an inert atmosphere, preferably argon. The reaction mixture was allowed to stir for 15 min, then an organic base such as pyridine (1/4 v / v) was added and the progress of the reaction monitored using HPŁC. The linker is typically consumed within 16 hours. The reaction mixture is then concentrated in vacuo and the resulting residue purified using, for example, HPLC to afford carbamate AM.

171 [0501] An alternative method of preparing drug-linker compounds is outlined in the scheme

14. Using the method of Scheme 14, the drug is attached to a partial linker unit (e.g., ZA) that does not have a stretch unit attached. This provides an intermediate AP that has an amino acid unit having a B end protected by Fmoc. The Fmoc group is then removed and the resulting amine intermediate AQ is then attached to the tensile unit via a catalyzed coupling reaction using PyBrop or DEPC. The construction of drug-linker compounds containing a bromoacetamide AR stretch element or a PEG maleimide stretch AS element is illustrated in Scheme 14.

<img file="PL1725249T3_D0082.tif" />

wherein Q is -C1-C8 alkyl, -O- (C1-C8 alkyl), -halo, -nitro or -cyano; and m is an integer in the range of 0-4.

172 [0502] A methodology useful for obtaining a linker unit containing a branched spacer is shown in Scheme 15.

<img file="PL1725249T3_D0083.tif" />

[0503] Scheme 15 illustrates the synthesis of a val-cit linker dipeptide having a maleimide stretch unit and a bis (4-hydroxymethyl) styrene unit (BHMS). The synthesis of intermediate BHMS (AW) has been improved over procedures previously known from the literature (see International Publication No. WO 9813059 for Firestone et al., And Crozet, MP; Archaimbault, G .; Vanelle, P .; Nouguier, R. Tetrahedron Lett. (1985) 26: 5133-5134) and used as starting materials commercially available (4-nitrobenzyl) diethyl phosphonate (AT) and commercially available 2,2-dimethyl-1,3-dioxan-5-one (AU). AY and BA linkers can be obtained from intermediate AW using the methodology described in scheme 9.

4.6.3 DENDRITIC LINKERS

173 [0504] The linker may be a dendritic type linker, for the covalent attachment of more than one drug moiety via a branched, multifunctional linker moiety to the ligand, such as in a non-limiting manner an antibody (Sun et al. (2002) Bioorganic &amp; Medicinal Chemistry Letters 12: 2213- 2215; Sun et al. (2003) Bioorganic &amp; Medicinal Chemistry 11: 1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, i.e. occupancy, which is related to the strength of the drug-linkerligand conjugate. Thus, when the engineered antibody cysteine ​​carries only one cysteine ​​thiol reactive group, several drug moieties can be attached via a dendritic linker.

[0505] The following exemplary embodiments of dendritic linker reagents allow the coupling of a maximum of nine nucleophilic reagents of drug moieties by reaction with functional groups of chloroethyl nitrate:

<img file="PL1725249T3_D0084.tif" />

<img file="PL1725249T3_D0085.tif" />

<img file="PL1725249T3_D0086.tif" />

4.6.4 COUPLING DRUG AGENTS TO ANTIBODIES [0506] Scheme 16 illustrates a methodology useful for making drug-linkerligand conjugates having about 2 to about 4 [drug] moieties per antibody. The antibody was treated with a reducing agent such as dithiothreitol (DTT) to reduce some or

174

DTT

The antibody all disulfide bonds of cysteine ​​residues to form highly nucleophilic cysteine ​​thiol groups (-CH₂SH). The partially reduced antibody therefore reacts with drug-linker compounds, or linker reagents, with electrophilic functional groups such as maleimide or α-halo carbonyl, according to the coupling method on page 766 Klussman, et al. (2004), Bioconjugate Chemistry (4): 765-773.

Scheme 16 partially reduced drug-linker drug-linker-ligand conjugate antibody with reduced drug load

For example, an antibody, e.g. AC10, was dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 ° C for about 30 minutes, the buffer was exchanged by elution on Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol / Ab value was checked by determining the concentration of the reduced antibody based on the absorbance of the solution at 280 nm and the concentration of thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the absorbance at 412 nm. The reduced antibody was dissolved in PBS and cooled on ice. The drug linker, e.g. MC-val-cit-PAB-MMAE in DMSO, was dissolved in acetonitrile and water of known concentration, added to chilled reduced antibody in PBS. After about one hour, excess maleimide was added to quench the reaction and protect any unreacted antibody thiol groups. The reaction mixture was concentrated by centrifugal ultrafiltration and ADC, e.g. AC10-MC-vc-PAB-MMAE, was purified and desalted by eluting on G25 resin in PBS, filtered through 0.2 gm filters under sterile conditions, and frozen for storage.

[0507] A number of antibody drug (ADC) conjugates were prepared, with a number of linkers and drug moieties, MMAE and MMAF. The following table is an example of an ADC group that was prepared using the following protocol from Example 27, and was characterized by HPLC and drug load assay.

<td>Object (antigen)</td><td>target ADC</td><td>isolated (Mg)</td><td>quantity ratio drug / Ab</td>

<td>0772P</td><td>16E12-MC-vc-P AB-MM AE reference</td><td>Przykład1,75</td><td> 4</td>

<td>0772P</td><td>11D10-MC-vc-P AB-MM AE reference</td><td>Przykład46,8</td><td> 4,4</td>

<td>0772P</td><td>11D10-MC-vc-P AB-MM AF</td><td> 54,5</td><td> 3,8</td>

<td>Brevican</td><td>Brevican-MC-MMAF</td><td> 2</td><td> 6</td>

<td>Brevican</td><td>Brevican-MC-vc-MMAE</td><td> 2</td><td> 6</td>

<td>Brevican</td><td colspan="2">Brevican-MC-vc-PAB-MMAF 1,4</td><td> 6</td>

<td>CD21</td><td>CD21-MC-vc-PAB-MMAE</td><td>Example 38.1</td><td> 4,3</td>

175

<td>Object</td><td>target ADC</td><td>isolated</td><td>quantity ratio</td>

<td>(antigen)</td><td>reference</td><td>(Mg)</td><td>drug / Ab</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 reference</td><td>Przykład2,3</td><td> 4,6</td>

<td>E16</td><td>3B5-MC-vc-PAB-MMAE reference</td><td>Przykład2,9</td><td> 4,6</td>

<td>E16</td><td>12B9-MC-vc-PAB-MMAE reference</td><td>Przykład1,4</td><td> 3,8</td>

<td>E16</td><td>12B9-MC-vc-PAB-MMAE reference</td><td>Przykład5,1</td><td> 4</td>

<td>E16</td><td>12G12-MC-vc-PAB-MMAE reference</td><td>Example 3</td><td> 4,6</td>

<td>E16</td><td>3B5-MC-vc-PAB-M MAE reference</td><td>Przykład4,8</td><td> 4,1</td>

<td>E16</td><td>385-MC-vc-PAB-MMAF</td><td> 24,7</td><td> 4,4</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAE reference</td><td>Przykład29,9</td><td> 7,1</td>

<td>EphB2R</td><td>2H9-MC-fk-PAB-MMAE reference</td><td>Przykład25</td><td> 7,5</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAE reference</td><td>Przykład175</td><td> 4,1</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAF</td><td> 150</td><td> 3,8</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAF</td><td> 120</td><td> 3,7</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAE reference</td><td>For example 10.7</td><td> 4,4</td>

<td>IL-20Ra</td><td>IL20Ra-fk-MMAE reference</td><td>Example 26</td><td> 6,7</td>

<td>IL-20Ra</td><td>IL20Ra-vc-MMAE reference</td><td>Przykład27</td><td> 7,3</td>

<td>EphB2</td><td>IL8-MC-vc-PAB-MMAE reference</td><td>Przykład251</td><td> 3,7</td>

176

<td>Object (antigen)</td><td>target ADC</td><td>isolated (Mg)</td>

<td>MDP</td><td>MDP-vc-MMAE reference</td><td>Przykład32</td>

<td>MPF</td><td>19C3-vc-MMAE reference</td><td>Przykład1,44</td>

<td>MPF</td><td colspan="2">7D9-vc-MMAE Reference example 4.3</td>

<td>MPF</td><td>19C3-vc-MMAE reference</td><td>Przykład7,9</td>

<td>MPF</td><td>7D9-MC-vc-PAB-MMAF</td><td> 5</td>

<td>Napi3b</td><td>10H1-vc-MMAE reference</td><td>Przykład4,5</td>

<td>Napi3b</td><td colspan="2">4C9-vc-MMAE Reference example 3.0</td>

<td>Napi3b</td><td>10H1-vc-MMAE reference</td><td>Przykład4,5</td>

<td>Napi3b</td><td>10H1-vc-MMAF</td><td> 6,5</td>

<td>NCA</td><td>3E6-MC-fk-PAB-MMAE reference</td><td>Przykład49,6</td>

<td>NCA</td><td>3E6-MC-vc-PAB-M MAE reference</td><td>Przykład56,2</td>

<td>PSCA</td><td>PSCA-fk-MMAE reference</td><td>Przykład51,7</td>

<td>PSCA</td><td>PSCA-vc-MMAE reference</td><td>Przykład61,1</td>

<td>Napi3b</td><td>10H1-MC-vc-PAB-MMAE</td><td> 75</td>

<td>Napi3b</td><td>10H1-MC-vc-PAB-MMAF</td><td> 95</td>

<td>Napi3b</td><td>10H1-MC-MMAF</td><td> 92</td>

<td>EphB2R</td><td>2H9-MC-vc-PAB-MMAE reference</td><td>Przykład79</td>

<td>EphB2R</td><td>2H9-MC-MMAF</td><td> 92</td>

quantity Drug / Ab. ratio

0772P 11D10 (Fc-chimera) -MC-vc-PAB-79

MMAE Reference example

<td>0772P</td><td>11D10 (Fc-chimera) -MC-vc-PABMMAF</td><td> 70</td>

6.5

3.8

4.3

4.6

5.4

4.8

5.4

6.4

8.9

8.6

4.2

4.4

4.9

4.3

4.5

177

<td>Object</td><td>target ADC</td><td>isolated</td><td>quantity ratio</td>

<td>(antigen)</td><td></td><td>(Mg)</td><td>drug / Ab</td>

<td>0772P</td><td>11D10 (Fc-chimera) -MC-MMAF</td><td> 23</td><td> 4,5</td>

<td>Brevican</td><td>6D2-MC-vc-PAB-MMAF</td><td> 0,3</td><td> 4,5</td>

<td>Brevican</td><td>6D2-MC-MMAF</td><td> 0,36</td><td> 4,5</td>

<td>EphB2R</td><td colspan="2">2H9 (Fc-chimera) -MC-vc-PAB-MMAE 1983 Reference example</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 colspan="2">12G12-MC-vc-PAB-MMAE Example 10.5 reference</td><td> 4,1</td>

<td>E16</td><td>12G12-MC-vc-PAB-MMAF</td><td> 10,2</td><td> 3,8</td>

<td>E16</td><td colspan="2">3B5-MC-vc-PAB- MMAE Example 58.6 reference</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>11D10 (Fc-chimera) -MC-vc-PABMMAE Reference</td><td> 340</td><td> 3,9</td>

<td>Steap1</td><td>example (Steap1-92) -MC-vc-PAB-MMAE Reference example</td><td> 3,5</td><td> 4</td>

<td>Steap1</td><td>(Steap 1-92) -MC-vc-PAB-MMAF</td><td> 4,7</td><td> 4</td>

<td>Steap1</td><td>(Steap 1 -120) -MC-vc-PAB-MMAE Reference example</td><td> 2</td><td> 4</td>

<td>Steap1</td><td>(Steap 1 -120) -MC-vc-PAB-MMAF</td><td> 2,3</td><td> 4</td>

<td>E16</td><td>3B5-MC-vc-PAB-MMAF</td><td> 52,2</td><td> 4,5</td>

4.7 COMPOSITIONS AND METHODS OF ADMINISTRATION [0508] In other embodiments, a composition is described comprising effective amounts of an exemplary compound and / or exemplary conjugate and a pharmaceutically acceptable carrier or excipient. For convenience, drug units and drug-linker compounds can be referred to as exemplary compounds, while drug-ligand conjugate and drug-linker-ligand conjugates can be referred to as exemplary conjugates. The compositions are suitable for veterinary or human administration.

[0509] The present compositions may be in any form that allows the composition to be administered to a patient. For example, the composition may be in the form of a solid,

178 liquid or gas (aerosol). Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, intraocular, intratumoral and intranasal routes. Parenteral administration includes subcutaneous, intravenous, intramuscular, intrasternal or infusion techniques. In one aspect, the compositions are administered parenterally. In yet another aspect, exemplary compounds and / or exemplary conjugates or compositions are administered intravenously.

[0510] Pharmaceutical compositions may be formulated to allow the exemplary compound and / or exemplary conjugate to be bioavailable upon administration of the composition to a patient. The compositions may take the form of one or more dosage units, wherein, for example, the tablet may be a single dosage unit, and the container of the exemplary compound and / or the exemplary aerosol conjugate may contain a number of dosage units.

[0511] Materials used in the preparation of pharmaceutical compositions may be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in a pharmaceutical composition will depend on a number of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the exemplary compound or exemplary conjugate, the mode of administration, and the composition used.

[0512] The pharmaceutically acceptable carrier or excipient may be composed of particles, and therefore the compositions may be in the form of tablets or a powder, for example. The carrier (s) may be a liquid, with compositions meaning, for example, oral syrup or an injectable liquid. In addition, the carrier (s) may be gaseous or composed of particles so as to provide an aerosol composition useful in, e.g., inhalation administration.

[0513] When oral administration is intended, the composition is preferably in solid or liquid form, wherein the semi-solid, semi-liquid, suspension and gel form are included within the forms considered as solid or liquid as described herein.

[0514] As a solid composition for oral administration, the composition may be formulated as a powder, granules, compressed tablets, pills, capsules, chewing gums, cachets or the like. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrants such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin, a flavoring such as peppermint, methyl salicylate or orange flavor, and a coloring agent.

[0515] When the composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

179 [0516] The composition may be in the form of a liquid, e.g., a potion, syrup, solution, emulsion or suspension. Liquids can be useful for oral administration or for injection delivery. When oral administration is planned, the composition may contain one or more sweeteners, preservatives, dyes / coloring agents and an aroma enhancer. One or more surfactants, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may also be included in compositions for administration by injection.

[0517] Liquid compositions, whether solutions, suspensions or other similar forms, may also contain one or more of the following: sterile diluents such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, hardened oils, such as synthetic mono or diglycerides that can serve as a solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The parenteral composition can be enclosed in an ampoule, disposable syringe or multi dose dose made of glass, plastic or other material. Saline means an exemplary adjuvant. The injectable composition is preferably sterile.

[0518] The amount of therapeutic compound or exemplary conjugate that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition, and this can be assessed by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose to be used in the compositions will also depend on the route of administration, and the severity of the disease or disorder, and should be selected at the discretion of the practitioner and the circumstances of each patient.

[0519] The compositions contain effective amounts 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 wt. compositions for an exemplary compound and / or exemplary conjugate. When oral administration is planned, the amount may range from about 0.1% to about 80% by weight of the composition. In one aspect, the oral compositions may contain from about 4% to about 50% by weight. compositions for an exemplary compound and / or exemplary conjugate. In yet a further aspect, the present compositions are prepared so that the parenteral dosage unit contains from about 0.01% to about 2% by weight of an exemplary compound and / or exemplary conjugate.

[0520] For intravenous administration, the composition may contain from about 0.01 to about 100 mg of an exemplary Compound and / or Exemplary conjugate per kg animal body weight. In one aspect, the composition may contain from about 1 to about 100 mg of an exemplary compound and / or exemplary conjugate per kg animal body weight. In another aspect

180 the amount administered will range from about 0.1 to about 25 mg / kg body weight of the exemplary compound and / or exemplary conjugate.

[0521] Generally, the dosage of the exemplary compound and / or exemplary conjugate administered to a patient is typically about 0.01 mg / kg to about 2000 mg / kg body weight of the animal. In one aspect, the dosage administered to the patient is between about 0.01 mg / kg to about 10 mg / kg of the body weight of the animal, in another aspect, the dosage administered to the patient is between about 0.1 mg / kg and about 250 mg / kg of the body weight of the animal. in yet another aspect, the dosage administered to the patient is between about 0.1 mg / kg and about 20 mg / kg of the body weight of the animal, in yet another aspect, the dosage administered is between about 0.1 mg / kg to about 10 mg / kg of the body weight of the animal . 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.

[0522] Exemplary compounds and / or exemplary conjugate or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous lining (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 and the like, 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.

[0523] In certain embodiments, it may be desirable to administer one or more exemplary compounds and / or exemplary conjugate or composition locally to an area in need of treatment. This can be achieved, for example, without limitation, by local infusion during surgery; topical application, e.g. in conjunction with a wound dressing; by injection; with a catheter; using a suppository; or by means of an implant, wherein the implant means porous, non-porous or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, the administration may be carried out as a direct injection at the site (or former site) of the cancer, cancer or cancerous or precancerous tissue. In another embodiment, administration may be as a direct injection at the site (or former site) of the onset of the autoimmune disease.

[0524] In certain embodiments, it may 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.

181 [0525] Pulmonary administration may also be used, e.g., by using an inhaler or nebulizer, and formulation with a spray, or by perfusion in a fluorine hydrocarbon derivative or synthetic lung surfactant.

[0526] In yet another embodiment, exemplary compounds and / or exemplary conjugate or compositions may be provided by means of a controlled release system, such as, but not limited to, a pump, or various polymeric materials may be used. In yet another embodiment, a controlled release system may be placed adjacent to the target of exemplary compounds and / or exemplary conjugate or composition, e.g. of the brain, hence only part of the systemic dose will be required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems may be used as discussed in the review by Langer (Science 249: 1527-1533 (1990)).

[0527] The term "carrier" refers to a diluent, adjuvant or excipient with which an exemplary compound and / or exemplary conjugate are administered. Such pharmaceutical carriers can be sterile 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. Carriers may include saline, acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. In one embodiment, for administration to a patient, an exemplary compound and / or exemplary conjugate or compositions and pharmaceutically acceptable carriers are sterile. Water is an exemplary carrier when exemplary compounds and / or exemplary conjugates are administered intravenously. Saline solutions, and aqueous dextrose and glycerol solutions may also be used 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, skimmed milk powder, glycerol, propylene glycol , water, ethanol and the like. The present compositions, if desired, may also contain minor amounts of wetting agents or emulsifying agents, or pH buffering agents.

[0528] The present compositions may take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0529] In an embodiment, exemplary compounds and / or exemplary conjugates are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, especially humans. Typically, carriers or excipients for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions may also contain an agent

182 solubilizing. Compositions for intravenous administration may optionally contain local anesthesia, such as lignocaine, to ease pain at the injection site. In general, the ingredients are provided 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 sachet, indicating the amount of active agent. When the exemplary compound and / or exemplary conjugate are to be administered by infusion, it may be dispensed, e.g., via an infusion bottle containing sterile sterile water or pharmaceutical grade physiological saline. When the exemplary compound and / or exemplary conjugate are to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

[0530] Oral delivery compositions may be, e.g., in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavorings such as peppermint, wintergreen oil, or cherry oil; coloring agents; and preservatives to provide a tasty pharmaceutical preparation. In addition, in the form of tablets or pills, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a prolonged effect over an extended period of time. Selectively permeable membranes surrounding an osmotically active lead compound are also suitable for orally administered compounds. In these latter platforms, liquid from the environment surrounding the capsule is absorbed by a lead compound that swells to displace the agent or composition of agents through the opening. These delivery platforms can generally provide a zero order delivery profile, as opposed to peak-rich immediate release formulation profiles. Time delay material such as glycerol monostearate or glycerol stearate may also be used.

[0531] The compositions may be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment or gel base. For planned transdermal administration, the composition may be in the form of a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of an exemplary compound and / or exemplary conjugate from about 0.05% to about 50% w / v. (weight per unit volume of the composition), in another aspect from 0.1% to 10% w / v.

[0532] The composition may be for rectal administration, in the form, e.g., a suppository, which will melt in the rectum and release the exemplary compound and / or exemplary conjugate.

[0533] The composition may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may contain materials that form a coating around the active ingredients. The materials that make up the coating are typically inert, and can be selected from, for example, sugar, shellac, and

183 other coating agents for release in the intestine. Alternatively, the active ingredients may be enclosed in a gelatin capsule.

[0534] The compositions may consist of gas dosage units, e.g. they may be in the form of an aerosol. The term aerosol is used to mean a variety of systems that range from colloidal to systems consisting of pressurized packaging. Delivery can take place via liquefied or compressed gas, or through a suitable pump system that distributes the active ingredients.

[0535] Whether in solid, liquid or gaseous form, the present compositions may contain a pharmacological agent used to treat cancer, an autoimmune disease or an infectious disease.

4.8. THERAPEUTIC APPLICATIONS OF EXAMPLE CONGRATULATES [0536] Exemplary compounds and / or exemplary conjugates are useful in the treatment of cancer, an autoimmune disease or an infectious disease in a patient.

4.8.1 TREATMENT OF CANCER [0537] Exemplary compounds and / or exemplary conjugates are useful for inhibiting tumor cell or cancer cell proliferation, resulting in apoptosis in a cancer cell or cancer cell, or in treating cancer in a patient. Exemplary compounds and / or exemplary conjugates may be used accordingly in a number of systems for the treatment of cancers in animals. Drug-linker-ligand conjugates can be used to deliver a drug or drug unit to a cancer cell or cancer cell. Without being bound by theory, in one embodiment the ligand unit of the exemplary conjugate binds to or binds to the antigen associated with the cancer cell or cancer cell, and the exemplary conjugate can be taken into the interior of the cancer cell or cancer cell via receptor dependent endocytosis. The antigen may attach to the cancer cell or cancer cell, or may be an extracellular matrix protein associated with the cancer cell or cancer cell. After entering the cell, one or more specific peptide sequences within the linker unit are hydrolytically cleaved by one or more proteases associated with the cancer cell or cancer cell, resulting in the release of the drug or drug-linker compound. The released drug or drug-linker compound is then free during intracellular migration, and induces cytotoxic or cytostatic activities. In an alternative embodiment, the drug or drug unit is excised from the exemplary conjugate outside the cancer cell or cancer cell, and the drug or drug-linker compound further penetrates the cell.

[0538] In one embodiment, the ligand unit binds to the cancer cell or cancer cell.

[0539] In another embodiment, the ligand unit binds to a tumor cell or cancer cell antigen that is on the surface of the cancer cell or cancer cell.

184 [0540] In another embodiment, the ligand unit binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with the cancer cell or cancer cell.

[0541] The specificity of the ligand unit for a particular cancer cell or cancer cell may be important in determining those cancers or cancers that are most effectively treated. For example, exemplary conjugates having a BR96 ligand unit may be useful in the treatment of antigen-positive tumors, including those of the lung, breast, colon, ovary and pancreas. Exemplary conjugates having an anti-CD30 ligand unit or an anti-CD40 ligand unit may be useful in the treatment of hematologic malignancies.

[0542] Other specific types of cancers that may be treated by exemplary conjugates include, but are not limited to, those disclosed in Table 3.

Table 3

Solid tumors, including but not limited to:

fibrosarcoma sarcoma liposarcoma cartilage sarcoma osteosarcoma osteosarcoma hemangioma endothelial sarcoma sarcoma lymphosarcoma lymphosarcoma lymphosarcoma lymphosarcoma sarcoma sarcoma mesothelioma carcinoma carcinoma myocarcinoma carcinoma myosarcoma

185 bone cancer breast cancer ovarian cancer prostate cancer esophageal cancer stomach cancer oral cancer nasal cancer throat cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma cancer of sweat gland cancer sebaceous gland cancer papillary carcinoma papillary cystoma carcinoma carcinoma carcinoma carcinoma carcinoma carcinoma cancer Wilms embryonic cancer cervical cancer uterine cancer small cell nucleus lung cancer bladder cancer

186 lung cancer epithelial carcinoma glioblastoma glioblastoma multiforme astrocytoma neuralgia craniocephaloma ependymoma pineal gland hemangioblastoma neuroma of the auditory nerve tumor of brain glial cells meningioma skin cancer melanoma neuroma retinoblastoma retinoblastoma, including but not limited to:

acute ALL lymphoblastic leukemia acute B cell lymphoblastic leukemia acute T cell lymphoblastic leukemia acute AML myeloblastic leukemia acute APL leukemic acute leukemia CML "chronic lymphocytic leukemia" CLL "

187 hairy cell leukemia multiple myeloma acute and chronic leukemia:

lymphoblastic myeloid origin lymphocytic myelocytic leukemia

lymphomas:

Hodgkin's disease non-Hodgkin's lymphoma multiple myeloma Waldenstrom macroglobulinemia heavy chain disease polycythemia vera polycythemia [0543] Exemplary conjugates provide tumor-specific coupling or targeting cancer, hence reducing the overall toxicity of these compounds. Linkeras units stabilize exemplary conjugates in the blood, but are cleavable by tumor-specific proteases within the cell, releasing the drug.

4.8.2 MULTIMODAL TREATMENT OF CANCER [0544] Cancers, including, but not limited to, cancer, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of an exemplary conjugate and / or exemplary compound.

[0545] Methods for treating or preventing cancer are described herein, including administering to a patient in need thereof an effective amount of an exemplary conjugate and chemotherapeutic agent. In one method, a chemotherapeutic agent is one for which cancer treatment has not proved to be refractory to treatment. In another method, a chemotherapeutic agent is one for which cancer treatment has proved to be refractory to treatment. Exemplary conjugates can be administered to a patient who has also undergone surgery as a treatment for cancer.

[0546] In one method, the additional method of treatment is radiation therapy.

[0547] In a specific method, an exemplary conjugate is administered simultaneously with a chemotherapeutic agent or with radiation therapy. In another particular method, the chemotherapeutic agent or radiation therapy is administered before or after the administration of exemplary conjugates, in one aspect at least an hour, five hours, 12 hours, day, tobacco.

188 a month, in further aspects several months (e.g., up to three months), before or after administration of the exemplary conjugate.

[0548] The chemotherapeutic agent may be administered in a number of sessions. Any or a combination of the chemotherapeutic agents listed in Table 4 can be administered. For radiotherapy, any radiotherapy protocol can be used, depending on the type of cancer to be treated. For example, but not limited to, X-ray irradiation; in particular, high-energy megavolt therapy (radiation greater than 1 MeV energy) can be used for deep tumors, and electron beam and ortovoltaic X-ray irradiation for skin cancers. Gamma-emitting radioactive isotopes, such as radioactive isotopes of radium, cobalt and other elements, may also be administered.

[0549] In addition, methods of treating cancer with an exemplary compound and / or exemplary conjugate have been described as alternatives to chemotherapy or radiotherapy, wherein the chemotherapy or radiation therapy has proved to be too toxic, e.g. resulting in unacceptable or unbearable adverse effects on the subject being treated. The treated animal can optionally be treated with subsequent cancer treatments such as surgery, radiation or chemotherapy, depending on which treatment proves to be acceptable or tolerable.

[0550] Exemplary compounds and / or exemplary conjugates may also be used in an in vitro or ex vivo manner, such as for the treatment of certain cancers, including, but not limited to, leukemia and lymphoma, such treatment involves autologous stem cell transplantation. This may involve a multi-stage process in which animal autologous hematopoietic stem cells are removed and purified from all cancer cells, the remaining animal bone marrow population is then removed by high dose administration of the exemplary compound and / or exemplary conjugate, with or without radiotherapy at high doses, and the stem cell transplant is infused back into the animal. Supportive treatment will then be provided while bone marrow function is restored and the animal returns to form.

4.8.3 MULTIMODAL TREATMENT OF CANCER [0551] Methods for treating cancer are disclosed, 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. Suitable anti-cancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxycarbamide, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, nitacarbazin, dacotarbazin 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, fileamycin, mitoxantrone, asparaginase,

189 vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel. In one aspect, the anti-cancer agent includes, but is not limited to, the drug listed in Table 4. TABLE 4

<td>Alkylating agents</td><td></td>

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

<td>nitrosoureas:</td><td>carmustine (BCNU) Lomustine (CCNU)</td>

<td>alkyl</td><td>busulfan</td>

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

<td>triazenes:</td><td>dacarbazine</td>

<td>Compounds containing platinum:</td><td>cisplatin carboplatin</td>

<td>Plant alkaloids</td><td></td>

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

<td>taxanes:</td><td>paclitaxel docetaxel</td>

<td>DNA topoisomerase inhibitors</td><td></td>

<td>epipodophyllotoxins:</td><td>etoposide teniposide topotecan 9-aminocamptothecin camptothecin crisnatol</td>

<td>mi tin volumes:</td><td>mitomycin C.</td>

190

<td>Anti-metabolites</td><td></td>

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

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

<td>IMP dehydrogenase inhibitors:</td><td>mycophenolic acid tiazofurin ribavirin EICAR</td>

<td>Ribonucleotide reductase inhibitors:</td><td>hydroxyurea deferoxamine</td>

<td>Pyrimidine analogues:</td><td></td>

<td>Uracil analogues</td><td>5-fluorouracil floxuridine doxifluridine ratitreksed</td>

<td>Anti-metabolites</td><td></td>

<td>Cytosine analogues</td><td>cytarabine (macaw C) cytosine arabinoside fludarabine</td>

<td>Purine analogues:</td><td>mercaptopurine thioguanine</td>

<td>Hormone therapies:</td><td></td>

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

<td>Anti-estrogen</td><td>tamoxifen raloksfen megestrol</td>

<td>LHRH agonists:</td><td>goserelin leuprolide acetate</td>

<td>Anti-androgens:</td><td>flutamide bicalutamide</td>

191

<td>Retinoids / Deltoids</td><td></td>

<td>Vitamin D3 analogues:</td><td>EB 1089 CB 1093 KH 1060</td>

<td>Photodynamic therapies:</td><td>verteporfin (BPD-MA) phthalocyanine Pc4 photosensitizer demethoxy-hypocrellin A (2BA-2-DMHA)</td>

<td>cytokines:</td><td>Interferon-α Interferon-γ tumor necrosis factor</td>

<td>Other:</td><td>gemcitabine Velcade Rewamid Thalamid</td>

<td>Isoprenylation inhibitors:</td><td>lovastatin</td>

<td>dopaminergic neurotoxins:</td><td>1-methyl-4-phenylpyridinium ion</td>

<td>Cell cycle inhibitors:</td><td>staurosporine</td>

<td>actinomycin:</td><td>Actinomycin D dactinomycin</td>

<td>bleomycin:</td><td>bleomycin A2 bleomycin B2 peplomycin</td>

<td>anthracyclines:</td><td>daunorubicin doxorubicin (Adriamycin) idarubicin epirubicin</td>

192

<td>Retinoids / Deltoidy</td><td></td>

<td></td><td>pirarubicin</td>

<td></td><td>zorubicin</td>

<td></td><td>mitoxantrone</td>

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

<td>Ca2 + ATPase inhibitors:</td><td>thapsigargin</td>

4.8.4 TREATMENT OF AUTOIMMUNOLOGICAL DISEASES [0552] Exemplary conjugates are useful for killing or inhibiting cell replication that causes an autoimmune disease or in the treatment of an autoimmune disease. Exemplary conjugates can be used accordingly in a number of systems for treating an autoimmune disease in a patient. Drug-linker-ligand conjugates can be used to deliver the drug to the target cell. Without being bound by theory, in one embodiment the drug-linker-ligand conjugate combines with an antigen on the surface of the target cell, and the exemplary conjugate is then taken up inside the target cell via receptor dependent endocytosis. Once inside the cell, one or more specific peptide sequences within the linker unit are enzymatically or hydrolytically cleaved, resulting in drug release. The released drug is then free during intra-cytosol migration, and induces cytotoxic or cytostatic activities. In an alternative embodiment, the drug is excised from the exemplary conjugate outside the target cell, and the drug then penetrates the cell.

[0553] In one embodiment, the ligand unit binds to the immunogenic antigen. In one aspect, the antigen is present on the cell surface by participating in an autoimmune state.

[0554] In another embodiment, the ligand unit binds to an autoimmune antigen that is on the surface of the cell.

[0555] In one embodiment, the ligand binds to an activated lymphocyte that is associated with an autoimmune disease state.

[0556] In a further embodiment, exemplary conjugates kill or inhibit the proliferation of cells that produce an autoimmune antibody associated with a particular autoimmune disease.

[0557] Certain types of autoimmune diseases that can be treated by exemplary conjugates include, but are not limited to, Th2 lymphocyte-related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis and graft versus host disease); disorders related to Th1 lymphocytes (e.g. rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Graves disease, primary biliary cirrhosis, Wegener's granulomatosis and

193 tuberculosis); disorders associated with activated B lymphocytes (e.g. systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis and type I diabetes); and those disclosed in table 5.

TABLE 5 active chronic hepatitis Addison's disease allergic alveolitis allergic allergic allergic rhinitis Alport syndrome anaphylaxis ankylosing spondylitis antiphospholipid syndrome arthritis asparagus aspergillosis atopic allergy atopic dermatitis atrophic rhinitis rhizophyll rhinitis nasophagitis disease Behcta disease cardiomyopathy, celiac disease, Chagas disease, chronic glomerulonephritis Cogan syndrome cold illness agglutinin congenital rubella infection CREST syndrome

194 Crohn's disease cryoglobulinemia Cushing's syndrome dermatomyositis lupus Dressler syndrome Eaton-Lambert syndrome echovirus infection encephalomyelitis endocrine ophthalmopathy Epstein-Barr virus infection chronic obstructive pulmonary disease in conjunctivoid disease Evan syndrome Fibromyalgia Fibromyialgia cervical inflammation gastrointestinal giant cell arteritis glomerulonephritis Goodpasture syndrome graft versus host disease Graves disease Guillain-Barre disease Hashimoto's thyroiditis hemolytic anemia Henoch-Schonlein purpura idiopathic adrenal atrophy idiopathic pulmonary fibrosis IgA nephropathy

195 inflammatory bowel disease insulin-dependent juvenile arthritis juvenile diabetes (type I) Lambert-Eaton syndrome plaque lupus lupoid hepatitis Lupus lymphopenia Meniere's disease mixed connective tissue disease multiple sclerosis myasthenia gravis severe pseudoprotective anemia pre-ridiculoma multigranular polyarthritis psoriasis psoriasis psoriatic arthritis phenomenon Raynauds recurrent miscarriage Reiter's syndrome rheumatic fever rheumatoid arthritis Sampter's syndrome schistosomiasis Schmidt's syndrome scleroderma Shulman's syndrome

196 Sjorgen's syndrome generalized stiffness syndrome sympathetic ophthalmitis lupus erythematosus systemic arteritis Takayasu temporal arteritis inflammation of the thyroid gland thrombocytopenia thyrotoxicosis toxic epidermal necrolysis type B insulin resistance type I ulcerative colitis uveitis uveitis granulomatosis papuloma papilloma maculoplasm

4.8.5 MULTI-DRUG THERAPIES FOR AUTOIMMUNOLOGICAL DISEASES [0558] Also disclosed are methods for treating autoimmune diseases, including administering to a patient in need thereof an effective amount of an exemplary conjugate and other therapeutic agent known to treat autoimmune disease. In one embodiment, the anti-immune disease agent is, without limitation, the agents listed in Table 6.

Table 6 cyclosporine cyclosporine A mycophenylate mofetil sirolimus tacrolimus enanercept prednisone azatioprine

197 methotrexate cyclophosphamide prednisone aminocaproic acid chloroquine hydroxychloroquine hydrocortisone dexamethasone chlorambucil

DHEA danazol bromocriptine meloxicam infliximab

4.8.4 TREATMENT OF INFECTIOUS DISEASES [0559] Exemplary conjugates are useful for killing or inhibiting the proliferation of cells that cause an infectious disease or for the treatment of an infectious disease. Exemplary conjugates can be used accordingly in a number of systems for treating an infectious disease in a patient. Drug-linker-ligand conjugates can be used to deliver the drug to the target cell. In one embodiment, the ligand unit binds to an infectious disease cell.

[0560] In one embodiment, the conjugates kill or inhibit the proliferation of cells that cause a particular infectious disease.

[0561] Specific types of infectious diseases that can be treated by exemplary conjugates include, but are not limited to, those disclosed in Table 7.

TABLE 7 Bacterial diseases:

diphtheria pertussis latent bacteraemia urinary tract infection gastroenteritis cellulitis

198 epiglottitis tracheitis adenoid hypertrophy abscess, impetigo, impetigo, pneumothorax, endocarditis, septic arthritis, pneumococcal infection peritonitis bacteremia meningitis acute suppurative meningitis urethritis cervicitis proctitis inflammation of the pharynx inflammation pharynx inflammation throat inflammation listeriosis anthrax nocardiosis salmonella typhoid fever diarrhea conjunctivitis sinusitis brucellosis

199 tularemia cholera bubo necrotizing necrotizing enterocolitis actinomycosis mixed anaerobic infections syphilis major recurrent leptospirosis Lyme disease fever after rat bite tuberculosis lymphadenitis leprosy chlamydia chlamydial pneumonia trachoma conjunctivitis systemic fungal diseases:

histoplasmosis coccidioidomycosis blastomycosis sporotrichosis cryptococcosis

Systemic yeast aspergillosis mucormycosis mycelium chromomycosis

Rickettsial diseases:

200 typhus

Rocky Mountain Spotted Fever

Rickettsiae: Eastern erlichiosis Rickettsiosis transmitted by ticks Rickettsiapox fever Q Bartonellosis

Parasitic diseases: malaria babesiosis, coma, African disease, Chagas, leishmaniasis, fever, Dum-Dum, toxoplasmosis, meningitis and encephalitis, keratitis, infection, Dientamoeba fragilis, lambliosis, cryptosporidiosis, isosporidosis, cyclosporiaza, microsporidosis, asparidosis, pinworm infection, hookworm infection, hookworm infection, tetanus infection.

201 lymphatic filaria disease loaosis river blindness dirofilariosis schistosomiasis pruritus of the float infection with oriental fluke infection with pulmonary fluke infection with oriental fluke infection fasciolosis fasciolopsidosis opistorchosis tapeworm infections alveolar echinococcosis

Viral diseases:

measles subacute sclerosing encephalitis common cold

Viral diseases: mumps rubella chickens Fifth disease chickenpox infection syncytial respiratory virus croup bronchiolitis infectious mononucleosis paralysis herpes herpes pharyngitis hand, foot and mouth disease

202 Bornholm's disease genital herpes genital warts aseptic meningitis myocarditis pericarditis gastroenteritis acquired immunodeficiency syndrome (AIDS) human immunodeficiency virus (HIV) Reye's syndrome Kawasaki syndrome flu bronchitis viral pneumonia not requiring hospitalization acute respiratory disease with fever acute fever with sore throat and conjunctivitis epidemic keratitis Herpes Simplex 1 virus (HSV-1) Herpes Simplex 2 virus (HSV-2) herpes zoster cytomegaly with inclusions rabies progressive multifocal leukoencephalopathy hen fatal insomnia familial Creutzfeldt-Jakob disease Gerstmann-Straussler-Scheinker disease western low calf inflammation encephalitis Louis

203 yellow fever dengue lymphocytic meningitis Lassa fever haemorrhagic fever pulmonary syndrome hantavirus viral infection Marburg virus infection Ebola pox

4.8.7 MULTI-DRUG THERAPIES FOR INFECTIOUS DISEASES [0562] Methods for treating infectious diseases are disclosed, including administering to a patient in need thereof an exemplary conjugate and another therapeutic agent that is an agent against an infectious disease. In one embodiment, the agent against infectious disease is, without limitation, the agents listed in Table 8.

TABLE 8

Β-lactam antibiotics:

Penicillin G

Penicillin V

cloxacillin

dicloxacillin

methicillin

nafcillin

oxacillin

ampicillin

amoxicillin

bacampicillin

azlocillin

carbenicillin

Mezlocylna

Piperacillin

ticarcillin

aminoglycosides:

204

amikacin

gentamicin

kanamycin

neomycin

netilmicin

Streptomycin

tobramycin

macrolides:

azithromycin

clarithromycin

Erythromycin

lincomycin

clindamycin

tetracycline:

demeclocycline

doxycycline

minocycline

oxytetracycline

tetracycline:

tetracycline

quinolones:

Cinoxacin Nalidixic acid

fluoroquinolones:

ciprofloxacin

enoxacin

grepafloxacin

levofloxacin

lomefloxacin

norfloxacin

ofloxacin

205

sparfloxacin

trovafloxacin

polypeptides:

Bacitracin Kolistyna Polymyxin B

sulfonamides:

sulfisoxazole

sulfamethoxazole

sulfadiazine

Sulfametyzol

sulfacetamide

Various antibacterial agents: Trymetoprim Sulfamethamol Chloramphenicol Vancomycin Metronidazole Quinupristine Dalfopristine Rifampicin Spectinomycin Nitrofurantoin Antiviral agents:

General antiviral agents: Idoxuridine Vidarabine Trifluridine Acyclovir Famciclovir Penxiclovir

206

valacyclovir

ganciclovir

Foscarnet

ribavirin

amantadine

rimantadine

cidofovir

Antisense oligonucleotides

immunoglobulins

Inteferony

Drugs for HIV infection:

tenofovir

emtricitabine

zidovudine

didanosine

zalcitabine

stavudine

lamivudine

nevirapine

delavirdine

saquinavir

ritonavir

indinavir

nelfinavir

5. EXAMPLES

Example 1 - Preparation of compound AB [0563]

207

<img file="PL1725249T3_D0087.tif" />

[0564] Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 equiv, US Patent No. 6214345 for Firestone et al.) Was diluted with DMF (120 mL, 0.2 M) and diethylamine (60 mL) was added to this solution. The reaction was monitored by HPLC and was completed after 2 hours. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 ml) with sonication for over 10 min. Ether (200 ml) was added and the precipitate continued to be sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring, then filtered and dried under high vacuum to afford 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.). The reaction was completed by HPLC after 2 hours. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonication for 15 min, ether (400 ml) was added and sonication of the mixture was continued until all large particles were broken. The solution was then filtered and the solid dried to obtain an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m / z 757.9 [MH] [0565] Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 equiv, US Patent No. 6214345 to Firestone et al. ) was diluted with DMF (120 ml, 0.2 M) and diethylamine (60 ml) was added to this solution. The reaction was monitored by HPLC and was completed after 2 hours. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 ml) with sonication for over 10 min. Ether (200 ml) was added and the precipitate continued to be sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring, then filtered and dried under high vacuum to afford 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.). The reaction was completed by HPLC after 2 hours. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonication for 15 min, ether (400 ml) was added and sonication of the mixture continued until disintegrated

208 all large particles. The solution was then filtered and the solid dried to obtain an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m / z 757.9 [MH] [0566] An off-white solid of intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and bis (4 was added to the resulting solution) -nitrophenyl) carbonate (8.5 g, 28.0 mmol, 2.0 equiv) and DIEA (3.66 ml, 21.0 mmol, 1.5 equiv). The reaction ended after 1 hour. according to HPLC. The reaction mixture was concentrated to an oil, which 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 afford compound AB, which was 93% pure by HPLC and used in the next step without further purification. Yield: 9.7 g (94%).

Example 2 - Preparation of compound 1 [0567]

<img file="PL1725249T3_D0088.tif" />

[0568] HCl salt of phenylalanine t-butyl ester (868 mg, 3 mmol), N-Boc-dolaproin (668 mg, 1 equiv), DEPC (820 ml, 1.5 equiv) and DIEA (1.2 ml ) was diluted with dichloromethane (3 mL). After 2 hours (h) at room temperature (about 28 degrees Celsius), the reaction mixture was diluted with dichloromethane (20 ml), washed successively with saturated aqueous (aq) NaHCO₃ (2 x 10 ml), saturated aq. NaCl (2 x 10 mL). The organic layer was separated and concentrated. The resulting residue was reslurried in ethyl acetate and purified by flash chromatography in ethyl acetate. the relevant fractions were combined and concentrated to give the dipeptide as a white solid: 684 mg (46%). ES-MS m / z 491.3 [M + H] +.

[0569] For selective Boc cleavage, in the presence of t-butyl ester, the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 mL). 4 M HCl / dioxane (960 g, 3 equiv) was added and the reaction mixture was stirred overnight at room temperature. In RP-HPLC, almost complete removal of the Boc protecting group was observed, with a minimal amount of t-butyl ester cleavage. The mixture was cooled in an ice bath, and triethylamine (500 g) was added. After 10 min the mixture was removed from the cooling bath, diluted with dichloromethane (20 mL), washed successively with saturated water. NaHCO3 (2 x 10 mL), saturated aq. NaCl (2 x 10 ml). The organic layer was concentrated to a yellow foam: 287 mg (57%). The intermediate was used without further purification.

[0570] Fmoc-Meval-val-dil-Ot-Bu tripeptide (prepared as described in WO 02/088172, "Pentapeptide Compounds and Uses Related Thereto" _ 0.73 mmol) was treated with TFA (3

209 ml), dichloromethane (3 ml) for 2 hours. in room temperature. The mixture was concentrated to dryness, the residue co-evaporated with toluene (3 x 20 mL), and dried in vacuo overnight. The residue was diluted with dichloromethane (5 mL) and added to the deprotected dipeptide (287 mg, 0.73 mmol) followed by DIEA (550 g, 4 eq), DEPC (201 g, 1.1 eq). After 2 hours at room temperature, the reaction mixture was diluted with ethyl acetate (50 mL), washed successively with 10% aq. citric acid (2 x 20 ml), saturated aq. NaHCO3 (2 x 10 mL), saturated aq. NaCl (10 ml). The organic layer was separated and concentrated. The resulting residue was reslurried in ethyl acetate and purified by flash chromatography in ethyl acetate. The relevant fractions were combined and concentrated to give Fmoc-Meval-val-dil-dap-phe-Ot-Bu as a white solid: 533 mg (71%). R_f 0.4 (EtOAc). ES-MS m / z 1010.6 [M + H] +.

[0571] 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 give an oil, which was purified by flash chromatography on silica gel using a 0-10% MeOH step gradient in dichloromethane to afford compound 1 as a white solid: 137 mg (87%). R_f 0.3 (10% MeOWCH2Cl2). ES-MS m / z 788.6 [M + H] +.

Example 3 - Preparation of compound 2 [0572]

<img file="PL1725249T3_D0089.tif" />

[0573] Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol) by treatment with 4 M HCl / dioxane (4 mL) for 7 hours. in room temperature. The solvent was removed and the residue was dried in vacuo overnight to obtain compound 2 as a hygroscopic white solid: 35 mg (120% calculated for the HCl salt). ES-MS m / z 732.56 [M + H] +.

Example 4 - Preparation of compound 3 [0574]

210

<img file="PL1725249T3_D0090.tif" />

[0575] Fmoc-Meval-val-dil-dap-phe-Ot-Bu (Example 2, 50 mg) was treated with 4 M HCl / dioxane (4 mL) for 16 hours. in room temperature. The solvent was removed and the residue was dried in vacuo overnight to obtain 50 mg of intermediate in the form of a hygroscopic white solid.

[0576] The white solid of intermediate (20 mg, 0.02 mmol) was diluted with dichloromethane (1 mL); DEPC (5 g, 0.03 mmol, 1.5 eq) was added followed by DIEA (11 g, 0.06 mmol, 3 eq), and t-butylamine (3.2 g, 0.03 mmol, 1.5 eq.). After 2 hours at room temperature it was found by RP-HPLC that the reaction was not complete. More DEPC (10 g) and t-butylamine (5 g) were added and the reaction stirred for an additional 4 hours. The reaction mixture was diluted with dichloromethane (15 mL), washed successively with water (5 mL), 0.1 M aq. HCl (10 mL), saturated aq. NaCl (10 ml). The organic layer was separated and concentrated. The resulting residue was diluted with dichloromethane and purified by flash chromatography using a 0-5% MeOH step gradient in dichloromethane. The relevant fractions were combined and concentrated to afford the Fmoc protected intermediate as a white solid: 7.3 mg (36%). R_f 0.75 (10% MeOH / CH2Cl2).

[0577] The Fmoc protected intermediate was diluted with dichloromethane (0.5 mL) and treated with diethylamine (0.5 mL) for 3 hours. in room temperature. The reaction mixture was concentrated to dryness. The product was isolated by flash chromatography on silica gel using a 0-10% MeOH in dichloromethane step gradient to afford compound 3 as a white solid: 4 mg (70%). Rf 0.2 (10% MeOH / CH₂cl₂). ES-MS m / z 787 [M + H] +, 809 [M + Na] +.

Example 5 - Preparation of compound 4

<img file="PL1725249T3_D0091.tif" />

triethylene (164 mL, 1 mmol, 1 eq.) was diluted with dichloromethane (5 mL). Next

211 DCC (412 mg, 2 mmol, 2 equiv) was added followed by DMAP (10 mg). The reaction mixture was stirred overnight at room temperature. Oad was filtered off. The solvent was removed in vacuo, the residue was diluted with ethyl acetate and purified by flash chromatography on silica gel in ethyl acetate. The product containing fractions was drawn, concentrated and dried in vacuo to give a white solid: 377 mg (91%). Rf 0.5 (EtOAc). ES-MS m / z 434 [M + Na] +.

[0580] Removal of the Boc protecting group was carried out by treatment of the above material in dioxane (10 ml) with 4 M HCl / dioxane (6 ml) for 6 hours. in room temperature. The solvent was removed in vacuo, the residue was dried in vacuo to give a white solid.

[0581] HCl salt of phenylalanine ester-triethylene glycol monomethyl ether (236 mg, 0.458 mmol, 1 equiv) and N-Boc-dolaproin (158 mg, 0.55 mmol, 1.2 equiv) was diluted with dichloromethane (3 ml) , DEPC (125 g, 1.5 eq) and added to the mixture followed by DIEA (250 g, 3 eq). After 2 hours at room temperature, the reaction mixture was diluted with ethyl acetate (30 mL), washed successively with saturated aq. NaHCO3 (2 x 10 mL), 10% aq. citric acid (2 x 10 ml), saturated aq. NaCl (10 ml). The organic layer was separated and concentrated. The resulting residue was resuspended in ethyl acetate and purified by flash chromatography on silica gel in ethyl acetate. The relevant fractions were combined and concentrated to give a white foam intermediate: 131 mg (50%). Rf 0.25 (EtOAc). ES-MS m / z 581.3 [M + H] +.

[0582] Removal of the Boc protecting group was carried out in dichloromethane (2 ml), TFA (0.5 ml) at room temperature for 2 hours. The solvent was removed in vacuo and the residue co-evaporated with toluene (3 x 25 mL), then dried in vacuo to give 138 mg of dipeptide TFA salt.

[0583] Fmoc-Meval-val-dil-OH (example 2, 147 mg, 0.23 mmol, 1 equiv) and the dipeptide TFA salt (138 mg) were diluted with dichloromethane (2 mL). DEPC (63 g, 1.5 eq.) Was added to the mixture followed by DIEA (160 g, 4 eq.). After 2 hours at room temperature, the reaction mixture was diluted with ethyl acetate (30 mL), washed successively with 10% aq. citric acid (2 x 20 ml), saturated aq. NaCl (20 ml). The organic layer was separated and concentrated. The resulting residue was resuspended in dichloromethane and purified by flash chromatography on silica gel using a 0-5% MeOH step gradient in dichloromethane. The relevant fractions were combined and concentrated to a white foam: 205 mg (81%). R_f 0.4 (10% MeOH / CH₂cl₂). ES-MS m / z 1100.6 [M + H] +, 1122.4 [M + Na] +.

[0584] The Fmoc protecting group was removed by treatment with diethylamine (2 mL) in dichloromethane (6 mL). After 6 hours at room temperature the solvent was removed in vacuo, the product was isolated by flash chromatography on silica gel using a 0-10% MeOH step gradient in dichloromethane. The relevant fractions were combined and concentrated. After evaporation from dichloromethane / hexane, 1: 1, compound 4 is obtained

212 as white foam: 133 mg (80%). R_f 0.15 (10% MeOH / CH₂cl₂). ES-MS m / z 878.6 [M + H] +.

Example 6 - Preparation of compound 5 [0585]

<img file="PL1725249T3_D0092.tif" />

[0586] Fmoc-Meval-val-dil-OH (example 2, 0.50 g, 0.78 mmol) and dap-phe-OMeHCl (0.3 g, 0.78 mmol, prepared according to Pettit, GR, et al. Anti-Cancer Drug Design 1998, 13, 243277) was dissolved in CH2Cl2 (10 mL) followed by the addition of diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (0.20 ml, 1.17, 1.5 eq) was added and the content was allowed to stand over Ar. The reaction was completed by HPLC after 1 hour. The mixture was concentrated to an oil and purified by SiO chromatography₂ (300 x 25 mm column) and eluted with 100% EtOAc. The product was isolated as a white foamy solid. Yield: 0.65 g (87%). ES-MS m / z 968.35 [M + H] +, 991.34 [M + Na] +; UV / max 215, 265 nm.

[0587] Fmoc protected peptide (0.14 g, 0.14 mmol) in methylene chloride (5 ml) was treated with diethylamine (2 ml) and the contents allowed to stand at room temperature for 2 hours. The reaction, complete by HPLC, was concentrated to an oil, taken up in 2 mL DMSO and injected onto a preparative HPLC column (column C₁₂-RP, 5 μ, 100 A, MeCN linear gradient in water (containing 0.1% TFA) 10 to 100% in 40 min and then 20 min in 100%, with a flow rate of 25 ml / min). Product containing fractions were evaporated to give a white powder for the trifluoroacetate salt. Yield: 0.126 g (98%). Rf 0.28 (100% EtOAc); ES-MS m / z 746.59 [M + H] +, 768.51 [M + Na] +; UV /., "_ax 215 nm.

Example 7 - Preparation of compound 6

<img file="PL1725249T3_D0093.tif" />

213 [0589] Trifluoroacetate salt of compound 5 (0.11 g, 0.13 mmol), compound AB (0.103 g, 0.14 mmol, 1.1 equiv) and HOBt (3.4 mg, 26 mmol, 0.2 equiv) were suspended in DMF / pyridine (2 ml / 0.5 ml, respectively). Diisopropylethylamine (22.5 g, 0.13 mmol, 1.0 eq) was added and the yellow solution was stirred under argon. After 3 hours an additional 1.0 eq. was added DIEA. 24 hours later, 0.5 eq. activated linker was attached to the reaction mixture. After a total of 40 hours, the reaction was completed. The contents were evaporated, taken up in DMSO and injected onto a prep-HPLC column (column C₁₂-RP, 5 μ, 100 A, MeCN linear gradient in water (containing 0.1% TFA) 10 to 100% in 40 min and then 20 min in 100%, with a flow rate of 50 ml / min). The desired fractions were evaporated to give the product as a yellow oil. Methylene chloride (approx. 2 ml) and excess ether were added to obtain compound 6 as a white solid which was filtered and dried. Yield: 90 mg (52%). ES-MS m / z 1334.32 [M + H] +, 1366.29 [M + Na] +; UV λ ^_χ215.248 nm.

Example 8 - Preparation of compound 7

<img file="PL1725249T3_D0094.tif" />

[0591] Compound 4 (133 mg, 0.15 mmol, 1 equiv), compound AB, (123 mg, 0.167 mmol, 1.1 equiv), and HOBt (4 mg, 0.2 equiv) were diluted with DMF (1.5 ml). After 2 min, pyridine (5 mL) was added and the reaction monitored by RP-HPLC. It was shown that the reaction ended after 18 hours. The reaction mixture was diluted with dichloromethane (20 mL), washed successively with 10% aq. citric acid (2 x 10 ml), water (10 ml), saturated aqueous NaCl (10 ml). The organic layer was separated and concentrated. The resulting residue was resuspended in dichloromethane and purified by flash chromatography on silica gel using a 0-10% MeOH step gradient in dichloromethane. The relevant fractions were combined and concentrated to give compound 7 as a white foam: 46 mg (21%). R_f 0.15 (10% MeOH / CH₂cl₂). ES-MS m / z 1476.94 [M + H] +.

Example 9 - Preparation of MC-Val-Cit-PAB-MMAF 8 t-butyl ester [0592]

214

<img file="PL1725249T3_D0095.tif" />

[0593] Compound 1 (83 mg, 0.11 mmol), compound AB (85 mg, 0.12 mmol, 1.1 equiv) and HOBt (2.8 mg, 21 gmol, 0.2 equiv) were taken in anhydrous DMF (1.5 mL) and pyridine (0.3 mL) under argon. After 30 hours it was found by HPLC that the reaction was essentially complete. The mixture was evaporated, taken up in a minimal amount of DMSO and purified by prep-HPLC (column C₁₂-RP, 5 μ, 100 A, MeCN linear gradient in water (containing 0.1% TFA) 10 to 100% in 40 min and then 20 min in 100%, with a flow rate of 25 ml / min) to obtain compound 8 in the form of a white solid. Yield: 103 mg (71%). ES-MS m / z 1387.06 [M + H] +, 1409.04 [M + Na] +; UV /.',ax 205, 248 nm.

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

<img file="PL1725249T3_D0096.tif" />

TFA (3 mL) was added. The resulting solution was allowed to stand for 2 hours. The reaction mixture was concentrated in vacuo and purified by prep-HPLC (column C₁₂-RP, 5 μ, 100 A, MeCN linear gradient in water (containing 0.1% TFA) 10 to 100% in 40 min and then 20 min in 100%, with a flow rate of 25 ml / min). The desired fractions were concentrated to afford maleimidocaprooyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF (MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MS m / z 1330.29 [M + H] +, 1352.24 [M + Na] +; UV /., 'max 205, 248 nm.

Example 11 - Preparation of MC-val-cit-PAB-MMAF tert-butylamide [0596]

215

<img file="PL1725249T3_D0097.tif" />

[0597] Compound 3 (217 mg, 0.276 mmol, 1.0 equiv), compound AB (204 mg, 0.276 mmol, 1.0 equiv) and HOBt (11 mg, 0.0828 mmol, 0.3 equiv ) was diluted with pyridine / DMF (6 ml). DIEA (0.048 ml) was added to this mixture, and the mixture was stirred for about 16 hours. Volatile organic compounds were evaporated in vacuo. The crude residue was purified by Chromatotron® (radial thin layer chromatography) using a step gradient (0-5-10% methanol in DCM) to afford MC-val-cit-PABMMAF 10 tert-butylamide . Yield: 172 mg (45%); ES-MS m / z 1386.33 [M + H] +, 1408.36 [M + Na] +; UV Xm_ax 215, 248 nm.

Example 12 - Preparation of AC10-MC-MMAE by coupling of AC10 and MC-MMAE [0598] AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0, was treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 ° C for about 30 minutes, the buffer was exchanged by elution on Sephadex G25 resin and eluted with PBS with 1mM DTPA. The thiol / Ab value was checked by determining the concentration of the reduced antibody based on the absorbance of the solution at 280 nm and the concentration of thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the absorbance at 412 nm. The reduced antibody was dissolved in PBS and cooled on ice.

[0599] The drug linker reagent, maleimidocaproyl monomethylauristatin E, i.e. MC-MMAE, was dissolved in DMSO, diluted in acetonitrile and water of known concentration, and added to chilled reduced AC 10 antibody in PBS. After about one hour, excess maleimide was added to quench the reaction and protect any unreacted antibody thiol groups. The reaction mixture was concentrated by centrifugal ultrafiltration and purified and desalted AC10-MC-MMAE by elution on G25 resin in PBS, filtered through 0.2 gm filters under sterile conditions, and frozen for storage.

Example 13 - Preparation of AC10-MC-MMAF by coupling of AC10 and MC-MMAF [0600] AC10-MC-MMAF was prepared by coupling of AC 10 and MC-MMAF by following the procedure of Example 12.

Example 14 - Preparation of AC10-MC-val-cit-PAB-MMAE by coupling of AC10 and MCval-cit-PAB-MMAE [0601] AC10-MC-val-cit-PAB-MMAE was prepared by coupling of AC10 and MC-val-citPAB -MMAE by following the procedure in Example 12.

216

Example 15 - Preparation of AC10-MC-val-cit-PAB-MMAF by coupling of AC10 and MCval-cit-PAB-MMAF (9) [0602] AC10-MC-val-cit-PAB-MMAF was prepared by coupling of AC10 and MC- val-citPAB-MMAF (9) by following the procedure in Example 12.

Example 16 - Determination of cytotoxicity of selected compounds [0603] The cytotoxic activity of MMAF and compounds 1-5 were evaluated on Lewis Y positive OVCAR-3 cell lines, H3396 from breast cancer, L2987 from lung cancer and LS174t from colon cancer. Lewis Y positive cell lines can be assayed for cytotoxicity. To assess the cytotoxicity of compounds 1-5, cells can be plated, approximately 5-10,000, per well in 150 g culture medium, and then treated with graduated doses of compounds 1-5 in four replicates, at the initiation of the assay. Cytotoxicity assays are usually carried out for 96 hours after the addition of test compounds. Fifty gl of resazurin dye can be added to each well during the last 4 to 6 hours of incubation to assess cell viability at the end of the culture. Dye reduction can be determined by fluorescence spectrometry using excitation and emission wavelengths of 535 nm and 590 nm, respectively. For analysis, the extent of resazurin reduction by treated cells can be compared to that of untreated control cells.

[0604] For 1 hour. cell exposure assays can be pulsed with the drug for 1 hour and then washed; the cytotoxic effect can be determined after 96 hours. incubation. EXAMPLE 17 - in vitro cytotoxicity data for selected compounds [0605] Table 10 shows the cytotoxic effect for cAC10 conjugates of compounds 7-10, determined as described generally in procedure I carried out on the CD30 + Karpas 299 cell line. Data from two separate experiments are presented. The cAC10 conjugates of compounds 7 and 9 were found to be slightly more active than cAC10-val-cit-MMAE.

TABLE 10

<td>conjugate</td><td>IC50 (Ng / ml)</td>

<td>cAC10-val-citMMAE</td><td> 6</td>

<td>cAC10-7</td><td> 1,0</td>

<td>cAC10-8</td><td> 15</td>

<td>cAC10-9</td><td> 0,5</td>

<td>cAC10-10</td><td> 20</td>

[0606] In other experiments, BR96-val-cit-MMAF were at least 250-fold stronger than free MMAF.

217 [0607] General procedure I - cytotoxicity determination. To assess the cytotoxicity of exemplary 7-10 conjugates, cells were seeded, approximately 5-10,000 per well in 150 g of culture medium, and then treated with graduated doses of exemplary 7-10 conjugates in quadruplicate at initial initiation. Cytotoxicity assays were performed for 96 hours after the addition of test compounds. Fifty gl of resazurin dye was added to each well during the last 4 to 6 hours of incubation to assess cell viability at the end of the culture. Dye reduction was determined by fluorescence spectrometry, using excitation and emission wavelengths of 535 nm and 590 nm, respectively. For analysis, the extent of resazurin reduction by treated cells was compared to that of untreated control cells.

Example 18 - in vitro cell proliferation assay [0608] The efficacy of ADC can be measured using a cell proliferation assay using the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62: 5485-5488):

1. A 100 g aliquot of cell culture containing about 10⁴ cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in the medium were deposited in each well of a 96-well opaque wall plate.

2. Control wells containing medium without cells were prepared.

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 CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added.

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

7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.

8. Luminescence was recorded and recorded as graphs as RLU = relative luminescence units.

Example 19 - plasma clearance in rats [0609] Plasma clearance pharmacokinetics for antibody drug and total antibody conjugates were tested in Sprague-Dawley rats (Charles River Laboratories, 250-275 g each). The animals were given a bolus by injecting a large dose into the tail vein (direct intravenous injection). Approximately 300 g whole blood was collected via the cervical or tail cannula, into lithium / heparin anticoagulant tubes, at each time point: 0 (before dose), 10 and 30 minutes; 1, 2, 4, 8, 24 and 36 hours; and 2, 3, 4, 7, 14, 21, 28 days after dosing. Total antibody was measured for

218 by ELISA - ECD / GxhuFc-HRP. Drug antibody conjugate was measured by ELISA-MMAE / MMAF / ECD-Bio / SA-HRP.

Example 20 - Plasma clearance in monkeys [0610] The pharmacokinetics of plasma clearance for antibody drug and total antibody conjugates can be tested in Cynomolguss monkeys. Figure 12 shows a two-step plasma clearance test after administration of H-MC-vc-MMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5 and 3.0 mg / kg administered on day 1 and day 21 Total antibody and ADC concentrations were measured over time. (H = Trastuzumab).

Example 21 - Tumor volume and in vivo efficacy in transgenic mice, from explant [0611] Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, NY). Many strains are suitable, but female FVB mice are preferred because of their greater susceptibility to tumor formation. Male FVB mating and reproduction males CD.1 after vasectomy can be used to stimulate pseudo-pregnancy. Vasectomy mice can be obtained from any commercial supplier. The founders of the line can be reproduced with FVB mice or with heterozygous 129 / BL6 x FVB p53 mice. Mice heterozygous for the p53 allele can be used to potentially increase tumor formation. Some F1 tumors come from mixed strains. Founder tumors can only be FVB.

[0612] Cancer animals (allograft propagated from Fo5 mmtv transgenic mice) can be treated with single or multiple doses by intravenous ADC injection. Tumor volume can be assessed at various time points after injection.

Example 22 - Synthesis of MC-MMAF via t-butyl ester

Synthesis 1:

[0613]

219

<img file="PL1725249T3_D0098.tif" />

[0614] MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163 mmol) was suspended in CH2Cl2 (0.500 mL). 6-maleimidocaproic acid (68.9 mg, 0.326 mmol) and 1,3-diisopropylcarbodiimide (0.0505 mL, 0.326 mmol) were added followed by pyridine (0.500 mL). The reaction mixture was allowed to stir for 1.0 h. HPLC analysis indicated complete consumption of starting compound 1. The volatile organic compounds were evaporated under reduced pressure. The product was isolated by flash column chromatography using a gradient of 0 to 5% methanol in CH2Cl2. A total of 96 mg of pure MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) was recovered (60% yield). ES-MS m / z 981.26 [M + H] +; 1003.47 [M + Na] +; 979.65 [MH]^-.

[0615] MC-MeVal-Val-Dil-Dap-Phe-OtBu (compound 12.74 mg, 0.0754 mmol) was suspended in CH2Cl2 (2.0 mL) and TFA (1 mL) at room temperature. After 2.5 hours HPLC analysis indicated total consumption of starting material. The volatile organic compounds were evaporated under reduced pressure and the product was isolated by preparative RP-HPLC using a Phenomenex C column₁₂ Synergi Max-RP 80A (250 x 21.20 mm). Eluent: linear gradient 10% to 90% MeCN / 0.05% TFA (aq) over 30 minutes, followed by isocratic 90% MeCN / 0.05% TFA (aq) over an additional 20 minutes. ESMS m / z 925.33 [M + H] +; 947.30 [M + Na] +; 923.45 [MH]^-.

Example 23a - Synthesis of MC-MMAF (11) via dimethoxybenzyl ester

Synthesis 2:

[0616]

220

<img file="PL1725249T3_D0099.tif" />

Preparation of Fmoc-L-phenylalanine-2,4-dimethoxybenzyl (Fmoc-Phe-ODMB) ester , 4-dimethoxybenzyl (95.4 g, 567 mmol, Aldrich) and CH2Cl2 (2.0 L). N, N-Dimethylformamide t-butyl acetal (155 mL, 586 mmol, Fluka) was added to the resulting suspension for 20 min under N2, resulting in a clear solution. The reaction was then stirred at room temperature overnight, at which time TLC (0.42, heptane / EtOAc = 2: 1) indicated completion of the reaction. The reaction mixture was concentrated under reduced pressure to obtain a slightly yellow oil, which was redissolved in CH2Cl2 (200 mL) and

221 purified through a short compacted silica gel pad (25 cm x 25 cm, CH 2 Cl 2) to give a colorless foam (250 g). MeCN (1 L) was added to the resulting foam, which completely 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 washed with a MeCN filter cake (2 x 200 mL) to obtain the Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester in forms of a white solid (113.58 g, 41%, 95.5% AUC, according to HPLC analysis). Data: HPLC

Preparation of L-phenylalanine-2,4-dimethoxybenzyl ester (Phe-ODMB) [0618] A 500 ml round bottom flask was charged with Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (26.00g, 48.3 mmol), CH2Cl2 (150 ml) and diethylamine (75 ml, Acros). The mixture was stirred at room temperature and monitored for completion by HPLC. After 4 hours, the mixture was concentrated (bath temperature &lt;30 ° C). The residue was resuspended in CH₂cl₂ (200 ml) and concentrated. This was repeated once. MeOH (20 ml) was added to the residue, which resulted in the formation of a gel. This residue was diluted with CH 2 Cl 2 (200 mL), concentrated and left a cloudy oil in vacuo overnight. The residue was suspended in CH2Cl2 (100 mL), followed by the addition of toluene (120 mL). The mixture was concentrated and the residue was left under vacuum overnight.

[0619] Data: HPLC, 1H NMR.

Preparation of Fmoc-dolaproin (Fmoc-Dap) [0620] Boc-dolaproin (58.8 g, 0.205 mol) was suspended in 4 N HCl in 1,4-dioxane (256 ml, 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC analysis showed that the reaction was complete (10% MeOH / CH2Cl2) and the mixture was concentrated to near dryness. Additional 1,4-dioxane (50 mL) was charged and the mixture was concentrated to dryness and dried in vacuo overnight. The resulting white solid was dissolved in H2O (400 mL) and transferred to a 3L round-bottomed, 3-liter flask, with a mechanical stirrer and temperature probe. N, N-Diisopropylethylamine (214.3 mL, 1.23 mol, Acros) was added over one minute, causing an exotherm of 20.5 to 28.2 ° C (internal). The mixture was cooled in an ice bath and 1,4-dioxane (400 ml) was added. A solution of Fmoc-OSu (89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 ml) was added through an additional dropping funnel over 15 minutes keeping the reaction temperature below 9 ° C. The mixture was allowed to warm to room temperature and stirred for 19 hours, after which the mixture was concentrated on a rotary evaporator to an aqueous slurry (390 g). The suspension was diluted with H2O (750 mL) and Et2O (750 mL), forming a large amount of white precipitate. The layers were separated, leaving solids with the organic layer. The aqueous layer was acidified with concentrated HCl (30 mL) and extracted with EtOAc (3 x 500 mL). the combined extracts were dried over MgSO4, filtered and concentrated to give 59.25 g of yellow oil A. The Et2O extract was extracted once with sat. NaHCO3 (200 ml), leaving solids with an aqueous layer. The aqueous suspension was acidified with concentrated HCl (50 mL) and extracted with Et2O (50 mL) to leave

222 solids with an organic layer. The organic layer was filtered and concentrated to give 32.33 g of yellow oil B. The two oils (A and B) were combined and purified by flash chromatography on silica gel, eluting with CH2Cl2 (3.5 L) followed by 3% MeOH / CH2Cl2 (9 L) to obtain 68.23 g of Fmoc-dolaproin as a white foam (81%, HPLC purity 97.5% (AUC)).

Preparation of Fmoc-Dap-Phe-ODMB [0621] Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 ml, Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3 mmol) was added . The mixture was cooled in an ice bath and TBTU (17.08 g, 53.20 mmol, Matrix Innovations) was added. N, N-diisopropylethylamine (25.3 mL, 145.0 mmol, Acros) was added via syringe for 3 min.

After 1 hour the ice bath was removed and the mixture was allowed to heat for 30 min. The mixture was poured into water (1 L) and extracted with ethyl acetate (300 mL). After separation, the aqueous layer was re-extracted with ethyl acetate (2 x 150 mL). The combined organic layers were washed with brine (150 ml), dried (MgSO4) and filtered (filter paper) to remove insoluble substances (inorganic and some dibenzofulvene). After concentration, the residue (41 g) was adsorbed onto 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 obtain 29.4 g of the product in the form of a white foam (86%, purity 92% by HPLC).

[0622] Data: HPLC, 1H NMR, TLC (1: 1 EtOAc / heptane Rf = 0.33, red staining in vanillin).

Preparation of Dap-Phe-ODMB [0623] A 1 L round bottom flask was charged with Fmoc-Dap-Phe-ODMB (27.66 g), CH2Cl2 (122 mL) and diethylamine (61 mL, Acros). The solution was stirred at room temperature and monitored for completion by HPLC. After 7 hours, the mixture was concentrated (bath temperature &lt;30 ° C). The residue was suspended in CH₂cl₂ (300 ml) and concentrated. This was repeated twice. MeOH (20 mL) and CH2Cl2 (300 mL) were added to the residue, and the solution was concentrated. The residue was suspended in CH2Cl2 (100 mL) and toluene (400ml), concentrated, and the residue was left under vacuum overnight to afford a cream-like residue.

[0624] Data: HPLC, 1 H NMR, MS.

Preparation of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB [0625] Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135 ml, Acros) for 5 minutes and Fmoc-MeVal-Val- was added Dil-OH (24.94g, 39.1 mmol, see Example 2 for preparation). The mixture was cooled in an ice bath and TBTU (13.81g, 43.0 mmol, Matrix Innovations) was added. N, N-diisopropylethylamine (20.5 mL, 117.3 mmol, Acros) was added via a syringe for 2 minutes. After 1 hour the ice bath was removed and the mixture was allowed to heat for 30 min. The mixture was poured into water (1.5 L) and diluted with ethyl acetate (480 mL). After standing for 15 minutes, the layers were separated and the aqueous layer was extracted with ethyl acetate (300 mL). Connected

223 the organic layers were washed with brine (200 ml), dried (MgSO4) and filtered (filter paper) to remove insoluble substances (inorganic and some dibenzofulvene). After concentration, the residue (49 g) was scraped off the flask, adsorbed onto silica (49 g) and purified by chromatography (15 cm x 10 cm diameter column; 2: 1 EtOAc / heptane (3 L), EtOAc (5 L); 250 ml fractions) to obtain 31.84 g FmocMeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%, 93% purity by HPLC (AUC)).

[0626] Data: HPLC, TLC (2: 1 EtOAc / heptane, Rf = 0.21, red staining in vanillin).

Preparation of MeVal-Val-Dil-Dap-Phe-ODMB [0627] A 1 L round bottom flask was charged with Fmoc-MeVal-Val-Dil-Dap-PheODMB (28.50 g), CH2Cl2 (80 ml) and diethylamine (40 ml) ). The mixture was stirred overnight at room temperature and then concentrated under reduced pressure. The residue was adsorbed onto silica (30 g) and purified by flash chromatography (15 cm x 8 cm diameter column; 2% MeOH / DCM (2 L), 3% MeOH / DCM (1 L), 6% MeOH / DCM ( 4 L); 250 mL fractions) to obtain 15.88 g MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (69%, 96% purity by HPLC (AUC)).

[0628] Data: HPLC, TLC (6% MeOH / DCM, Rf = 0.24, red staining in vanillin).

Preparation of MC-MeVal-Val-Dil-Dap-Phe-ODMB [0629] 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, 0.93 mmol, Matrix Innovations) at room temperature. N, N-Diisopropylethylamine (450 mL, 2.57 mmol) was added using a syringe. After 1.5 hours, the mixture was poured into water (50 mL) and diluted with ethyl acetate (30 mL). NaCl was added to improve 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 obtain MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg, 57%, 94% purity by HPLC (AUC)).

[0630] Data: 1 H NMR, HPLC.

Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF) (11) [0631] A 50ml round bottom flask was charged with MC-MeVal-Val-Dil-DapPhe-ODMB (compound 13.428 mg, 0, 39 mmol) and dissolved in 2.5% TFA / CH2Cl2 (20 mL).

The solution turned pink-purple within 2 min. Completion of the reaction was monitored by HPLC and TLC (6% MeOH / DCM, KMnO4 staining). After 40 min, three drops of water were added, and the cloudy pink-purple mixture was concentrated to obtain 521 mg of pink

224 residue. Purification by chromatography (15% IPA / DCM) gave 270 mg of MCMMAF (73%, purity 92% according to HPŁC) as a white solid.

Example 23b - Synthesis of mc-MMAF analogue [0632]

<img file="PL1725249T3_D0100.tif" />

[0633] MeVal-Val-Dil-Dap-Phe-OtBu (compound 1.35 mg, 0.044 mmol) was suspended in DMF (0.250 mL). 4- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -benzoic acid (11 mg, 0.049 mmol) and HATU (17 mg, 0.044 mmol) were added followed by DIEA (0.031 ml, 0 , 17 mmol). This reaction mixture was allowed to stir for 2.0 hours. HPLC analysis indicated complete consumption of starting compound 1.

[0634] The product was isolated by preparative RP-HPLC using a Phenomenex C column₁₂ Synergi Max-RP 80A (250 x 21.20 mm). Eluent: linear gradient 10% to 80% MeCN / 0.05% TFA (aq) over 8 minutes, followed by isocratic 80% MeCN / 0.05% TFA (aq) over an additional 12 minutes. A total of 20 mg of pure product (14) was isolated (0.02 mmol, 46% yield). ES-MS m / z 987.85 [M + H] +; 1019.41 [M + Na] +; 985.54 [MH]^-.

[0635] MB-MeVal-Val-Dil-Dap-Phe-OtBu (compound 14.38 mg, 0.0385 mmol) was suspended in CH₂cl₂ (1 ml) and TFA (1 ml). The mixture was stirred for 2.0 h and then the volatile organic compounds were evaporated under reduced pressure. The product was isolated by preparative RP-HPLC using a Phenomenex C12 Synergi MaxRP 80A column (250 x 21.20 mm). Eluent: linear gradient 10% to 80% MeCN / 0.05% TFA (aq) over 8 minutes, followed by isocratic 80% MeCN / 0.05% TFA (aq) over an additional 12

225 minutes. A total of 14.4 mg of MB-MMAF product (0.015 mmol, 40% yield) was isolated. ESMS m / z 930.96 [M + H] + 952.98 [M + Na] +; 929.37 [MH]^-.

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

<img file="PL1725249T3_D0101.tif" />

[0637] For suspension at room temperature, Fmoc-MeVal-OH (3.03 g, 8.57 mmol) and disuccinimidyl N, N'-carbonate (3.29 g, 12.86 mmol) in CH2Cl2 (80 mL ) DIEA (4.48 mL, 25.71 mmol) was added. This reaction mixture was allowed to stir for 3.0 h and then poured into a separatory funnel in which the organic mixture was extracted with 0.1 M HCl (aq). The crude organic residue was concentrated under reduced pressure, and the product was isolated by flash column chromatography on silica gel using a 20-100% ethyl acetate / hexanes linear gradient. A total of 2.18 g of pure Fmoc-MeVal-OSu (4.80 mmol, 56% yield) was recovered.

[0638] To a suspension at room temperature, 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 NaHCO3 (0.41 g, 4.84 mmol) in H2O (13 mL). The suspension was allowed to stir at room temperature for 16 hours and then extracted with tert-BuOH / CHCl₃/ H₂O and acidified to pH = 2-3 with 1 M HCl. The organic phases were separated, dried and concentrated under reduced pressure. The residue was triturated with diethyl ether, which gave 2.01 g of Fmoc-MeVal-Cit-COOH, which was used without further purification.

[0639] Crude Fmoc-MeVal-Cit-COOH was suspended in 2: 1 CH₂cl₂/ MeOH (100 mL), and p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g, 7.9 mmol) were added thereto. This suspension was allowed to stir for 125 h, then the volatile organic compounds were removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel with 10% MeOH / CH2Cl2. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol, 18.5% yield) was recovered. ES-MS m / z 616.48 [M + H] +.

[0640] STRATOSPHERES was added to a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55g, 0.896 mmol) in CH2Cl2 (40 mL).^tm (piperizine bound resin) (&gt; 5 mmol / g, 150 mg). After stirring at room temperature for 16 hours, the mixture was filtered through celite (pre-washed with MeOH) and concentrated under reduced pressure. The residue was triturated with diethyl ether and hexanes. The resulting solid material, MeVal-Cit-PAB-OH, was suspended in CH2Cl2 (20 mL), and MC-OSu (0.28 g, 0.896 mmol) was added to this, DIEA

226 (0.17 mL, 0.99 mmol) and DMF (15 mL). This suspension was stirred for 16 hours, but HPLC analysis of the reaction mixture showed that the reaction was not complete, so the suspension was concentrated under reduced pressure to a volume of 6 ml, then 10% NaHCO solution was added₃ (aq) and the suspension was stirred for an additional 16 hours. The solvent was removed under reduced pressure, the residue was purified by flash column chromatography on silica gel using a gradient of 0-10% MeOH / CH2Cl2, which gave 42 mg (0.072 mmol, 8% yield) MC-MeVal-Cit-PAB OH.

[0641] For a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) and bis (nitrophenyl) carbonate (2.59 g, 8.52 mmol) in CH₂cl₂ (10 ml) DIEA (1.06 ml, 6.06 mmol) was added. This suspension was stirred for 5.5 hours, concentrated under reduced pressure and purified by trituration with diethyl ether. MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a 1: 5 pyridine / DMF solution (3 mL), and HOBt (5 mg, 0.039 mmol), DIEA (0.17) was added to this. ml, 0.978 mmol) and MMAF (compound 2, 150 mg, 0.205 mmol). This reaction mixture was stirred for 16 hours. at room temperature and then purified by preparative RP-HPLC (x3) using a Phenomenex C column₁₂ Synergi Max-RP 80A (250 x 21.20 mm). Eluent: linear gradient 10% to 90% MeCN / 0.05% TFA (aq) over 30 minutes, followed by isocratic 90% MeCN / 0.05% TFA (aq) over an additional 20 minutes. MC-MeVal-Cit-PAB-MMAF (16) were obtained as a yellowish solid (24.5 mg, 0.0182, yield 0.45%). ES-MS m / z 1344.95 [M + H] +; 1366.94 [M + Na] +.

Example 23d - Preparation of suberyl-Val-Cit-PABMMAF succinic acid imide ester (17)

<img file="PL1725249T3_D0102.tif" />

[0643] Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol, 1.5 eq) was suspended in anhydrous pyridine, 5 mL. HOBt (10 mg, 0.076 mmol, 0.2 equiv) was added followed by DIEA (199 μ ^ 114 mmol, 3 equiv). The reaction mixture was sonicated for 10 min and then stirred overnight at room temperature. Pyridine was removed under reduced pressure, the residue was resuspended in CH2Cl2. The mixture was separated by flash chromatography on silica gel using a MeOH step gradient, from 0 to 10%, in CH2Cl2. Product containing fractions

227 drawn, concentrated, dried in vacuo overnight to afford 317 mg (59% yield) Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m / z 1415.8 [M + H] +.

[0644] Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20% TFA / CH₂cl₂ (10 ml) for 2 hours. The mixture was diluted with CH₂cl₂ (50 ml). The organic layer was washed successively with water (2 x 30 ml) and brine (1 x 30 ml). The organic phase was concentrated, loaded onto a silica gel cartridge in 10% MeOH / CH2Cl2. The product was eluted with 30% MeOH / CH2Cl2. After drying under vacuum overnight, Fmoc-Val-CitPAB-MMAF was obtained as a white solid, 38 mg, 40% yield. ES-MS m / z 1357.7 [MH]^-.

[0645] Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH 2 Cl 2 (2 mL) diethylamine (2 mL) and DMF (2 mL). The mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure. The residue was co-evaporated with pyridine (2 mL) followed by toluene (2 x 5 mL), dried in vacuo. ValCit-PAB-MMAF was obtained as a brownish oil, and used without further purification.

[0646] All Val-Cit-PAB-MMAF obtained from 67 mg Fmoc-Val-Cit-PAB-MMAF was suspended in pyridine (2 mL), and added to the solution of disuccinimidyl suberane (74 mg, 0.2 mmol, 4 eq. ), in pyridine (1 ml). The reaction mixture was stirred at room temperature. After 3 hours ether (20 ml) was added. The precipitate was collected, washed with additional ether. The reddish solid was suspended in 30% MeOH / CH₂cl₂, filtered through a silica gel pad, with 30% MeOH / CH₂cl₂ as eluent. Compound 17 was obtained as a white solid, 20 mg (29% yield). ES-MS m / z 1388.5 [MH]Example 24 - In vivo efficacy of antibody-drug mcMMAF conjugates [0647] Effectiveness of cAC10-mcMMAF in Karpas-299ALCL xenografts: To assess the in vivo efficacy of cAC10-mcMMAF having an average of 4 drug moieties per antibody (cAC10-mcF4), subcutaneous human ALC cells were implanted Karpas-299 immunocompromised mice: CB-17 SCID (5x10⁶ cells per mouse). Tumor volumes were calculated using the formula (0.5 x L x W²) in which L and W are the longer and shorter of two bidirectional measurements. When the mean tumor volume in the test animals reached approximately 100 mm³ (range 48-162) mice were divided into 3 groups (5 mice per group) and or left untreated or given a single intravenous injection via tail vein at 1 or 2 mg / kg cAC10-mcF4 (Figure 1). Tumors in untreated mice grew rapidly to an average volume&gt; 1,000 mm³ within 7 days of starting treatment. In contrast, all cAC10-mcF4-treated tumors showed a rapid regression of 3/5 in the 1 mg / kg group and 5/5 in the 2 mg / kg group, which meant a full tumor response. While the tumor returned in one of the individuals that showed full response, in the 2 mg / kg group, approximately 4 weeks later, there were no detectable tumors in the remaining 4/5 individuals that showed full response in this group and in 3 individuals that showed a full response in the 1 mg / kg group 10 weeks after the end of treatment.

228 [0648] Efficacy of cBR96-mcMMAF in L2987 NSCLC xenografts: cBR96 is a chimeric antibody that recognizes the Le antigen^Y. To assess the in vivo efficacy of cBR96-mcMMAF with 4 drug moieties (cBR96-mcF4), a fragment of L2987 non-small cell lung tumor (NSCLC) was implanted in Nude mice. When the tumors reached an average size of approximately 100 mm³ mice were divided into 3 groups: untreated and 2 treatment groups. For treatment as shown in Figure 3a, mice were given cBR96-mcF4 at 3 or 10 mg / kg / injection every 4 days, resulting in a total of 4 injections (q4dx4). As shown in Figure 3b, mice were treated with cBR96mcF4 or non-binding control conjugate, cAC10-mcF4, 10 mg / kg / injection every 4 days, resulting in a total of 4 injections (q4dx4). As shown in Figures 3a and 3b, BR96-mcF4 caused a clear delay in tumor growth compared to controls.

[0649] Figure 2 shows an in vivo assay for the efficacy of a single dose of cAC10mcMMAF in subcutaneous L540CY. In this study there were 4 mice in the untreated group and 10 in each of the treatment groups.

Example 25 - in vitro efficacy of antibody-drug MC-MMAF conjugates [0650] Activity of cAC10-antibody-drug conjugates against CD30 + cell lines. Figures 4a and 16b show dose response curves from a representative experiment in which the cultures Karpas 299 (anaplastic large cell lymphoma) and L428 (Hodgkin's lymphoma) were incubated with serially diluted cAC10-mcMMAF (Figure 4a) or cAC10-vcMMAF (Figure 4b) hours. Cultures were labeled for 4 hours with 50 mM resazurin [7-hydroxy-3H-phenoxazin-3-one 10-oxide] and fluorescence measured. Data was reduced in GraphPad Prism version 4.00 software using a 4-parameter dose response curve fitting procedure. IC50 values ​​are defined as the concentration at which growth is reduced by 50% compared to untreated control cultures. Each concentration was tested in four replications.

[0651] Activity of cBR96-antibody-drug conjugates against Le cell lines^s+. Figures 5a and 5b show dose response curves from a representative experiment in which the cultures H3396 (breast cancer) and L2987 (non-small cell lung cancer) were incubated with serially diluted cBR96-mcMMAF (Figure 5a) or -vcMMAF (Figure 5b) for 96 hours. Cultures were labeled for 4 hours with 50 mM resazurin and fluorescence was measured. Data was reduced in GraphPad Prism version 4.00 software using a 4-parameter dose response curve fitting procedure. IC50 values ​​are defined as the concentration at which growth is reduced by 50% compared to untreated control cultures. Each concentration was tested in four replications.

[0652] Activity of c1F6-antibody-drug conjugates against CD70 + renal cell carcinoma cell lines. Figures 6a and 6b show dose response curves from a representative experiment in which Caki-1 and 786-O cell cultures were incubated with serially diluted c1F6-mcMMAF (Figure 6a) or -vcMMAF (Figure 6b) for 96

229 hours. Cultures were labeled for 4 hours with 50 mM resazurin and fluorescence was measured. Data was reduced in GraphPad Prism version 4.00 software using a 4-parameter dose response curve fitting procedure. IC50 values ​​are defined as the concentration at which growth is reduced by 50% compared to untreated control cultures. Each concentration was tested in four replications.

Examples 26 - Purification of Trastuzumab [0653] One vial of 440 mg HERCEPTIN® (huMAb4D5-8 antibody, rhuMAb HER2, US Patent No. 5821337) was dissolved in 50 ml MES buffer (25 mM MES, 50 mM NaCl, pH 5.6) and applied to a cation exchange column (Sepharose S, 15 cm x 1.7 cm) which was equilibrated in the same buffer. The column was then washed with the same buffer (5 column volumes). Trastuzumab was eluted by raising the NaCl buffer concentration to 200 mM. The fractions containing the antibody were pooled, diluted to 10 mg / ml, and dialyzed in buffer containing 50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.

Example 27 - Preparation of Trastuzumab-MC-MMAE by coupling Trastuzumab and MCMMAE [0654] Trastuzumab, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 was treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37 ° C for about 30 minutes, the buffer was exchanged by elution on Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol / Ab value was checked by determining the concentration of the reduced antibody based on the absorbance of the solution at 280 nm and the concentration of thiol by reaction with DTNB (Aldrich, Milwaukee, WI) and determining the absorbance at 412 nm. The reduced antibody was dissolved in PBS and cooled on ice.

[0655] The drug linker reagent, maleimidocaproyl monomethylauristatin E (MMAE), i.e. MCMMAE, was dissolved in DMSO, diluted in acetonitrile and water of known concentration, and added to chilled reduced Trastuzumab antibody in PBS. After about one hour, excess maleimide was added to quench the reaction and protect any unreacted antibody thiol groups. The reaction mixture was concentrated by centrifugal ultrafiltration and purified and desalted Trastuzumab-MC-MMAE by elution on G25 resin in PBS, filtered through 0.2 Pm filters under sterile conditions, and frozen for storage.

Example 28 - Preparation of Trastuzumab-MC-MMAF by conjugation of Trastuzumab and MCMMAF [0656] Trastuzumab-MC-MMAF was prepared by conjugation of Trastuzumab and MC-MMAF following the procedure of Example 27.

Example 29 - Preparation of Trastuzumab-MC-val-cit-PAB-MMAE by coupling

Trastuzumab and MC-val-cit-PAB-MMAE

230 [0657] Trastuzumab-MC-val-cit-PAB-MMAE was prepared by coupling Trastuzumab and MC-val-cit-PAB-MMAE by following the procedure of Example 27.

Example 30 - Preparation of Trastuzumab-MC-val-cit-PAB-MMAF by coupling

Trastuzumab and MC-val-cit-PAB-MMAF 9 [0658] Trastuzumab-MC-val-cit-PAB-MMAF was prepared by coupling Trastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of Example 27.

Example 31 - toxicity in rats [0659] The acute toxicity profile of free drugs and ADCs were assessed in adolescent Sprague-Dawley rats (75-125 g each, Charles River Laboratories (Hollister, CA). Injection was done on animals on day 1, full chemical and hematological profiles were obtained at the beginning of the study, on day 3 and day 5, and complete autopsy was performed on day 5. Liver enzyme tests were performed on all animals, and routine histology was performed on three random animals per group for the following tissues: sternum, liver, kidney, thymus, spleen, small and large intestine. Experimental groups were as follows: _

<td>Group</td><td>given</td><td>mg / kg</td><td>μg MMAF / _m2</td><td>MMAF / Has b</td><td>N / Sex</td>

<td> 1</td><td>excipient</td><td> 0</td><td> 0</td><td> 0</td><td>2 / F</td>

<td> 2</td><td>Trastuzumab-MC-val-cit-MMAF</td><td> 9,94</td><td> 840</td><td> 4,2</td><td>6 / F</td>

<td> 3</td><td>Trastuzumab-MC-val-cit-MMAF</td><td> 24,90</td><td> 2105</td><td> 4,2</td><td>6 / F</td>

<td> 4</td><td>Trastuzumab-MC (Me) -Val-citPAB-MMAF</td><td> 10,69</td><td> 840</td><td> 3,9</td><td>6 / F</td>

<td> 5</td><td>Trastuzumab-MC (Me) -Val-citPAB-MMAF</td><td> 26,78</td><td> 2105</td><td> 3,9</td><td>6 / F</td>

<td> 6</td><td>Trastuzumab-MC-MMAF</td><td> 10,17</td><td> 840</td><td> 4,1</td><td>6 / F</td>

<td> 7</td><td>Trastuzumab-MC-MMAF</td><td> 25,50</td><td> 2105</td><td> 4,1</td><td>6 / F</td>

<td> 8</td><td>Trastuzumab-MC-val-cit-PABMMAF</td><td> 21,85</td><td> 2105</td><td> 4,8</td><td>6 / F</td>

[0660] For Trastuzumab-MC-val-cit-MMAF, Trastuzumab-MC (Me) -val-cit-PAB-MMAF, Trastuzumab-MC-MMAF and Trastuzumab-MC-val-cit-PAB-MMAF, μg MMAF / m² calculated using 731.5 as MW [molecular weight] MMAF and 145167 as MW Herceptin.

[0661] Body surface was calculated as follows: [{(body weight in g to 0.667) x 11.8} / 10,000]. (Guidance for Industry and Reviewers, 2002).

[0662] Dose solutions were administered by a single intravenous bolus injection into the tail vein on study day 1 in a dose volume of 10 ml / kg. Animal body weights were measured before dosing on study day 1 and then daily. Whole blood was collected into

231 test tubes containing EDTA for hematological analysis. Whole blood was collected in serum separation tubes for clinical chemistry analysis. Blood samples were taken prior to dose administration on study day -4, study day 3 and study day 5. Whole blood was also collected in tubes containing sodium heparin during autopsy, and plasma was frozen at -70 ° C for potential subsequent analysis. The following tissues were collected and placed in neutral buffered formalin during autopsy: liver, kidneys, heart, thymus, spleen, brain, sternum and gastrointestinal tract, including stomach, small and large intestine. The sternum, small intestine, large intestine, liver, thymus, spleen and kidneys were examined.

[0663] Levels of liver-associated enzymes in the serum were compared for each time point to the range (5th and 95th percentile) for healthy female SpragueDawley rats. The counts of white blood cells and platelets for each time point were compared to the range (5th and 95th percentile) for healthy SpragueDawley rats.

[0664] High dose study in healthy female SpragueDawley rats: _

Group 1: Excipient

Group 2: Trastuzumab-MC-MMAF, 52.24 mg / kg, 4210 mg / m²

Group 3: Trastuzumab-MC-MMAF, 68.25 mg / kg, 5500 mg / m²

Group 4: Trastuzumab-MC-MMAF, 86.00 mg / kg, 6930 mg / m² [0665] Tissues from 11 animals underwent routine histology. These animals were part of an acute dose range toxicity study using the TrastuzumabMC-MMAF immunoconjugate. Animals were observed for 12 days after dosing.

Example 32 - toxicity / safety in Cynomolgus monkeys [0666] Three groups of four (2 males, 2 females) previously untreated Macaca fascicularis monkeys (Cynomolgus monkeys) were tested for Trastuzumab-MC-vc-PAB-MMAE and Trastuzumab-MC-vc- PAB-MMAF. Intravenous administration was carried out on days 1 and 22 of the study.

<td>A sample</td><td>Group</td><td>Dose</td>

<td>excipient</td><td> 1</td><td>day 1</td>

<td></td><td>1M / 1F</td><td>day 22</td>

<td>H-MC-vc-PAB-MMAE</td><td> 2</td><td>180 gg / m2² (0.5 mg / kg) in</td>

<td>Reference example</td><td>2M / 2F</td><td>on day 1 1100 gg / m2² (3.0 mg / kg) on ​​day 22</td>

<td>H-MC-vc-PAB-MMAE</td><td> 3</td><td>550 gg / m² (1.5 mg / kg) in</td>

<td>Reference example</td><td>2M / 2F</td><td>on the 8th</td>

232

<td></td><td></td><td>550 μg / m² (1.5 mg / kg) on ​​day 29</td>

<td>H-MC-vc-PAB-MMAE Reference example</td><td>4 2M / 2F</td><td>880 μg / m² (2.5 mg / kg) on ​​day 15 880 μg / m² (2.5 mg / kg) on ​​day 36</td>

<td>excipient</td><td>1 1M / 1F</td><td>day 1 day 22</td>

<td>H-MC-vc-PAB-MMAF</td><td>2 2M / 2F</td><td>180 μg / m² (0.5 mg / kg) on ​​day 1 1100 μg / m² (3.0 mg / kg) on ​​day 22</td>

<td>H-MC-vc-PAB-MMAF</td><td>3 2M / 2F</td><td>550 μg / m² (1.5 mg / kg) on ​​day 1 550 μg / m² (1.5 mg / kg) on ​​day 22</td>

<td>H-MC-vc-PAB-MMAF</td><td>4 2M / 2F</td><td>880 μg / m² (2.5 mg / kg) on ​​day 1 880 μg / m² (2.5 mg / kg) on ​​day 22</td>

<td colspan="3">H = Trastuzumab</td>

[0667] Dosages were expressed through the animal's surface area to be appropriate for other species, ie dosages in μg / m² it is independent of the species and hence comparable between species. The ADC formulations contained PBS, 5.4 mM sodium phosphate, 4.2 mM potassium phosphate, 140 mM sodium chloride, pH 6.5.

[0668] Blood was collected for hematological analysis before dose administration, and after 5 min, 6 h, 10 h, and 1, 3, 5, 7, 14, 21 days after each dose. Erythrocytes (RBC) and platelets (PLT) were counted by light scattering. Leukocyte counts (WBC) were carried out by the peroxidase / basophil method. Reticulocyte count testing was performed by light scattering with a cationic dye. Cell counts were carried out using an Advia 120 device. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels in U / L were measured by UV / NADH; IFCC methodology on the Olympus AU400, and using ECD / GxhuFc-HRP Total Ab ELISA tests. Conjugate. Ab - ELISA-MMAE / MMAF // ECD-Bio / SA-HRP tests.

Example 33 - production, characterization and humanization of anti-ErbB2 4D5 monoclonal antibody

233 [0669] Mouse monoclonal antibody 4D5 that specifically binds the extracellular domain of ErbB2 was prepared as described in Fendly et al. (1990) Cancer Research 50: 15501558. Briefly, NIH 3T3 / HER2-3 cells₄₀₀ (expressing approximately 1 x 10⁵ ErbB2 molecules / cell) obtained as described in Hudziak et al. Natl. Acad. Sci. (USA) 84: 7158-7163 (1987) was harvested using phosphate buffered saline (PBS) containing 25mM EDTA and used to immunize BALB / c mice. Mice were given ip injections after 10⁷ cells in 0.5 ml PBS at weeks 0, 2, 5 and 7. Mice with antiserum that have immunoprecipitated labeled ³²P ErbB2 was administered ip injection of wheat germ Sepghose (WGA) agglutinin and the ErbB2 membrane extract was purified at weeks 9 and 13. Intravenous injection of 0.1 ml ErbB2 was then followed by fusion of splenocytes with the mouse myeloma line X63-Ag8,653. The hybridoma supernatants were screened for ErbB2 binding by ELISA and radioimmunoprecipitation.

Epitope mapping and characterization [0670] The ErbB2 epitope bound by the 4D5 monoclonal antibody was determined by competitive binding analysis (Fendly et al. Cancer Research 50: 1550-1558 (1990)). Cross-blocking studies were carried out by direct fluorescence on intact cells using a PANDEX ™ Screen Machine for fluorescence quantification. Monoclonal antibody was conjugated to fluorescein isothiocyanate (FITC) using recognized procedures (Wofsy et al. Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi (eds.) San Francisco: WJ Freeman Co. (1980)). Confluent monolayers of NIH 3T3 / HER2-3400 cells were trypsinized, washed once, and resuspended after 1.75 x 10⁶ cells / ml in cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN₃. A final concentration of 1% latex particles (IDC, Portland, OR) was added to reduce clogging of PANDEX ™ membrane plates. Cells in suspension, 20 g, and 20 g of purified monoclonal antibodies (100 gg / ml to 0.1 gg / ml) were added to PANDEX ™ wells and incubated on ice for 30 minutes. A predetermined dilution of FITC-labeled monoclonal antibody in 20 g was added to each well, incubated for 30 minutes, washed, and fluorescence quantified by PANDEX ™. Monoclonal antibodies were considered to share the epitope if each blocked the binding of the other by 50% or more compared to an irrelevant control monoclonal antibody. In this experiment, the 4D5 monoclonal antibody was assigned epitope I (amino acid residues from about 529 to about 625, including the ErbB2 extracellular domain.

[0671] Characterization of the 4D5 monoclonal antibody with growth inhibitors was performed using a breast cancer cell line, SK-BR-3 (see Hudziak et al. (1989) Molec. Cell. Biol. 9 (3): 1165-1172) . Briefly, SK-BR-3 cells were detached using 0.25% (v / v) trypsin and suspended in complete medium at a density of 4 x 10⁵cells per ml. Portions of 100 gl (4 x 10⁴ cells) were applied to 96-well microdilution plates, allowed cells to adhere to the medium, and then added

234

100 gl media, alone or media containing monoclonal antibody (final concentration 5 gg / ml). After 72 hours, the plates were washed twice with PBS (pH 7.5), stained with crystal violet (0.5% in methanol) and analyzed for relative cell proliferation as described in Sugarman et al. (1985) Science 230: 943-945. Monoclonal antibody 4D5 inhibited the relative proliferation of SK-BR-3 cells by approximately 56%.

[0672] Monoclonal antibody 4D5 has also been evaluated for its ability to inhibit HRG-stimulated protein tyrosine phosphorylation, in the range of Mr 180,000, for whole cell lysates for MCF7 cells (Lewis et al. (1996) Cancer Research 56: 14571465). MCF7 cells have been reported to express all known ErbB receptors, but at relatively low levels. As ErbB2, ErbB3 and ErbB4 have almost identical molecular sizes, it is not possible to distinguish which protein is subject to tyrosine phosphorylation when assessing whole cell lysates by Western blot analysis. However, these cells are ideal for HRG tyrosine phosphorylation assays because, under the conditions of the assay used, in the absence of exogenously added HRG, they show low levels to undetectable protein tyrosine phosphorylation in the Mr 180,000 range.

[0673] MCF7 cells were seeded in 24-well plates, anti-ErbB2 monoclonal antibodies were added to each well, and incubated for 30 minutes at room temperature; then rHRGp 1 was added₁₇7-2₄₄ to each well to a final concentration of 0.2 nM, and incubation continued for 8 minutes. The cultures were carefully aspirated from each well and the reactions quenched by the addition of 100 g SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 g) was subjected to gel electrophoresis with a 4-12% gradient (Novex) and then electrophoretically transferred to a polyvinylidene difluoride membrane. Immunoblots for anti-phosphotyrosine (4G10, from UBI, used at 1 gg / ml) were developed, and the intensity of the main reactive band at Mr 180,000 was quantified using reflectance densitometry as described previously (Holmes et al. (1992) Science 256: 1205-1210; Sliwkowski et al. J. Biol. Chem. 269: 14661-14665 (1994)).

[0674] Monoclonal antibody 4D5 significantly inhibited signal generation for HRG-induced tyrosine phosphorylation at Mr 180,000. In the absence of HRG, however, it was unable to stimulate protein tyrosine phosphorylation in the Mr 180,000 range. Also, this antibody does not cross-react with EGFR (Fendly et al. Cancer Research 50: 1550-1558 (1990)), ErbB3 or ErbB4. The 4D5 monoclonal antibody was able to block HRG stimulated tyrosine phosphorylation by 50%.

[0675] The effect of growth inhibitors on monoclonal antibody 4D5 was assessed using MDA-MB-175 and SK-BR-3 cells in the presence or absence of exogenous rHRGp1 (Schaefer et al. Oncogene 15: 1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6 times higher than those found in normal breast epithelial cells, and the ErbB2-ErbB4 receptor has constitutively phosphorylated tyrosines in MDA-MB-175 cells. The 4D5 monoclonal antibody was able to inhibit cell proliferation with respect to MDA-MB-175 cells in both the presence and

235 in the absence of exogenous HRG. Inhibition of cell proliferation by 4D5 depended on the level of ErbB2 expression (Lewis et al. Cancer Immunol. Immunother. 37: 255-263 (1993)). It was possible to detect maximum inhibition, in the order of 66%, in SK-BR-3 cells. However, this effect could be overcome by means of exogenous HRG.

[0676] The murine 4D5 monoclonal antibody was humanized using the "gene conversion mutagenesis" strategy as described in US Patent No. 5821337, which disclosure is hereby expressly incorporated by reference in its entirety. The humanized 4D5 monoclonal antibody used in the following experiments was designated huMAb4D5-8. This antibody represents the IgG1 isotype.

List of sequences [0677] &lt;110&gt; Doronina, Svetlana O.

Toki, Brian E.

Senter, Peter D.

Ebens, Allen J.

Polakis, Paul

Sliwkowski, Mark X.

Spencer, Susan D.

Kline, Toni Beth &lt;120&gt; MONOMETHYL-VALLEY COMPOUNDS CAPABLE OF COUPLING FOR

LIGANDS &lt;130&gt; 018891-001020PC &lt;141&gt; 2004-11-05 &lt;150&gt; US 60 / 598,899 &lt;151&gt; 2004-08-04 &lt;150&gt; US 60 / 557,116 &lt;151&gt; 2004-03-26 &lt;150&gt; US 60 / 518,534 &lt;151&gt; 2003-11-06 &lt;160&gt; 35 &lt;210&gt; 1 &lt;211&gt; 502 &lt;212&gt; PRT

236 &lt;213&gt; Homo sapiens &lt;400&gt; 1

<td>Underworld 1</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Cheese 5</td><td>ala</td><td>Gly</td><td>lys</td><td>Leu</td><td>own 10</td><td>val</td><td>Gly</td><td>Thr</td><td>lys</td><td>lys 15</td>

<td>Glu</td><td>Asp</td><td>Gly</td><td>Glu</td><td>Cheese twenty</td><td>Thr</td><td>ala</td><td>Pro</td><td>Thr</td><td>Pro 25</td><td>Arg</td><td>Pro</td><td>lys</td><td>val</td><td>Leu thirty</td>

<td>Arg</td><td>Cys</td><td>lys</td><td>Cys</td><td>His 35</td><td>His</td><td>His</td><td>Cys</td><td>Pro</td><td>Glu 40</td><td>Asp</td><td>Cheese</td><td>val</td><td>own</td><td>own 45</td>

<td>How much</td><td>Cys</td><td>Cheese</td><td>Thr</td><td>Asp 50</td><td>Gly</td><td>Tyr</td><td>Cys</td><td>phe</td><td>Thr 55</td><td>Underworld</td><td>How much</td><td>Glu</td><td>Glu</td><td>Asp 60</td>

<td>Asp</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro .65</td><td>val</td><td>val</td><td>Thr</td><td>Cheese</td><td>Gly 70</td><td>Cys</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Glu 75</td>

<td>Gly</td><td>Cheese</td><td>Asp</td><td>phe</td><td>Gin 80</td><td>Cys</td><td>Arg</td><td>Asp</td><td>Thr</td><td>Pro 85</td><td>How much</td><td>Pro</td><td>His</td><td>Gin</td><td>Arg 90</td>

<td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Cys 95</td><td>Cys</td><td>Thr</td><td>Glu</td><td>Arg</td><td>own 100</td><td>Glu</td><td>Cys</td><td>own</td><td>lys</td><td>Asp 105</td>

<td>Leu</td><td>His</td><td>Pro</td><td>Thr</td><td>Leu 110</td><td>Pro</td><td>Pro</td><td>Leu</td><td>lys</td><td>own 115</td><td>Arg</td><td>Asp</td><td>phe</td><td>val</td><td>Asp 120</td>

<td>Gly</td><td>Pro</td><td>How much</td><td>His</td><td>His 125</td><td>Arg</td><td>ala</td><td>Leu</td><td>Leu</td><td>How much 130</td><td>Cheese</td><td>val</td><td>Thr</td><td>val</td><td>Cys 135</td>

237

<td colspan="5">Cheese Leu Leu Leu Val</td><td colspan="4" rowspan="2">Leu Ile Ile Leu</td><td colspan="6">Phe Cys Tyr Phe Arg Tyr</td>

<td colspan="4"></td><td> 140</td><td colspan="5"> 145 '</td><td> 150</td>

<td>lys</td><td>Arg</td><td>Gln</td><td>Glu</td><td>Thr</td><td>Arg</td><td>Pro</td><td>Arg</td><td>Tyr</td><td>Cheese</td><td>How much</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Gln</td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td>

<td>Asp</td><td>Glu</td><td>Thr</td><td>Tyr</td><td>How much</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Asp</td><td>Leu</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td>

<td>Glu</td><td>Gln</td><td>Cheese</td><td>Gln</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td>

<td>val</td><td>Gln</td><td>Arg</td><td>Thr</td><td>How much</td><td>ala</td><td>lys</td><td>Gln</td><td>How much</td><td>Gln</td><td>Underworld</td><td>val</td><td>lys</td><td>Gln</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td>

<td>Gly</td><td>lys</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>Glu</td><td>val</td><td>Trp</td><td>Underworld</td><td>Gly</td><td>lys</td><td>Trp</td><td>Arg</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td>

<td>Glu</td><td>lys</td><td>val</td><td>ala</td><td>val</td><td>lys</td><td>val</td><td>phe</td><td>phe</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Glu</td><td>ala</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Trp</td><td>phe</td><td>Arg</td><td>Glu</td><td>Thr</td><td>Glu</td><td>How much</td><td>Tyr</td><td>Gln</td><td>Thr</td><td>val</td><td>Leu</td><td>Underworld</td><td>Arg</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td>Glu</td><td>own</td><td>How much</td><td>Leu</td><td>Gly</td><td>phe</td><td>How much</td><td>ala</td><td>ala</td><td>Asp</td><td>How much</td><td>lys</td><td>Gly</td><td>Thr</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td>

<td>Cheese</td><td>Trp</td><td>Thr</td><td>Gln</td><td>Leu</td><td>Tyr</td><td>Leu</td><td>How much</td><td>Thr</td><td>Asp</td><td>Tyr</td><td>His</td><td>Glu</td><td>own</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td>

<td>Cheese</td><td>Leu</td><td>φ '&gt;&gt; · ^Α Ύ * ·</td><td>Asp</td><td>Ti iv « And JJ-</td><td>T at</td><td>T wo AJ JO</td><td>Cheese</td><td>Th 2?</td><td>Thr</td><td>Leu</td><td>Asp</td><td>ala</td><td>lys</td><td>X</td>

<td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td>

<td>Underworld</td><td>Leu</td><td>lys</td><td>Leu</td><td>ala</td><td>Tyr</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Cys</td><td>His</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td>

<td>His</td><td>Thr</td><td>Glu</td><td>How much</td><td>phe</td><td>Cheese</td><td>Thr</td><td>Gln</td><td>Gly</td><td>lys</td><td>Pro</td><td>ala</td><td>How much</td><td>ala</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 320</td><td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td>

<td>Arg</td><td>Asp</td><td>Leu</td><td>lys</td><td>Cheese</td><td>lys</td><td>own</td><td>How much</td><td>Leu</td><td>val</td><td>lys</td><td>lys</td><td>own</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td>

<td>Cys</td><td>Cys</td><td>How much</td><td>ala</td><td>Asp</td><td>Leu</td><td>Gly</td><td>Leu</td><td>ala</td><td>val</td><td>lys</td><td>phe</td><td>How much</td><td>Cheese</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td>

<td>Thr</td><td>own</td><td>Glu</td><td>val</td><td>Asp</td><td>How much</td><td>Pro</td><td>Pro</td><td>own</td><td>Thr</td><td>Arg</td><td>val</td><td>Gly</td><td>Thr</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td>

<td>Arg</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>Pro</td><td>Glu</td><td>val</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Cheese</td><td>Leu</td><td>own</td><td>Arg</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 385</td><td></td><td></td><td></td><td></td><td> 390</td>

<td>His</td><td>phe</td><td>Gln</td><td>Cheese</td><td>Tyr</td><td>How much</td><td>Underworld</td><td>ala</td><td>Asp</td><td>Underworld</td><td>Tyr</td><td>Cheese</td><td>phe</td><td>Gly</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td><td></td><td></td><td></td><td> 405</td>

<td>How much</td><td>Leu</td><td>Trp</td><td>Glu</td><td>val</td><td>ala</td><td>Arg</td><td>Arg</td><td>Cys</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td><td></td><td></td><td> 420</td>

<td>Glu</td><td>Glu</td><td>Tyr</td><td>Gln</td><td>Leu</td><td>Pro</td><td>Tyr</td><td>His</td><td>Asp</td><td>Leu</td><td>val</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td><td></td><td></td><td> 435</td>

<td>Cheese</td><td>Tyr</td><td>Glu</td><td>Asp</td><td>Underworld</td><td>Arg</td><td>Glu</td><td>How much</td><td>val</td><td>Cys</td><td>How much</td><td>lys</td><td>lys</td><td>Leu</td><td>Arg</td>

238

<td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td><td> 450</td>

<td>Pro</td><td>Cheese</td><td>phe</td><td>Pro</td><td>own 455</td><td>Arg</td><td>Trp</td><td>Cheese</td><td>Cheese</td><td>Asp 460</td><td>Glu</td><td>Cys</td><td>Leu</td><td>Arg</td><td>Gin 4 65</td>

<td>Underworld</td><td>Gly</td><td>lys</td><td>Leu</td><td>Underworld 470</td><td>Thr</td><td>Glu</td><td>Cys</td><td>Trp</td><td>ala 4 75</td><td>His</td><td>own</td><td>Great</td><td>ala</td><td>Cheese 480</td>

<td>Arg</td><td>Leu</td><td>Thr</td><td>ala</td><td>Leu 485</td><td>Arg</td><td>val</td><td>lys</td><td>lys</td><td>Thr 490</td><td>Leu</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cheese 4 95</td>

<td>Glu</td><td>Cheese</td><td>Gin</td><td>Asp</td><td>rie 500</td><td>lys</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 2 &lt;211&gt; 507 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 2

239

<td colspan="5">Met Ala Gly Ala Gly</td><td rowspan="2">Pro</td><td colspan="2" rowspan="2">Lys Arg</td><td rowspan="2">Arg</td><td rowspan="2">ala 10</td><td rowspan="2">Leu</td><td rowspan="2">ala</td><td rowspan="2">ala</td><td rowspan="2">Pro</td><td rowspan="2">ala 15</td>

<td colspan="3"> 1</td><td colspan="2"> 5</td>

<td>ala</td><td>Glu</td><td>Glu</td><td>lys</td><td>Glu</td><td>Glu</td><td>ala</td><td>Arg</td><td>Glu</td><td>lys</td><td>Underworld</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Cheese</td><td>ala</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>ala</td><td>Pro</td><td>ala</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Gly</td><td>val</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td>

<td>Leu</td><td>Gin</td><td>Arg</td><td>own</td><td>How much</td><td>Thr</td><td>Leu</td><td>Leu</td><td>own</td><td>Gly</td><td>val</td><td>ala</td><td>How much</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td>

<td>Gly</td><td>Thr</td><td>How much</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>How much</td><td>phe</td><td>val</td><td>Thr</td><td>Pro</td><td>Thr</td><td>Gly</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 7 5</td>

<td>Leu</td><td>lys</td><td>Glu</td><td>ala</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Leu</td><td>ala</td><td>Leu</td><td>val</td><td>val</td><td>Trp</td><td>ala</td>

<td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td>

<td>ala</td><td>Cys</td><td>Gly</td><td>val</td><td>phe</td><td>Cheese</td><td>How much</td><td>val</td><td>Gly</td><td>ala</td><td>Leu</td><td>Cys</td><td>Tyr</td><td>ala</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td>

<td>Leu</td><td>Gly</td><td>Thr</td><td>Thr</td><td>How much</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Asp</td><td>Tyr</td><td>ala</td><td>Tyr</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td>

<td>Leu</td><td>Glu</td><td>val</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Leu</td><td>Pro</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Leu</td><td>Trp</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td>

<td>Glu</td><td>Leu</td><td>Leu</td><td>How much</td><td>How much</td><td>Arg</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Gin</td><td>Tyr</td><td>How much</td><td>vai</td><td>ala</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td>

<td>val</td><td>phe</td><td>ala</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Leu</td><td>lys</td><td>Pro</td><td>Leu</td><td>phe</td><td>Pro</td><td>Thr</td><td>Cys</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td>

<td>val</td><td>Pro</td><td>Glu</td><td>Glu</td><td>ala</td><td>ala</td><td>lys</td><td>Leu</td><td>val</td><td>ala</td><td>Cys</td><td>Leu</td><td>Cys</td><td>val</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td>

<td>Leu</td><td>Leu</td><td>Thr</td><td>ala</td><td>val</td><td>own</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>val</td><td>lys</td><td>ala</td><td>ala</td><td>Thr</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td>

<td>val</td><td>Gin</td><td rowspan="2">Asp</td><td>ala</td><td>phe</td><td>ala</td><td>ala</td><td>ala</td><td>lys</td><td>Leu</td><td>Leu</td><td>ala</td><td>Leu</td><td>ala</td><td>Leu</td>

<td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td>

240

<td>How much</td><td colspan="2">How much leu</td><td colspan="2">Leu Gly 215</td><td>phe</td><td colspan="2">Val Gin</td><td>How much</td><td colspan="4">Gly Lys Gly Val 220</td><td>val</td><td>Cheese 225</td>

<td>own</td><td>Leu</td><td>Asp</td><td>Pro</td><td>own</td><td>phe</td><td>Cheese</td><td>phe</td><td>Glu</td><td>Gly</td><td>Thr</td><td>lys</td><td>Leu</td><td>Asp</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Gly</td><td>own</td><td>How much</td><td>val</td><td>Leu</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>phe</td><td>ala</td><td>Tyr</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td rowspan="2">Gly</td><td rowspan="2">Trp</td><td>own</td><td>Tyr</td><td>Leu</td><td>own</td><td>phe</td><td>val</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Underworld</td><td>How much</td><td>own</td><td>Pro</td>

<td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td>

<td>Tyr</td><td>Arg</td><td>own</td><td>Leu</td><td>Pro</td><td>Leu</td><td>ala</td><td>How much</td><td>How much</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Pro</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td>

<td>Thr</td><td>Leu</td><td>val</td><td>Tyr</td><td>val</td><td>Leu</td><td>Thr</td><td>own</td><td>Leu</td><td>ala</td><td>Tyr</td><td>phe</td><td>Thr</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td>

<td>Cheese</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Underworld</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Glu</td><td>ala</td><td>val</td><td>ala</td><td>val</td><td>Asp</td><td>phe</td>

<td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td>

<td>Gly</td><td>own</td><td>Tyr</td><td>His</td><td>Leu</td><td>Gly</td><td>val</td><td>Underworld</td><td>Cheese</td><td>Trp</td><td>How much</td><td>How much</td><td>Pro</td><td>val</td><td>phe</td>

<td></td><td></td><td></td><td></td><td> 320</td><td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td>

<td>val</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>phe</td><td>Gly</td><td>Cheese</td><td>val</td><td>own</td><td>Gly</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td>

<td>Cheese</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>phe</td><td>phe</td><td>val</td><td>Gly</td><td>Cheese</td><td>Arg</td><td>Glu</td><td>Gly</td><td>His</td><td>Leu</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td>

<td>Cheese</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Underworld</td><td>How much</td><td>His</td><td>Pro</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Pro</td><td>val</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td>

<td>Cheese</td><td>Leu</td><td>val</td><td>phe</td><td>Thr</td><td>Cys</td><td>val</td><td>Underworld</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>ala</td><td>phe</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 385</td><td></td><td></td><td></td><td></td><td> 390</td>

<td>lys</td><td>Asp</td><td>How much</td><td>phe</td><td>Cheese</td><td>val</td><td>How much</td><td>own</td><td>phe</td><td>phe</td><td>Cheese</td><td>phe</td><td>phe</td><td>own</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td><td></td><td></td><td></td><td> 405</td>

<td>Leu</td><td>Cys</td><td>val</td><td>ala</td><td>Leu</td><td>ala</td><td>How much</td><td>How much</td><td>Gly</td><td>Underworld</td><td>How much</td><td>Trp</td><td>Leu</td><td>Arg</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td><td></td><td></td><td> 4 20</td>

<td>Arg</td><td>lys</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Pro</td><td>How much</td><td>lys</td><td>val</td><td>own</td><td>Leu</td><td>ala</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td><td></td><td></td><td> 435</td>

<td>Pro</td><td>val</td><td>phe</td><td>phe</td><td>How much</td><td>Leu</td><td>ala</td><td>Cys</td><td>Leu</td><td>phe</td><td>Leu</td><td>He</td><td>ala</td><td>val</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td><td> 450</td>

<td>phe</td><td>Trp</td><td>lys</td><td>Thr</td><td>Pro</td><td>val</td><td>Glu</td><td>Cys</td><td>Gly</td><td>How much</td><td>Gly</td><td>phe</td><td>Thr</td><td>How much</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td> 465</td>

<td>Leu</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro</td><td>val</td><td>Tyr</td><td>phe</td><td>phe</td><td>Gly</td><td>val</td><td>Trp</td><td>Trp</td><td>lys</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>lys</td><td>Pro</td><td>lys</td><td>Trp</td><td>Leu</td><td>Leu</td><td>Gin</td><td>Gly</td><td>How much</td><td>phe</td><td>Cheese</td><td>Thr</td><td>Thr</td><td>val</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 4 90</td><td></td><td></td><td></td><td></td><td> 4 95</td>

<td>Cys</td><td>Gin</td><td>lys</td><td>Leu</td><td>Underworld</td><td>Gin</td><td>val</td><td>val</td><td>Pro</td><td>Gin</td><td>Glu</td><td>Thr</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td></td>

241 &lt;210&gt; 3 &lt;211&gt; 339 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 3

242

<td rowspan="2">Underworld 1</td><td rowspan="2">Glu</td><td colspan="12">Cheese Arg Lys Asp Ile Thr Asn Gin Glu Glu Leu Trp</td><td rowspan="2">lys 15</td>

<td colspan="7"> 5</td><td colspan="5"> 10</td>

<td>Underworld</td><td>lys</td><td>Pro</td><td>Arg</td><td>Arg</td><td>own</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Asp</td><td>Asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Asp</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Underworld</td><td>Leu</td><td>lys</td><td>Arg</td><td>Pro</td><td>val</td><td>Leu</td><td>Leu</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td>

<td>Leu</td><td>His</td><td>Gin</td><td>Thr</td><td>ala</td><td>His</td><td>ala</td><td>Asp</td><td>Glu</td><td>phe</td><td>Asp</td><td>Cys</td><td>Pro</td><td>Cheese</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td>

<td>Leu</td><td>Gin</td><td>His</td><td>Thr</td><td>Gin</td><td>Glu</td><td>Leu</td><td>phe</td><td>Pro</td><td>Gin</td><td>Trp</td><td>His</td><td>Leu</td><td>Pro</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td>

<td>lys</td><td>How much</td><td>ala</td><td>ala</td><td>How much</td><td>How much</td><td>ala</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>phe</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td>

<td>Leu</td><td>Arg</td><td>Glu</td><td>val</td><td>How much</td><td>His</td><td>Pro</td><td>Leu</td><td>ala</td><td>Thr</td><td>Cheese</td><td>His</td><td>Gin</td><td>Gin</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td>

<td>phe</td><td>Tyr</td><td>lys</td><td>How much</td><td>Pro</td><td>How much</td><td>Leu</td><td>val</td><td>How much</td><td>own</td><td>lys</td><td>val</td><td>Leu</td><td>Pro</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td>

<td>val</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Leu</td><td>Leu</td><td>ala</td><td>Leu</td><td>val</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>Gly</td><td>val</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td>

<td>ala</td><td>m =</td><td>How much</td><td>val</td><td>Gin</td><td>TDH Π¹- ll</td><td>s</td><td>he</td><td>mw '- «j-jr</td><td>Thy i.</td><td>T.tle</td><td>'Tiry *</td><td>T</td><td>t-łf</td><td>t and IC</td>

<td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td>

<td>Pro</td><td>His</td><td>Trp</td><td>Leu</td><td>Asp</td><td>lys</td><td>Trp</td><td>Underworld</td><td>Leu</td><td>Thr</td><td>Arg</td><td>lys</td><td>Gin</td><td>phe</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td>

<td>Leu</td><td>Leu</td><td>Cheese</td><td>phe</td><td>phe</td><td>phe</td><td>ala</td><td>val</td><td>Leu</td><td>His</td><td>ala</td><td>How much</td><td>Tyr</td><td>Cheese</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td>

<td>Cheese</td><td>Tyr</td><td>Pro</td><td>Underworld</td><td>Arg</td><td>Arg</td><td>Cheese</td><td>Tyr</td><td>Arg</td><td>Tyr</td><td>lys</td><td>Leu</td><td>Leu</td><td>own</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td>

<td>ala</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>val</td><td>Gin</td><td>Gin</td><td>own</td><td>lys</td><td>Glu</td><td>Asp</td><td>ala</td><td>Trp</td><td>How much</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td>

<td>His</td><td>Asp</td><td>val</td><td>Trp</td><td>Arg</td><td>Underworld</td><td>Glu</td><td>How much</td><td>Tyr</td><td>val</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td>

<td>Gly</td><td>Leu</td><td>ala</td><td>How much</td><td>Leu</td><td>ala</td><td>Leu</td><td>Leu</td><td>ala</td><td>val</td><td>Thr</td><td>Cheese</td><td>How much</td><td>Pro</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>val</td><td>Cheese</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Trp</td><td>Arg</td><td>Glu</td><td>phe</td><td>His</td><td>Tyr</td><td>How much</td><td>Gin</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td>lys</td><td>Leu</td><td>Gly</td><td>How much</td><td>val</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Thr</td><td>How much</td><td>His</td><td>ala</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td>

<td>How much</td><td>phe</td><td>ala</td><td>Trp</td><td>own</td><td>lys</td><td>Trp</td><td>How much</td><td>Asp</td><td>How much</td><td>lys</td><td>Gin</td><td>phe</td><td>val</td><td>Trp</td>

243

<td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td>

<td>Tyr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Thr 290</td><td>phe</td><td>Underworld</td><td>How much</td><td>ala</td><td>val 295</td><td>phe</td><td>Leu</td><td>Pro</td><td>How much</td><td>val 300</td>

<td>val</td><td>Leu</td><td>How much</td><td>phe</td><td>lys 305</td><td>Cheese</td><td>How much</td><td>Leu</td><td>phe</td><td>Leu 310</td><td>Pro</td><td>Cys</td><td>Leu</td><td>Arg</td><td>lys 315</td>

<td>lys</td><td>How much</td><td>Leu</td><td>lys</td><td>How much 320</td><td>Arg</td><td>His</td><td>Gly</td><td>Trp</td><td>Glu 325</td><td>Asp</td><td>val</td><td>Thr</td><td>lys</td><td>How much 330</td>

<td>own</td><td>lys</td><td>Thr</td><td>Glu</td><td>How much 335</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 4 &lt;211&gt; 6995 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 4

244

<td colspan="11">Pro Val Thr Ser Leu Leu Thr Pro Gly Leu Val</td><td colspan="2" rowspan="2">How much thr</td><td rowspan="2">Thr</td><td rowspan="2">Asp 15</td>

<td colspan="2"> 1</td><td colspan="3"> 5</td><td colspan="6"> 10</td>

<td>Arg</td><td>Underworld</td><td>Gly</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Leu</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>His</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Asp</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td>

<td>val</td><td>Asp</td><td>Thr</td><td>Glu</td><td>ala</td><td>Underworld</td><td>Gin</td><td>Pro</td><td>Cheese</td><td>Thr</td><td>His</td><td>Thr</td><td>ala</td><td>val</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td>

<td>own</td><td>val</td><td>Arg</td><td>Thr</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Gly</td><td>His</td><td>Glu</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>Cheese</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td>

<td>Leu</td><td>Cheese</td><td>Asp</td><td>Cheese</td><td>Glu</td><td>Thr</td><td>Pro</td><td>lys</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Underworld</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td>

<td>Thr</td><td>Tyr</td><td>Thr</td><td>Underworld</td><td>Gly</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>val</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Thr.</td><td>Cheese</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td>

<td>phe</td><td>phe</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Arg</td><td>How much</td><td>Gin</td><td>How much</td><td>Glu</td><td>Pro</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td>

<td>Thr</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Glu</td><td>Arg</td><td>How much</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td>

<td>ala</td><td>Thr</td><td>Glu</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>val</td><td>Leu</td><td>Cheese</td><td>Glu</td><td>val</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>ala</td>

<td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td>

<td>Thr</td><td>Thr</td><td>Glu</td><td>val</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Glu</td><td>val</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td>

<td>Cheese</td><td>Underworld</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Asp</td><td>Gin</td><td>phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Pro</td><td>Asp</td><td>How much</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td>

<td>Thr</td><td>Glu</td><td>ala</td><td>How much</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>How much</td><td>Underworld</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td>

<td>Cheese</td><td>ala</td><td>Glu</td><td>Cheese</td><td>ala</td><td>How much</td><td>Thr</td><td>How much</td><td>Glu</td><td>Thr</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>ala</td>

<td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td>

245

<td>Thr</td><td>Cheese</td><td colspan="2">Glu Gly</td><td>Thr 215</td><td colspan="4">Leu Thr Leu Asp</td><td>Thr 220</td><td colspan="4">Ser Thr Thr Thr</td><td>phe 225</td>

<td>Trp</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>His</td><td>Cheese</td><td>Thr</td><td>ala</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>phe</td><td>Cheese</td><td>His</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Glu</td><td>Underworld</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Asp</td><td>val</td><td>Pro</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td>Pro</td><td>Cheese</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>val</td><td>Glu</td><td>Glu</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td>

<td>Leu</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>ala</td><td>Underworld</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>phe</td><td>phe</td><td>Cheese</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td>

<td>Pro</td><td>Glu</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>His</td><td>Pro</td><td>val</td><td>Thr</td><td>ala</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td>

<td>Thr</td><td>Leu</td><td>Gly</td><td>Pro</td><td>val</td><td>lys</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Underworld</td><td>Leu</td><td>Arg</td><td>Thr</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td>

<td>Glu</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Pro</td><td>own</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>ala</td>

<td></td><td></td><td></td><td></td><td> 320</td><td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td>

<td>Glu</td><td>How much</td><td>Leu</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Glu</td><td>val</td><td>Thr</td><td>lys</td><td>Asp</td><td>Arg</td><td>Glu</td><td>lys</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td>

<td>His</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>own</td><td>Thr</td><td>Pro</td><td>val</td><td>val</td><td>own</td><td>val</td><td>Gly</td><td>Thr</td><td>val</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td>

<td>Tyr</td><td>lys</td><td>His</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Leu</td><td>ala</td><td>Asp</td><td>Leu</td><td>val</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td>

<td>Thr</td><td>lys</td><td>Pro</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Underworld</td><td>ala</td><td>Thr</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Gly</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 385</td><td></td><td></td><td></td><td></td><td> 390</td>

<td>Thr</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Pro</td><td>ala</td><td>phe</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Underworld</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td><td></td><td></td><td></td><td> 405</td>

<td>Thr</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Glu</td><td>How much</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td><td></td><td></td><td> 420</td>

<td>Thr</td><td>Cheese</td><td>Gin</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Glu</td><td>Arg</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td><td></td><td></td><td> 435</td>

<td>Cheese</td><td>Gly</td><td>Underworld</td><td>Pro</td><td>Thr</td><td>Gly</td><td>ala</td><td>Thr</td><td>Thr</td><td>lys</td><td>val</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td><td> 450</td>

<td>ala</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>Arg</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Ala</td><td>Gin</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td> 4 65</td>

<td>Thr</td><td>Ile</td><td>Ser</td><td>Pro</td><td>Glu</td><td>Ile</td><td>Ser</td><td>Thr</td><td>Glu</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Arg</td><td>Ile</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>Thr</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Ser</td><td>Ala</td><td>Glu</td><td>Met</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 4 90</td><td></td><td></td><td></td><td></td><td> 4 95</td>

<td>Lys</td><td>Thr</td><td>Gly</td><td>His</td><td>Ser</td><td>Gly</td><td>Ala</td><td>Ser</td><td>Ser</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Phe</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td>

246

<td>Asp</td><td colspan="3">Thr Ser Ser</td><td>Arg 515</td><td>Ala</td><td colspan="3">Ser Trp Pro</td><td colspan="2">Gly Thr 520</td><td>His</td><td>Ser</td><td>Ala</td><td>Ala 525</td>

<td>Thr</td><td>His</td><td>Arg</td><td>Ser</td><td>Pro</td><td>His</td><td>Ser</td><td>Gly</td><td>Met</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Met</td><td>Ser</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td>

<td>Gly</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Val</td><td>Ser</td><td>Trp</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Val</td><td>Glu</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 555</td>

<td>Thr</td><td>Ser</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Leu</td><td>Val</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ala</td><td>Val</td><td>Thr</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 560</td><td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td><td> 570</td>

<td>Pro</td><td>Ser</td><td>Pro</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Glu</td><td>Ser</td><td>Ser</td><td>His</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 575</td><td></td><td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td>

<td>Pro</td><td>Leu</td><td>Arg</td><td>Val</td><td>Thr</td><td>Ser</td><td>Leu</td><td>Phe</td><td>Thr</td><td>Pro</td><td>Val</td><td>Met</td><td>Met</td><td>Lys</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td><td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td>

<td>Thr</td><td>Asp</td><td>Met</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Ser</td><td>Leu</td><td>Glu</td><td>Pro</td><td>Val</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td><td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td>

<td>Pro</td><td>Ser</td><td>Met</td><td>Asn</td><td>Ile</td><td>Thr</td><td>Ser</td><td>Asp</td><td>Glu</td><td>Ser</td><td>Leu</td><td>Ala</td><td>Thr</td><td>Ser</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td><td> 625</td><td></td><td></td><td></td><td></td><td> 630</td>

<td>Ala</td><td>Thr</td><td>Met</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Ala</td><td>Ile</td><td>Gin</td><td>Leu</td><td>Ser</td><td>Glu</td><td>Asn</td><td>Thr</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td><td></td><td></td><td></td><td></td><td> 645</td>

<td>Val</td><td>Thr</td><td>Gin</td><td>Met</td><td>Gly</td><td>Thr</td><td>Ile</td><td>Ser</td><td>Ala</td><td>Arg</td><td>Gin</td><td>Glu</td><td>Phe</td><td>Tyr</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td><td></td><td></td><td></td><td> 660</td>

<td>O</td><td></td><td></td><td>Cl &gt;</td><td>T ZM,</td><td>ϋν·Λ</td><td>Clii</td><td>Pito</td><td>Ca v</td><td>T.VP</td><td>Val</td><td>Thr</td><td>Ser</td><td></td><td>Val</td>

<td>Od</td><td></td><td>L 4. U</td><td>j- y</td><td>OO UŁ</td><td>4. O. *_/</td><td>OJ-U</td><td></td><td></td><td>J-.J U»</td><td></td><td></td><td></td><td>ί Λ.</td><td></td>

<td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td></td><td></td><td></td><td> 675</td>

<td>Val</td><td>Thr</td><td>Ser</td><td>Ser</td><td>Thr</td><td>Ile</td><td>Lys</td><td>Asp</td><td>Ile</td><td>Val</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Ile</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td><td></td><td> 690</td>

<td>Ala</td><td>Ser</td><td>Ser</td><td>Glu</td><td>Ile</td><td>Thr</td><td>Arg</td><td>Ile</td><td>Glu</td><td>Met</td><td>Glu</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td><td> 705</td>

<td>Leu</td><td>Thr</td><td>Pro</td><td>Thr</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Gin</td><td>Glu</td><td>Ile</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td>Ser</td><td>Ala</td><td>Thr</td><td>Lys</td><td>Pro</td><td>Ser</td><td>Thr</td><td>Val</td><td>Pro</td><td>Tyr</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td>

<td>Ala</td><td>Thr</td><td>Ile</td><td>Glu</td><td>Asp</td><td>Ser</td><td>Met</td><td>Thr</td><td>Gin</td><td>Val</td><td>Met</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td>

<td>Gly</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Asp</td><td>Gin</td><td>Ser</td><td>Thr</td><td>Met</td><td>Ser</td><td>Gin</td><td>Asp</td><td>Ile</td><td>Ser</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td>

<td>Glu</td><td>Val</td><td>Ile</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Pro</td><td>Ile</td><td>Lys</td><td>Thr</td><td>Glu</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td>

<td>Thr</td><td>Glu</td><td>Met</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Ala</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td>

<td>Ser</td><td>Arg</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Phe</td><td>Met</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 800</td><td></td><td></td><td></td><td></td><td> 805</td><td></td><td></td><td></td><td></td><td> 810</td>

<td>Gly</td><td>Thr</td><td>His</td><td>Ser</td><td>Thr</td><td>Ala</td><td>Ser</td><td>Gin</td><td>Gly</td><td>Phe</td><td>Ser</td><td>His</td><td>Ser</td><td>Gin</td><td>Met</td>

247

<td></td><td>BIS</td><td> 820</td><td> 325</td>

<td>Thr Ala</td><td>Leu Met Ser Arg 830</td><td>Thr Pro Gly Glu Val Pro Trp 835</td><td>Leu Ser 840</td>

<td>His Pro</td><td>Ser Val Glu Glu 645</td><td>Ala Ser Ser Ala Ser Phe Ser 850</td><td>Leu Ser 855</td>

<td>Ser Pro</td><td>Val Met Thr Ser 860</td><td>Ser Ser Pro Val Ser Ser Thr 865</td><td>Leu Pro 870</td>

<td>Asp Ser</td><td>He His Ser Ser 875</td><td>Ser Leu Pro Val Thr Ser Leu 880</td><td>Leu Thr 885</td>

<td>Ser Gly</td><td>Leu Val Lys Thr 890</td><td>Thr Glu Leu Leu Gly Thr Ser 895</td><td>Ser Glu 900</td>

<td>Pro Glu</td><td>Thr Ser Ser Pro 905</td><td>Pro Asn Leu Ser Ser Thr Ser 910</td><td>Ala Glu 915</td>

<td>Ile Leu</td><td>Ala Thr Thr Glu 920</td><td>Val Thr Thr Asp Thr Glu Lys 925</td><td>Leu Glu 930</td>

<td>Met Thr</td><td>Asn Val Val Thr 935</td><td>Ser Gly Tyr Thr His Glu Ser 940</td><td>Pro Ser 945</td>

<td>Ser Val</td><td>Leu Ala Asp Ser 950</td><td>Val Thr Thr Lys Ala Thr Ser 955</td><td>Ser Met 960</td>

<td>Gly Ile</td><td>Thr Tyr Pro Thr 965</td><td>Gly Asp Thr Asn Val Leu Thr 970</td><td>Ser Thr 975</td>

<td>Pro Ala</td><td>Phe Ser Asp Thr 980</td><td>Ser Arg Ile Gin Thr Lys Ser 985</td><td>Lys Leu 990</td>

<td>Ser Leu</td><td>Thr Pro Giy Leu 995</td><td>Met Glu Thr Ser Ile Ser Glu 1000</td><td>Glu Thr 1005</td>

<td>Ser Ser</td><td>Ala Thr Glu Lys 1010</td><td>Ser Thr Val Leu Ser Ser Val 1015</td><td>Pro Thr 1020</td>

<td>Gly Ala</td><td>Thr Thr Glu Val 1025</td><td>Ser Arg Thr Glu Ala Ile Ser 1030</td><td>Ser Ser 1035</td>

<td>Arg Thr</td><td>Ser Ile Pro Gly 1040</td><td>Pro Ala Gin Ser Thr Met Ser 1045</td><td>Ser Asp 1050</td>

<td>Thr Ser</td><td>Met Glu Thr Ile 1055</td><td>Thr Arg Ile Ser Thr Pro Leu 1060</td><td>Thr Arg 1065</td>

<td>Lys Glu</td><td>Ser Thr Asp Met 1070</td><td>Ala Ile Thr Pro Lys Thr Gly 1075 ’</td><td>Pro Ser 1060</td>

<td>Gly Ala</td><td>Thr Ser Gin Gly 1085</td><td>Thr Phe Thr Leu Asp Ser Ser 1090</td><td>Ser Thr 1095</td>

<td>Ala Ser</td><td>Trp Pro Gly Thr 1100</td><td>His Ser Ala Thr Thr Gin Arg 1105</td><td>Phe Pro 1110</td>

<td>Arg Ser</td><td>Val Val Thr Thr</td><td>Pro Met Ser Arg Gly Pro Glu</td><td>Asp Val</td>

1115 1120 1125

248

<td>Ser</td><td>Trp</td><td>Pro</td><td>Ser Pro 1130</td><td>Leu</td><td>Ser</td><td>Val</td><td>Glu Lys 1135</td><td>Asn</td><td>Ser</td><td>Pro</td><td>Pro Ser 1140</td>

<td>Ser</td><td>Leu</td><td>Val</td><td>Ser Ser 1145</td><td>Ser</td><td>Ser</td><td>Val</td><td>Thr Ser 1150</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Leu Tyr 1155</td>

<td>Ser</td><td>Thr</td><td>Pro</td><td>Ser Gly 1160</td><td>Ser</td><td>Ser</td><td>His</td><td>Ser Ser 1165</td><td>Pro</td><td>Val</td><td>Pro</td><td>Val Thr 1170</td>

<td>Ser</td><td>Leu</td><td>Phe</td><td>Thr Ser 1175</td><td>Ile</td><td>Met</td><td>Met</td><td>Lys Ala 1180</td><td>Thr</td><td>Aap</td><td>Met</td><td>Leu Asp 1185</td>

<td>Ala</td><td>Ser</td><td>Leu</td><td>Glu Pro 1190</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Ser Ala 1195</td><td>Pro</td><td>Asn</td><td>Met</td><td>Asn Ile 1200</td>

<td>Thr</td><td>Ser</td><td>Asp</td><td>Glu Ser 1205</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Ser Lys 1210</td><td>Ala</td><td>Thr</td><td>Thr</td><td>Glu Thr 1215</td>

<td>Glu</td><td>Ala</td><td>Ile</td><td>His Val 1220</td><td>Phe</td><td>Glu</td><td>Asn</td><td>Thr Ala 1225</td><td>Ala</td><td>Ser</td><td>His</td><td>Val Glu 1230</td>

<td>Thr</td><td>Thr</td><td>Ser</td><td>Ala Thr 1235</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Tyr Ser 1240</td><td>Ser</td><td>Ser</td><td>Pro</td><td>Gly Phe 1245</td>

<td>Ser</td><td>Glu</td><td>Pro</td><td>Thr Lys 1250</td><td>Val</td><td>Ile</td><td>Ser</td><td>Pro Val 1255</td><td>Val</td><td>Thr</td><td>Ser</td><td>Ser Ser 1260</td>

<td>Ile</td><td>Arg</td><td>Asp</td><td>Asn Met 1265</td><td>Val</td><td>Ser</td><td>Thr</td><td>Thr Met 1270</td><td>Pro</td><td>Gly</td><td>Ser</td><td>Ser Gly 1275</td>

<td>Ile</td><td>Thr</td><td>Arg</td><td>Ile Glu 1280</td><td>Ile</td><td>Glu</td><td>Ser</td><td>Met Ser 1285</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Pro Gly 1290</td>

<td>Leu</td><td>Arg</td><td>Glu</td><td>Thr Arg 1295</td><td>Thr</td><td>Ser</td><td>Gin</td><td>Asp Tle 1300</td><td>Thr</td><td>Ser</td><td>Ser</td><td>Thr Glu 1305</td>

<td>Thr</td><td>Ser</td><td>Thr</td><td>Val Leu 1310</td><td>Tyr</td><td>Lys</td><td>Met</td><td>Pro Ser 1315</td><td>Gly</td><td>Ala</td><td>Thr</td><td>Pro Glu 1320</td>

<td>Val</td><td>Ser</td><td>Arg</td><td>Thr Glu 1325</td><td>Val</td><td>Met</td><td>Pro</td><td>Ser Set 1330</td><td>Arg</td><td>Thr</td><td>Ser</td><td>Ile Pro 1335</td>

<td>Gly</td><td>Pro</td><td>Ala</td><td>Gin Ser 1340</td><td>Thr</td><td>Met</td><td>Ser</td><td>Leu Asp 1345</td><td>Ile</td><td>Ser</td><td>Asp</td><td>Glu Val 1350</td>

<td>Val</td><td>Thr</td><td>Arg</td><td>Leu Ser 1355</td><td>Thr</td><td>Ser</td><td>Pro</td><td>Ile Met 1360</td><td>Thr</td><td>Glu</td><td>Ser</td><td>Ala Glu 1365</td>

<td>Ile</td><td>Thr</td><td>Ile</td><td>Thr Thr 1370</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Tyr Ser 1375</td><td>Leu</td><td>Ala</td><td>Thr</td><td>Ser Gin 1380</td>

<td>Val</td><td>Thr</td><td>Leu</td><td>Pro Leu 1385</td><td>Gly</td><td>Thr</td><td>Se£</td><td>Met Thr 1390</td><td>Phe</td><td>Leu</td><td>Ser</td><td>Gly Thr 1395</td>

<td>His</td><td>Ser</td><td>Thr</td><td>Met Ser 1400</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Ser His 1405</td><td>Ser</td><td>Glu</td><td>Met</td><td>Thr Asn 1410</td>

<td>Leu</td><td>Met</td><td>Ser</td><td>Arg Gly 1415</td><td>Pro</td><td>Glu</td><td>Ser</td><td>Leu Ser 1420</td><td>Trp</td><td>Thr</td><td>Ser</td><td>Pro Arg 1425</td>

249

<td>Phe</td><td>Val</td><td>Glu</td><td>Thr Thr 1430</td><td>Arg</td><td>Ser</td><td>Ser</td><td>Ser Ser 1435</td><td>Leu</td><td>Thr</td><td>Sar</td><td>Leu Pro 1440</td>

<td>Leu</td><td>Thr</td><td>Thr</td><td>Ser Leu 1445</td><td>Ser</td><td>Pro</td><td>Ual</td><td>Ser Ser 1450</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Asp Ser 1455</td>

<td>Ser</td><td>Pro</td><td>Ser</td><td>Ser Pro 1460</td><td>Leu</td><td>Pro</td><td>Val</td><td>Thr Ser 1465</td><td>Leu</td><td>Ile</td><td>Leu</td><td>Pro Gly 1470</td>

<td>Leu</td><td>Val</td><td>Lys</td><td>Thr Thr 1475</td><td>Glu</td><td>Val</td><td>Leu</td><td>Aap Thr 1480</td><td>Ser</td><td>Ser</td><td>Glu</td><td>Pro Lys 1405</td>

<td>Thr</td><td>Ser</td><td>Ser</td><td>Ser Pro 14 90</td><td>Asn</td><td>Leu</td><td>Ser</td><td>Ser Thr 14 95</td><td>Ser</td><td>Val</td><td>Glu</td><td>He Pro 1500</td>

<td>Ala</td><td>Thr</td><td>Ser</td><td>Glu Ile 1505</td><td>Met</td><td>Thr</td><td>Asp</td><td>Thr Glu 1510</td><td>Lys</td><td>Ile</td><td>His</td><td>Pro Ser 1515</td>

<td>Ser</td><td>Asn</td><td>Thr</td><td>Ala Val 1520</td><td>Ala</td><td>Lys</td><td></td><td>Arg Thr 1525</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Val His 1530</td>

<td>Glu</td><td>Ser</td><td>Hi3</td><td>Ser Ser 1535</td><td>Val</td><td>Leu</td><td>Ala</td><td>Asp Ser 1540</td><td>Glu</td><td>Thr</td><td>Thr</td><td>rie Thr 1545</td>

<td>Ile</td><td>Pro</td><td>Ser</td><td>Met Gly 1550</td><td>Ile</td><td>Thr</td><td>Ser</td><td>Ala Val 1555</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Thr Val 1560</td>

<td>Phe</td><td>Thr</td><td>Ser</td><td>Asn Pro 1565</td><td>Ala</td><td>Phe</td><td>Ser</td><td>Glu rhr 1570</td><td>Arg</td><td>Arg</td><td>Ile</td><td>Pro Thr 1575</td>

<td>Glu</td><td>Pro</td><td>Thr</td><td>Phe Ser 1580</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Gly Phe 1585</td><td>Arg</td><td>Glu</td><td>Thr</td><td>Sar Thr 1590</td>

<td>Ser</td><td>Glu</td><td>Glu</td><td>Thr Thr 1595</td><td>Ser</td><td>lla</td><td>Thr</td><td>Glu Thr 1600</td><td>Ser</td><td>Ala</td><td>vai</td><td>Leu Phe 1605</td>

<td>Gly</td><td>Val</td><td>Pro</td><td>Thr Ser 1610</td><td>Ala</td><td>Thr</td><td>Thr</td><td>Glu Val 1615</td><td>Ser</td><td>Met</td><td>Thr</td><td>Glu Ile 1620</td>

<td>Met</td><td>Ser</td><td>Ser</td><td>Asn Arg 1625</td><td>Thr</td><td>His</td><td>Ile</td><td>Pro Asp 1630</td><td>Sar</td><td>Asp</td><td>Gin</td><td>Ser Thr 1635</td>

<td>Met</td><td>Ser</td><td>Pro</td><td>Asp Ile 1640</td><td>Ile</td><td>Thr</td><td>Glu</td><td>Val Ile 1645</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Ser Ser 1650</td>

<td>Ser</td><td>Ser</td><td>Met</td><td>Met Ser 1655</td><td>Glu</td><td>Ser</td><td>Thr</td><td>Gin Mat 1660</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Thr Gin 1665</td>

<td>Lys</td><td>Ser</td><td>Ser</td><td>Pro Gly 1670</td><td>Ala</td><td>Thr</td><td>Ala</td><td>Gin Ser 1675</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu Ala 16B0</td>

<td>Thr</td><td>Thr</td><td>Thr</td><td>Ala Pro 1685</td><td>Leu</td><td>Ala</td><td>Arg</td><td>Thr His 1690</td><td>Sar</td><td>Thr</td><td>Val</td><td>Pro Pro 1695</td>

<td>Arg</td><td>Phe</td><td>Leu</td><td>His Ser 1700</td><td>Glu</td><td>Met</td><td>Thr</td><td>Thr Leu 1705</td><td>Met</td><td>Ser</td><td>Arg</td><td>Ser Pro 1710</td>

<td>Glu</td><td>Asn</td><td>Pro</td><td>Sar Trp 1715</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Pro Phe 1720</td><td>vai</td><td>Glu</td><td>LyS</td><td>Thr Ser 1725</td>

<td>Ser</td><td>Ser</td><td>Ser</td><td>Ser Leu</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Pro Val</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Pro Ser</td>

250

<td></td><td></td><td></td><td> 1730</td><td></td><td></td><td></td><td> 1735</td><td></td><td></td><td></td><td> 1740</td>

<td>Val</td><td>Ser</td><td>Ser</td><td>Thr Leu 1745</td><td>Pro</td><td>Gln</td><td>Ser</td><td>Ile Pro 1750</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Phe Ser 1755</td>

<td>val</td><td>Thr</td><td>Ser</td><td>Leu Leu 1760</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Met Val 17 65</td><td>Lys</td><td>Thr</td><td>Thr</td><td>ASp Thr 1770</td>

<td>Ser</td><td>Thr</td><td>Glu</td><td>Pro Gly 1775</td><td>Thr</td><td>Ser</td><td>Leu</td><td>ser Pro 1780</td><td>Asn</td><td>Leu</td><td>Ser</td><td>Gly Thr 1785</td>

<td>Ser</td><td>Val</td><td>Glu</td><td>Ile Leu 1790</td><td>Ala</td><td>Ala</td><td>Ser</td><td>Glu Val 1795</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Thr Glu 1800</td>

<td>Lys</td><td>Ile</td><td>His</td><td>Pro Ser 1B05</td><td>Ser</td><td>Sar</td><td>Met</td><td>Ala Val 1810</td><td>Thr</td><td>Asn</td><td>Val</td><td>Gly Thr 1815</td>

<td>Thr</td><td>Ser</td><td>Ser</td><td>Gly His 1620</td><td>Glu</td><td>Leu</td><td>Tyr</td><td>Ser Ser 1825</td><td>Val</td><td>Ser</td><td>Tle</td><td>His Ser 1830</td>

<td>Glu</td><td>Pro</td><td>Ser</td><td>Lys Ala 1835</td><td>Thr</td><td>Tyr</td><td>Pro</td><td>Val Gly 1840</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Ser Met 1845</td>

<td>Ala</td><td>Glu</td><td>Thr</td><td>Ser Ile 1850</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Met Pro 1855</td><td>Ala</td><td>Asn</td><td>Phe</td><td>Glu Thr 1860</td>

<td>Thr</td><td>Gly</td><td>Phe</td><td>Glu Ala 1865</td><td>Glu</td><td>Pro</td><td>Phe</td><td>Ser His 1070</td><td>Leu</td><td>Thr</td><td>Ser</td><td>Gly Leu 1875</td>

<td>Arg</td><td>Lys</td><td>Thr</td><td>Asn Met 1880</td><td>Ser</td><td>Leu</td><td>Asp</td><td>Thr Ser 1885</td><td>Ser</td><td>Val</td><td>Thr</td><td>Pro Thr 1890</td>

<td>Asn</td><td>Thr</td><td>Pro</td><td>Ser Ser 1095</td><td>Pro</td><td>Gly</td><td>Ser</td><td>Thr His 1900</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Ser Ser 1905</td>

<td>Lys</td><td>Thr</td><td>Asp</td><td>Phe Thr 1910</td><td>Ser</td><td>Ser</td><td>Ala</td><td>Lys Thr 1915</td><td>Ser</td><td>Ser</td><td>Pro</td><td>Asp Trp 1920</td>

<td>Pro</td><td>Pro</td><td>Ala</td><td>Ser Gln 1925</td><td>Tyr</td><td>Thr</td><td>Glu</td><td>Ile Pro 1930</td><td>Val</td><td>Asp</td><td>Ile</td><td>Ile Thr 1935</td>

<td>Pro</td><td>Phe</td><td>Asn</td><td>Ala Ser 1940</td><td>Pro</td><td>Ser</td><td>Ile</td><td>Thr Glu 1945</td><td>Ser</td><td>Thr</td><td>Gly</td><td>Ile Thr 1950</td>

<td>Ser</td><td>Phe</td><td>Pro</td><td>Glu Ser 1955</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Met Ser 1960</td><td>Val</td><td>Thr</td><td>Glu</td><td>Ser Thr 1965</td>

<td>His</td><td>His</td><td>Leu</td><td>Ser Thr 1970</td><td>Asp</td><td>Leu</td><td>Leu</td><td>Pro Ser 1975</td><td>Ala</td><td>Glu</td><td>Thr</td><td>Ile Ser 1980</td>

<td>Thr</td><td>Gly</td><td>Thr</td><td>Val Met 1935</td><td>Pro</td><td>Ser</td><td>Leu</td><td>Ser Glu 1990</td><td>Ala</td><td>Met</td><td>Thr</td><td>Ser Phe 1995</td>

<td>Ala</td><td>Thr</td><td>Thr</td><td>Gly Val 2000</td><td>Pro</td><td>Arg</td><td>Ala</td><td>Ile Ser 2005</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Ser Pro 2010</td>

<td>Phe</td><td>Ser</td><td>Arg</td><td>Thr Glu 2015</td><td>Ser</td><td>Gly</td><td>Pro</td><td>Gly Asp 2020</td><td>Ala</td><td>Thr</td><td>Leu</td><td>Ser Thr 2025</td>

<td>Ile</td><td>ΑΣ-3</td><td>Glu</td><td>Ser Leu 2030</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Thr Pro 2035</td><td>Val</td><td>Pro</td><td>Phe</td><td>Ser Ser 2040</td>

251

<td>Ser</td><td>Thr</td><td>Phe</td><td>Thr Thr 2045</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Ser Thr 2050</td><td>Ile</td><td>Pro</td><td>Ala</td><td>Leu His 2055</td>

<td>Glu</td><td>Ile</td><td>Thr</td><td>Ser Ser 2060</td><td>Ser</td><td>Ala</td><td>Thr</td><td>Pro Tyr 2065</td><td>Arg</td><td>Val</td><td>Asp</td><td>Thr Ser 2070</td>

<td>Leu</td><td>Gly</td><td>Thi</td><td>G1Q Ser 2075</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Glu Gly 2080</td><td>Arg</td><td>Leu</td><td>Val</td><td>Met Val 2085</td>

<td>ser</td><td>Thr</td><td>Leu</td><td>Asp Thr 2090</td><td>Ser</td><td>Ser</td><td>Gin</td><td>Pro Gly 2095</td><td>Arg</td><td>Thr</td><td>Ser</td><td>Ser Ser 2100</td>

<td>Pro</td><td>Ile</td><td>Leu</td><td>Asp Thr 2105</td><td>Arg</td><td>Met</td><td>Thr</td><td>Glu Ser 2110</td><td>Val</td><td>Glu</td><td>Leu</td><td>Gly Thr 2115</td>

<td>Val</td><td>Thr</td><td>Ser</td><td>Ala Tyr 2120</td><td>Gin</td><td>Ual</td><td>Pro</td><td>Ser Leu 2125</td><td>Ser</td><td>Thr</td><td>Arg</td><td>Leu Thr 2130</td>

<td>Arg</td><td>Thr</td><td>Asp</td><td>Gly Ile 2135</td><td>Met</td><td>Glu</td><td>His</td><td>Ile Thr 2140</td><td>Lys</td><td>Ile</td><td>Pro</td><td>Asn Glu 2145</td>

<td>Ala</td><td>Ala</td><td>His</td><td>Arg Gly 2150</td><td>Thr</td><td>Ile</td><td>Arg</td><td>Pro Val 2155</td><td>Lys</td><td>Gly</td><td>Pro</td><td>Gin Thr 2160</td>

<td>Ser</td><td>Thr</td><td>Ser</td><td>Pro Ala 2165</td><td>Ser</td><td>Pro</td><td>Lys</td><td>Gly Leu 2170</td><td>His</td><td>Thr</td><td>Gly</td><td>Gly Thr 2175</td>

<td>Lys</td><td>Arg</td><td>Met</td><td>Glu Thr 2100</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Ala Leu 21Ξ5</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Thr Thr 2190</td>

<td>Ala</td><td>Leu</td><td>Lys</td><td>Thr Thr 2195</td><td>Ser</td><td>Arg</td><td>Ala</td><td>Thr Leu 2200</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Val Tyr 2205</td>

<td>Thr</td><td>Pro</td><td>Thr</td><td>Leu Gly 2210</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Pro Leu 2215</td><td>Asn</td><td>Ala</td><td>Sec</td><td>Met Gin 2220</td>

<td>Met</td><td>Ala</td><td>Ser</td><td>Thr Ile 2225</td><td>Pro</td><td>Thr</td><td>Glu</td><td>Met Met 2230</td><td>Ile</td><td>Thr</td><td>Thr</td><td>Pro Tyr 2235</td>

<td>Val</td><td>Phe</td><td>Pro</td><td>Asp Val 2240</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Thr Ser 2245</td><td>Ser</td><td>Leu</td><td>Ala</td><td>Thr Ser 2250</td>

<td>Leu</td><td>Gly</td><td>Ala</td><td>Glu Thr 2255</td><td>Ser</td><td>Thr</td><td>Ala</td><td>Leu Pro 2260</td><td>Arg</td><td>Thr</td><td>Thr</td><td>Pro Ser 2265</td>

<td>Val</td><td>Phe</td><td>Asn</td><td>Arg Glu 2270</td><td>Ser</td><td>Glu</td><td>Thr</td><td>Thr Ala 2275</td><td>Ser</td><td>Leu</td><td>Val</td><td>Ser Arg 2280</td>

<td>Ser</td><td>Gly</td><td>Ala</td><td>Glu Arg 2285</td><td>Ser</td><td>Pro</td><td>Val</td><td>Ile Gin 2290</td><td>Thr</td><td>Leu</td><td>A3p</td><td>Val Ser 2295</td>

<td>Ser</td><td>Ser</td><td>Glu</td><td>Pro Asp 2300</td><td>Thr</td><td>Thr</td><td>Ala</td><td>Ser Trp 2305</td><td>Val</td><td>Ile</td><td>His</td><td>Pro Ala 2310</td>

<td>Glu</td><td>Thr</td><td>Ile</td><td>Pro Thr 2315</td><td>Val</td><td>Ser</td><td>Lys</td><td>Thr Thr 2320</td><td>Pro</td><td>ASn</td><td>Phe</td><td>Phe His 2325</td>

<td>Ser</td><td>Glu</td><td>Leu</td><td>Asp Thr 2330</td><td>Val</td><td>Ser</td><td>Ser</td><td>Thr Ala 2335</td><td>Thr</td><td>Set</td><td>His</td><td>Gly Ala 2340</td>

252

<td>Asp</td><td>Ual</td><td>Ser</td><td>Ser Ala 2345</td><td>Ile</td><td>Pro</td><td>Thr</td><td>Asn Ile 2350</td><td>Ser</td><td>Pro</td><td>Ser</td><td>Glu Leu 2355</td>

<td>Asp</td><td>Ala</td><td>Leu</td><td>Thr Pro 2360</td><td>Leu</td><td>Val</td><td>T,hr</td><td>Ile Ser 2365</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Thr Ser 2370</td>

<td>Thr</td><td>Thr</td><td>Phe</td><td>Pro Thr 2375</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Ser Pro 2380</td><td>His</td><td>Glu</td><td>Thr</td><td>Glu Thr 23Θ5</td>

<td>Arg</td><td>Thr</td><td>Thr</td><td>Trp Leu 2390</td><td>Thr</td><td>His</td><td>Pro</td><td>Ala Siu 2395</td><td>Thr</td><td>Ser</td><td>Ser</td><td>Thr Ile ' 2400</td>

<td>Pro</td><td>Arg</td><td>Thr</td><td>Ile Pro 2405</td><td>Asn</td><td>Phe</td><td>Ser</td><td>His His 2410</td><td>Glu</td><td>Ser</td><td>Asp</td><td>Ala Thr 2415</td>

<td>Pro</td><td>Ser</td><td>Ile</td><td>Ala Thr 2420</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Ala Glu 2425</td><td>Thr</td><td>Ser</td><td>Ser</td><td>Ala Ile 2430</td>

<td>Pro</td><td>Ile</td><td>Met</td><td>Thr Val 2435</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Ala Glu 2440</td><td>Asp</td><td>Leu</td><td>Val</td><td>Thr Ser 2445</td>

<td>Gin</td><td>Val</td><td>Thr</td><td>Ser Ser 2450</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Arg Asn 2455</td><td>Met</td><td>Thr</td><td>Ile</td><td>Pro Thr 2460</td>

<td>Leu</td><td>Thr</td><td>Leu</td><td>Ser Pro 2465</td><td>Gly</td><td>Glu</td><td>pro</td><td>Lys Thr 2470</td><td>Ile</td><td>Ala</td><td>Ser</td><td>Leu Val 2475</td>

<td>Thr</td><td>His</td><td>Pro</td><td>Glu Ala 2480</td><td>Gin</td><td>Thr</td><td>Ser</td><td>Ser Ala 2485</td><td>Ile</td><td>Pro</td><td>Thr</td><td>Ser Thr 24 90</td>

<td>Ile</td><td>Ser</td><td>Pro</td><td>Ala Val 2495</td><td>Ser</td><td>Arg</td><td>Leu</td><td>Val Thr 2500</td><td>Ser</td><td>Met</td><td>Ual</td><td>Thr Ser 2505</td>

<td>Leu</td><td>fila</td><td>Ala</td><td>Ly3 Thr 2510</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Asn Arg 2515</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Asn Ser 2520</td>

<td>Pro</td><td>Gly</td><td>Glu</td><td>Pro Ala 2525</td><td>Thr</td><td>Thr</td><td>Ual</td><td>Ser Leu 2530</td><td>Ual</td><td>Thr</td><td>His</td><td>Ser Ala 2535</td>

<td>Gin</td><td>Thr</td><td>Ser</td><td>Pro Thr 2540</td><td>Ual</td><td>Pro</td><td>Trp</td><td>Thr Thr 254 5</td><td>Ser</td><td>Tle</td><td>Phe</td><td>Phe His 2550</td>

<td>Ser</td><td>Lys</td><td>Ser</td><td>Asp Thr 2555</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Met Thr 2560</td><td>Thr</td><td>Ser</td><td>His</td><td>Gly Ala 2565</td>

<td>Glu</td><td>Ser</td><td>Ser</td><td>Ser Ala 2570</td><td>val</td><td>Pro</td><td>Thr</td><td>Pro Thr 2575</td><td>Val</td><td>Ser</td><td>Thr</td><td>Glu Ual 2580</td>

<td>Pro</td><td>Gly</td><td>Ual</td><td>Ual Thr 2535</td><td>Pro</td><td>Leu</td><td>Ual</td><td>Thr Ser 2590</td><td>Ser</td><td>Arg</td><td>Ala</td><td>Val Ile 2595</td>

<td>Ser</td><td>Thr</td><td>Thr</td><td>Ile Pro 2600</td><td>Ile</td><td>Leu</td><td>Thr</td><td>Leu Ser 2605</td><td>Pro</td><td>Gly</td><td>GlU</td><td>Pro Glu 2610</td>

<td>Thr</td><td>Thr</td><td>Pro</td><td>Ser Met 2615</td><td>Ala</td><td>Thr</td><td>Ser</td><td>His Gly 2620</td><td>Glu</td><td>Glu</td><td>Ala</td><td>Ser Ser 2625</td>

<td>Ala</td><td>Ile</td><td>Pro</td><td>Thr Pro 2630</td><td>Thr</td><td>Ual</td><td>Ser</td><td>Pro Gly 2635</td><td>Val</td><td>Pro</td><td>Gly</td><td>Ual Ual 2640</td>

<td>Thr</td><td>Ser</td><td>Leu</td><td>Ual Thr</td><td>Ser</td><td>Ser</td><td>Arg</td><td>Ala Ual</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Thr Ile</td>

253

<td colspan="2"></td><td colspan="3"> 2645</td><td colspan="3"> 2650</td><td colspan="4"> 2655</td>

<td>Pro</td><td>Ile</td><td>Leu</td><td>Thr Phe 2660</td><td>Ser</td><td>Leu</td><td>Gly</td><td>Glu Pro 2665</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Pro Ser 2670</td>

<td>Met</td><td>Ala</td><td>Thr</td><td>Ser His 2675</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Ala Gly 2600</td><td>Ser</td><td>Ala</td><td>Val</td><td>Pro Thr 2685</td>

<td>Val</td><td>Leu</td><td>Pro</td><td>Glu Val 2690</td><td>Pro</td><td>Gly</td><td>Met</td><td>Ual Thr 2695</td><td>Ser</td><td>Leu</td><td>Val</td><td>Ala Set 2700</td>

<td>Ser</td><td>Arg</td><td>Ala</td><td>Ual Thr 2705</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Leu Pro 2710</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu Ser £715</td>

<td>Pro</td><td>Gly</td><td>Glu</td><td>Pro Glu 2720</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Ser Met 2725</td><td>Ala</td><td>Thr</td><td>Ser</td><td>His Gly 2730</td>

<td>Ala</td><td>Glu</td><td>Ala</td><td>Ser Ser 2735</td><td>Thr</td><td>Val</td><td>Pro</td><td>Thr Val 2740</td><td>Ser</td><td>Pro</td><td>Glu</td><td>Ual Pro 2745</td>

<td>Gly</td><td>Ual</td><td>Val</td><td>Thr Ser 2750</td><td>Leu</td><td>Ual</td><td>Thr</td><td>Ser Ser 2755</td><td>Ser</td><td>Gly</td><td>Ual</td><td>Asn Ser 2760</td>

<td>Thr</td><td>Ser</td><td>Tle</td><td>Pro Thr 2765</td><td>Leu</td><td>Ile</td><td>Leu</td><td>Ser Pro 2770</td><td>Gly</td><td>Glu</td><td>Leu</td><td>Glu Thr 2775</td>

<td>Thr</td><td>Pro</td><td>Ser</td><td>Met Ala 2780</td><td>Thr</td><td>Ser</td><td>His</td><td>Gly Ala 2785</td><td>Glu</td><td>Ala</td><td>Ser</td><td>Ser Ala 2790</td>

<td>vai</td><td>Pro</td><td>Thr</td><td>Pro Thr 2795</td><td>Val</td><td>Ser</td><td>Pro</td><td>Gly Val 2800</td><td>Ser</td><td>Gly</td><td>Uel</td><td>Ual Thr 2805</td>

<td>Pro</td><td>Leu</td><td>Val</td><td>Thr Ser 2810</td><td>Ser</td><td>Arg</td><td>Ala</td><td>Val Thr 2815</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Ile Pro 2820</td>

<td>Ile</td><td>Leu</td><td>Thr</td><td>Leu Ser 2825</td><td>Ser</td><td>Ser</td><td>Glu</td><td>Pro Glu 2830</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Sar Met 2835</td>

<td>Ala</td><td>Thr</td><td>Ser</td><td>His Gly 2840</td><td>Ual</td><td>Glu</td><td>Ala</td><td>Ser Ser 2845</td><td>Ala</td><td>Val</td><td>Leu</td><td>Thr Val 2850</td>

<td>Ser</td><td>Pro</td><td>Glu</td><td>Val Pro 2855</td><td>Gly</td><td>Met</td><td>val</td><td>Thr Phe 28 60</td><td>Leu</td><td>Ual</td><td>Thr</td><td>Ser Ser 2865</td>

<td>Arg</td><td>Ale</td><td>val</td><td>Thr Ser 2870</td><td>Thr</td><td>Thr</td><td>Ile</td><td>Pro Thr 2875</td><td>Leu</td><td>Thr</td><td>Ile</td><td>Ser Ser 2880</td>

<td>Asp</td><td>Glu</td><td>Pro</td><td>Glu Thr 2885</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Leu Ual 2890</td><td>Thr</td><td>His</td><td>Ser</td><td>Glu Ala 2895</td>

<td>Lys</td><td>Met</td><td>Ile</td><td>Ser Ala 2900</td><td>Ile</td><td>Pro</td><td>Thr</td><td>Leu Gly 2905</td><td>Val</td><td>Ser</td><td>Pro</td><td>Thr Val 2910</td>

<td>Gin</td><td>Gly</td><td>Leu</td><td>Ual Thr 2915</td><td>Ser</td><td>Leu</td><td>Ual</td><td>Thr Ser 2920</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Glu Thr 2925</td>

<td>Ser</td><td>Ala</td><td>Phe</td><td>Ser Asn 2930</td><td>Leu</td><td>Thr</td><td>Vai</td><td>Ala Ser 2935</td><td>Ser</td><td>Gin</td><td>Pro</td><td>Glu Thr 2940</td>

<td>Ile</td><td>Asp</td><td>Sar</td><td>Trp Val 2945</td><td>Ala</td><td>His</td><td>Pro</td><td>Gly Thr 2950</td><td>Glu</td><td>Ala</td><td>Ser</td><td>Ser Val 2955</td>

254

<td>Val</td><td>Pro</td><td>Thr</td><td>Leu Thr 2960</td><td>Val</td><td>Ser</td><td>Thr</td><td>Gly Glu 2965</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Asn Ile 2970</td>

<td>ser</td><td>Leu</td><td>Val</td><td>Thr &gt;His 2975</td><td>Pro</td><td>Ala</td><td>Glu</td><td>Ser Ser 2980</td><td>Ser</td><td>Thr</td><td>Leu</td><td>Pro Arg 2 935</td>

<td>Thr</td><td>Thr</td><td>Ser</td><td>Arg Phe 2990</td><td>Ser</td><td>His</td><td>Ser</td><td>Glu Leu 2995</td><td>Asp</td><td>Thr</td><td>Met</td><td>Pro Ser 3000</td>

<td>Thr</td><td>Val</td><td>Thr</td><td>Ser Pro 3005</td><td>Glu</td><td>Ala</td><td>Glu</td><td>Ser Ser 3010</td><td>Ser</td><td>Ala</td><td>Ile</td><td>Ser Thr 3015</td>

<td>Thr</td><td>Ile</td><td>Ser</td><td>Pro Gly 3020</td><td>Ile</td><td>Pro</td><td>Gly</td><td>Val Leu 3025</td><td>Thr</td><td>Ser</td><td>Leu</td><td>Val Thr 3030</td>

<td>Ser</td><td>Ser</td><td>Gly</td><td>Arg Asp 3035</td><td>Ile</td><td>Ser</td><td>Ala</td><td>Thr Phe 3040</td><td>Pro</td><td>Thr</td><td>Val</td><td>Pro Glu 3045</td>

<td>Ser</td><td>Pro</td><td>His</td><td>Glu Ser 3050</td><td>Glu</td><td>Ala</td><td>Thr</td><td>Ala Ser 3055</td><td>Trp</td><td>Val</td><td>Thr</td><td>His Pro 3060</td>

<td>Ala</td><td>Val</td><td>Thr</td><td>Ser Thr 3065</td><td>Thr</td><td>Val</td><td>Pro</td><td>Arg Thr 3070</td><td>Thr</td><td>Pro</td><td>Asn</td><td>Tyr Ser 3075</td>

<td>His</td><td>Ser</td><td>Glu</td><td>Pro Asp 30B0</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Ser Ile 3085</td><td>Ala</td><td>Thr</td><td>Ser</td><td>Pro Gly 3090</td>

<td>Ala</td><td>Glu</td><td>Ala</td><td>Thr Ser 3095</td><td>Asp</td><td>Phe</td><td>Pro</td><td>Thr Ile 3100</td><td>Thr</td><td>Val</td><td>Ser</td><td>Pro Asp 3105</td>

<td>Val</td><td>Pro</td><td>Asp</td><td>Met Val 3110</td><td>Thr</td><td>Ser</td><td>Gin</td><td>Val Thr 3115</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Thr Asp 3120</td>

<td>Thr</td><td>Ser</td><td>Ile</td><td>Thr Ile 3125</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Thr Leu 3130</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Glu Pra 3135</td>

<td>Glu</td><td>Thr</td><td>Thr</td><td>Thr Ser 3140</td><td>Phe</td><td>Ile</td><td>Thr</td><td>Tyr Ser 3145</td><td>Glu</td><td>Thr</td><td>His</td><td>Thr Ser 3150</td>

<td>Ser</td><td>Ala</td><td>Ile</td><td>Pro Thr 3155</td><td>Leu</td><td>Pro</td><td>Val</td><td>Ser Pro 3160</td><td>Asp</td><td>Ala</td><td>Ser</td><td>Lys Met 3165</td>

<td>Leu</td><td>Thr</td><td>Ser</td><td>Leu Val 3170</td><td>Ile</td><td>Ser</td><td>Ser</td><td>Gly Thr 3175</td><td>Asp</td><td>Ser</td><td>Thr</td><td>Thr Thr 3180</td>

<td>Phe</td><td>Pro</td><td>Thr</td><td>Leu Thr 31B5</td><td>Glu</td><td>Thr</td><td>Pro</td><td>Tyr Glu 3190</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Thr Ala 3195</td>

<td>Ile</td><td>Gin</td><td>Leu</td><td>Ile His 3200</td><td>Pro</td><td>Ala</td><td>Glu</td><td>Thr Asn 3205</td><td>Thr</td><td>Met</td><td>Ual</td><td>Pro Arg 3210</td>

<td>Thr</td><td>Thr</td><td>Pro</td><td>Lys Phe 3215</td><td>Ser</td><td>His</td><td>Ser</td><td>Lys Ser 3220</td><td>Asp</td><td>Thr</td><td>Thr</td><td>Leu Pro 3225</td>

<td>Val</td><td>Alg</td><td>Ile</td><td>Thr Ser 3230</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Glu Ala 3235</td><td>Ser</td><td>Ser</td><td>Ala</td><td>Ual Ser 3240</td>

<td>Thr</td><td>Thr</td><td>Thr</td><td>Ile Ser 3245</td><td>Pro</td><td>Asp</td><td>Met</td><td>ser Asp 3250</td><td>Leu</td><td>Ual</td><td>Thr</td><td>Ser Leu 3255</td>

255

<td>Val</td><td>Pro</td><td>Ser</td><td>Ser Gly 3260</td><td>Thr</td><td>Asp</td><td>Thr</td><td>Ser Thr 3265</td><td>Thr</td><td>Phe</td><td>Pro</td><td>Thr Leu 3270</td>

<td>Ser</td><td>Glu</td><td>Thr</td><td>Pro Tyr 3275</td><td>Glu</td><td>Pro</td><td>Glu</td><td>Thr Thr 32B0</td><td>Ala</td><td>Thr</td><td>Trp</td><td>Leu Thr 3295</td>

<td>His</td><td>Pro</td><td>Ala</td><td>Glu Thr 3290</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Val Ser 3295</td><td>Gly</td><td>Thr</td><td>Ile</td><td>Pro Asn 3300</td>

<td>Phe</td><td>Ser</td><td>His</td><td>Arg Gly 3305</td><td>Ser</td><td>Asp</td><td>Thr</td><td>Ala Pro 3310</td><td>Ser</td><td>Met</td><td>Val</td><td>Thr Ser 3315</td>

<td>Pro</td><td>Gly</td><td>Val</td><td>Asp Thr 3320</td><td>Arg</td><td>Ser</td><td>Gly</td><td>Val pro 3325</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Ile Pro 3330</td>

<td>Pro</td><td>Ser</td><td>Ile</td><td>Pro Gly 3335</td><td>Val</td><td>Val</td><td>Thr</td><td>Ser Gin 3340</td><td>Val</td><td>Thr</td><td>Ser</td><td>Ser Ala 3345</td>

<td>Thr</td><td>Asp</td><td>Thr</td><td>Ser Thr 3350</td><td>Ala</td><td>Ile</td><td>Pro</td><td>Thr Leu 3355</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Pro Gly 3360</td>

<td>Glu</td><td>Pro</td><td>Glu</td><td>Thr Thr 3365</td><td>Ala</td><td>Ser</td><td>Ser</td><td>Ala Thr 3370</td><td>His</td><td>Pro</td><td>Gly</td><td>Thr Gin 3375</td>

<td>Thr</td><td>Gly</td><td>Phe</td><td>Thr Oal 3330</td><td>Pro</td><td>Ile</td><td>Arg</td><td>Thr vai 3305</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Glu Pro 3390</td>

<td>Asp</td><td>Thr</td><td>Met</td><td>Ala Ser 3395</td><td>Trp</td><td>val</td><td>Thr</td><td>His Pro 3400</td><td>Pro</td><td>Gin</td><td>Thr</td><td>Ser Thr 3405</td>

<td>Pro</td><td>Val</td><td>Ser</td><td>Arg Thr 3410</td><td>Thr</td><td>Ser</td><td>Ser</td><td>Phe Ser 3415</td><td>His</td><td>Ser</td><td>Ser</td><td>Pro Asp 3420</td>

<td>Ala</td><td>Thr</td><td>Pro</td><td>Val Met 3425</td><td>Ala</td><td>Thr</td><td>Ser</td><td>Pro Arg 3430</td><td>Thr</td><td>Glu</td><td>Ala</td><td>Ser Ser 34 35</td>

<td>Ala</td><td>Val</td><td>Leu</td><td>Thr Thr 3440</td><td>Ile</td><td>Ser</td><td>Pro</td><td>Gly Ala 3445</td><td>Pro</td><td>Glu</td><td>Met</td><td>vai Thr 3450</td>

<td>Ser</td><td>Gin</td><td>Ile</td><td>Thr Ser 3455</td><td>Ser</td><td>Gly</td><td>Ala</td><td>Ala Thr 3460</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Val Pro 34 65</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>His Ser 3470</td><td>Pro</td><td>Gly</td><td>Met</td><td>Pro Glu 3475</td><td>Thr</td><td>Thr</td><td>Ala</td><td>Leu Leu 3490</td>

<td>Ser</td><td>Thr</td><td>His</td><td>Pro Arg 3485</td><td>Thr</td><td>Glu</td><td>Thr</td><td>Ser Lys 3490</td><td>Thr</td><td>Phe</td><td>Pro</td><td>Ala Ser 34 95</td>

<td>Thr</td><td>Val</td><td>Phe</td><td>Pro Gin 3500</td><td>Val</td><td>Ser</td><td>Glu</td><td>Thr Thr 3505</td><td>Ala</td><td>Ser</td><td>Leu</td><td>Thr Ile 3510</td>

<td>Arg</td><td>Pro</td><td>Gly</td><td>Ala Glu 3515</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Ala Leu 3520</td><td>Pro</td><td>Thr</td><td>Gin</td><td>Thr Thr 3525</td>

<td>Ser</td><td>Ser</td><td>Leu</td><td>Phe Thr 3530</td><td>Leu</td><td>Leu</td><td>Val</td><td>Thr Gly 3535</td><td>Thr</td><td>Ser</td><td>Arg</td><td>Val Asp 3540</td>

<td>Leu</td><td>Ser</td><td>Pro</td><td>Thr Ala 3545</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Val Ser 3550</td><td>Ala</td><td>Lys</td><td>Thr</td><td>Ala Pro 3555</td>

<td>Leu</td><td>Ser</td><td>Thr</td><td>Hi3 Pro</td><td>Gly</td><td>Thr</td><td>GlU</td><td>Thr Ser</td><td>Thr</td><td>Met</td><td>Ile</td><td>Pro Thr</td>

256

<td colspan="3"></td><td> 3560</td><td colspan="5"> 3565</td><td colspan="3"> 3570</td>

<td>Ser</td><td>Thr</td><td>Leu</td><td>Ser Leu 3575</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Glu Thr 3580</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Leu Ala 3505</td>

<td>Thr</td><td>Ser</td><td>Ser</td><td>Ser Ala 3590</td><td>Glu</td><td>Thr</td><td>Ser</td><td>Thr Ser 3595</td><td>Thr</td><td>Leu</td><td>Thr</td><td>leu Thr 3600</td>

<td>Val</td><td>Ser</td><td>Pro</td><td>Ala Val 3605</td><td>Ser</td><td>Gly</td><td>Leu</td><td>Ser Ser 3610</td><td>Ala</td><td>Ser</td><td>Ile</td><td>Thr Thr 3615</td>

<td>Asp</td><td>Łys</td><td>Pro</td><td>Gin Thr 3620</td><td>Val</td><td>Thr</td><td>Ser</td><td>Trp Asn 3625</td><td>Thr</td><td>Glu</td><td>Thr</td><td>Ser Pro 3630</td>

<td>Ser</td><td>Val</td><td>Thr</td><td>Ser Val 3635</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Glu Phe 3S40</td><td>Ser</td><td>Arg</td><td>Thr</td><td>Val Thr 3645</td>

<td>Gly</td><td>Thr</td><td>Thr</td><td>Met Thr 3650</td><td>Leu</td><td>lla</td><td>Pro</td><td>Ser Glu 3655</td><td>Met</td><td>Pro</td><td>Thr</td><td>Pro Pro 3660</td>

<td>Lys</td><td>Thr</td><td>Ser</td><td>His Gly 3665</td><td>Glu</td><td>Gly</td><td>Val</td><td>Ser Pro 3670</td><td>Thr</td><td>Thr</td><td>Ile</td><td>Leu Arg 3675</td>

<td>rhr</td><td>Thr</td><td>Met</td><td>Vol Glu 3680</td><td>Ala</td><td>Thr</td><td>Asn</td><td>Leu Ala 3685</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Ser Ser 3690</td>

<td>Pro</td><td>Thr</td><td>val</td><td>Ala Lys 3695</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Thr Phe 3700</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Ala Gly 3705</td>

<td>Ser</td><td>Leu</td><td>Phe</td><td>Thr Pro 3710</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Pro Gly 3715</td><td>Met</td><td>Ser</td><td>Thr</td><td>Leu Ala 3720</td>

<td>Ser</td><td>Glu</td><td>Ser</td><td>Val Thr 3725</td><td>Ser</td><td>Arg</td><td>Thr</td><td>Ser Tyr 3730</td><td>Asn</td><td>His</td><td>Arg</td><td>Ser Trp 3735</td>

<td>Ile</td><td>Ser</td><td>Thr</td><td>Thr Ser 3740</td><td>Ser</td><td>Tyr</td><td>Asn</td><td>Arg Arg 3745</td><td>Tyr</td><td>Trp</td><td>Thr</td><td>Pro Ala 3750</td>

<td>Thr</td><td>Ser</td><td>Thr</td><td>Pro Val 3755</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Phe Ser 3760</td><td>Pro</td><td>Gly</td><td>Ile</td><td>Ser Thr 3765</td>

<td>Sar</td><td>Sar</td><td>Ile</td><td>Pro Ser 3770</td><td>Ser</td><td>Thr</td><td>Ala</td><td>Ala Thr 3775</td><td>val</td><td>Pro</td><td>Phe</td><td>Met Val 3780</td>

<td>Pro</td><td>Phe</td><td>Thr</td><td>Leu Asn 3785</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Thr Asn 3730</td><td>Leu</td><td>Gin</td><td>Tyr</td><td>Glu Glu 37 95</td>

<td>Asp</td><td>Met</td><td>Arg</td><td>His Pro 3300</td><td>Gly</td><td>Ser</td><td>Arg</td><td>Lys Phe 3805</td><td>Asn</td><td>Ala</td><td>Thr</td><td>Glu Arg 3810</td>

<td>Glu</td><td>Leu</td><td>Gin</td><td>Gly Leu 3815</td><td>Leu</td><td>Lys</td><td>Pro</td><td>Leu Phe 3020</td><td>Arg</td><td>Asn</td><td>Ser</td><td>Ser Leu 3825</td>

<td>Glu</td><td>Tyr</td><td>Leu</td><td>Tyr Ser 3830</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Leu Ala 3835</td><td>Ser</td><td>Leu</td><td>Arg</td><td>Pro Glu 3340</td>

<td>Lya</td><td>Asp</td><td>Sar</td><td>Ser ALa 3845</td><td>Thr</td><td>Ala</td><td>Val</td><td>Asp Ala 3050</td><td>Ile</td><td>Cys</td><td>Thr</td><td>His Arg 38 55</td>

<td>Pro</td><td>Asp</td><td>Pro</td><td>Glu Asp 3860</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Asp Arg 3865</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Tyr Trp 3870</td>

257

<td>Glu</td><td>Leu</td><td>Ser Asn Leu 3875</td><td>Thr Asn</td><td>Gly</td><td>Ile Gln Glu 3830</td><td>Leu Gly</td><td>Pro Tyr 3085</td>

<td>Thr</td><td>Leu</td><td>Asp Arg Asn 3890</td><td>Ser Leu</td><td>Tyr</td><td>Val Asn Gly 3895</td><td>Phe Thr</td><td>His Arg 3900</td>

<td>Ser</td><td>Ser</td><td>Met Pro Thr 3905</td><td>Thr Ser</td><td>Thr</td><td>Pro Gly Thr 3910</td><td>Ser Thr</td><td>Val Asp 3915</td>

<td>Val</td><td>Gly</td><td>Thr Ser Gly 3920</td><td>Thr Pro</td><td>Ser</td><td>Ser Ser Pro 3925</td><td>Ser Pro</td><td>Thr Thr 3930</td>

<td>Ala</td><td>Gly</td><td>Pro Leu Leu 3935</td><td>Met Pro</td><td>Phe</td><td>Thr Leu Asn 3940</td><td>Phe Thr</td><td>Ile Thr 3945</td>

<td>Asn</td><td>Leu</td><td>Gln Tyr Glu 3950</td><td>Glu Asp</td><td>Met</td><td>Arg Arg Thr 3955</td><td>Gly Ser</td><td>Arg Lys 3960</td>

<td>Phe</td><td>Asn</td><td>Thr Met Glu 3955</td><td>Ser Val</td><td>Leu</td><td>Gln Gly Leu 3970</td><td>Leu Lys</td><td>Pro Leu 3975</td>

<td>Phe</td><td>Lys</td><td>Asn Thr Ser 3980</td><td>Val Gly</td><td>Pro</td><td>Leu Tyr Ser 3985</td><td>Gly Cys</td><td>Arg Leu 3990</td>

<td>Thr</td><td>Leu</td><td>Leu Arg Pro 3995</td><td>Glu Lys</td><td>Asp</td><td>Gly Ala Ala 4000</td><td>Thr Gly</td><td>Val Asp 4005</td>

<td>Ala</td><td>Ile</td><td>Cys Thr His 4010</td><td>Arg Leu</td><td>Asp</td><td>Pro Lys Ser 4015</td><td>Pro Gly</td><td>Leu Asn 4020</td>

<td>Arg</td><td>Glu</td><td>Gln Leu Tyr 4025</td><td>Trp Glu</td><td>Leu</td><td>Ser Lys Leu 4030</td><td>Thr Asn</td><td>Asp He 4 035</td>

<td>Glu</td><td>Glu</td><td>Leu Gly Pro 4040</td><td>Tyr Thr</td><td>Leu</td><td>Asp Arg Asn 4045</td><td>Ser Leu</td><td>Tyr vai 4 050</td>

<td>Asn</td><td>Gly</td><td>Phe Thr His 4055</td><td>Gln Ser</td><td>Ser</td><td>Val Ser Thr 4060</td><td>The Ser</td><td>Thr Pro 4065</td>

<td>Gly</td><td>Thr</td><td>Ser Thr Val 4070</td><td>Asp Leu</td><td>Arg</td><td>Thr Ser Gly 4075</td><td>Thr Pro</td><td>Ser Ser 4080</td>

<td>Leu</td><td>Ser</td><td>Ser Pro Thr 4085</td><td>Ile Met</td><td>Ala</td><td>Ala Gly Pro 4 090</td><td>Leu Leu</td><td>Val Pro 4095</td>

<td>Phe</td><td>Thr</td><td>Leu Asn phe 4100</td><td>Thr Ile</td><td>Thr</td><td>Asn Leu Gln 4105</td><td>Tyr Gly</td><td>Glu Asp 4110</td>

<td>Met</td><td>Gly</td><td>His Pro Gly 4115</td><td>Ser Arg</td><td>Lys</td><td>Phe Asn Thr 4120</td><td>Thr Glu</td><td>Arg Val 4125</td>

<td>Leu</td><td>Gln</td><td>Gly Leu Leu ' 4130</td><td>Gly Pro</td><td>Ile</td><td>Phe Lys Asn 4135</td><td>Thr Ser</td><td>Val Gly 4140</td>

<td>Pro</td><td>Leu</td><td>Tyr Ser Gly 4145</td><td>Cys Arg</td><td>Leu</td><td>Thr Ser Leu 4150</td><td>Arg Ser</td><td>Glu Lys 4155</td>

<td>Asp</td><td>Gly</td><td>Ala Ala Thr 4160</td><td>Gly Val</td><td>Asp</td><td>Ala Ile Cys 4165</td><td>Ile Hi$</td><td>His Leu 4170</td>

258

<td>Asp</td><td>Pro</td><td>Lys</td><td>Ser Pro 4175</td><td>Gly</td><td>Leu</td><td>Asn</td><td>Arg Glu 4100</td><td>Arg</td><td>Leu</td><td>Tyr</td><td>Trp Glu 4105</td>

<td>Leu</td><td>Ser</td><td>Gin</td><td>Leu Thr 4190</td><td>Asn</td><td>Gly</td><td>ile</td><td>Lys Glu 4195</td><td>Leu</td><td>Gly</td><td>Pro</td><td>Tyr Thr 4 200</td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Asn Ser 4205</td><td>Leu</td><td>Tyr</td><td>Val</td><td>Asn Gly 4210</td><td>Phe</td><td>Thr</td><td>His</td><td>Arg Thr 4215</td>

<td>Sec</td><td>Ual</td><td>Pro</td><td>Thr Thr 4220</td><td>Ser</td><td>Thr</td><td>Pro</td><td>Gly Thr 4225</td><td>Ser</td><td>Thr</td><td>Val</td><td>Asp Leu 4230</td>

<td>Gly</td><td>Thr</td><td>Ser</td><td>Gly Thr 4235</td><td>Pro</td><td>Phe</td><td>Ser</td><td>Leu Pro 4240</td><td>Ser</td><td>Pro</td><td>Ala</td><td>Thr Ala 4245</td>

<td>Gly</td><td>Pro</td><td>Leu</td><td>Leu Ual 4250</td><td>Leu</td><td>Phe</td><td>Thr</td><td>Leu Asn 4255</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Thr Asn 4260</td>

<td>Leu</td><td>Lys</td><td>Tyr</td><td>Glu Glu 4265</td><td>Asp</td><td>Met</td><td>His</td><td>Arg Pro 4270</td><td>Gly</td><td>Ser</td><td>Arg</td><td>Lys Phe 4275</td>

<td>Asn</td><td>Thr</td><td>Thr</td><td>Glu Arg 4230</td><td>vai</td><td>Leu</td><td>Gin</td><td>Thr Leu 4235</td><td>val</td><td>Gly</td><td>Pro</td><td>Met Phe 4290</td>

<td>Lys</td><td>Asn</td><td>Thr</td><td>Ser Val 4295</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Tyr Ser 4300</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Leu Thr 4305</td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Ser Glu 4310</td><td>Lys</td><td>Asp</td><td>Gly</td><td>Ala Ala 4315</td><td>Thr</td><td>Gly</td><td>Ual</td><td>Asp Ala 4320</td>

<td>Ile</td><td>Cy3</td><td>Thr</td><td>His Arg 4325</td><td>Leu</td><td>Asp</td><td>Pro</td><td>Lya Ser 4330</td><td>Pro</td><td>Gly</td><td>Ual</td><td>Asp Arg 4335</td>

<td>GlU</td><td>Gin</td><td>LeU</td><td>Tyr Trp 4340</td><td>Glu</td><td>Leu</td><td>Ser</td><td>Gin Leu 4345</td><td>Thr</td><td>Asm</td><td>Gly</td><td>Ile Lys 4350</td>

<td>Glu</td><td>Leu</td><td>Gly</td><td>Pro Tyr 4355</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Arg Asn 4360</td><td>Ser</td><td>Leu</td><td>Tyr</td><td>Val Asn 4365</td>

<td>Gly</td><td>Phe</td><td>Thr</td><td>His Trp 4370</td><td>Ile</td><td>Pro</td><td>Val</td><td>Pro Thr 4375</td><td>Ser</td><td>Ser</td><td>Thr</td><td>Pro Gly 4380</td>

<td>Thr</td><td>Ser</td><td>Thr</td><td>Ual Asp 4395</td><td>Leu</td><td>Gly</td><td>Ser</td><td>Gly Thr 4390</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Leu Pro 4395</td>

<td>Ser</td><td>Pro</td><td>Thr</td><td>Ser Ala 4400</td><td>Thr</td><td>Ala</td><td>Gly</td><td>Pro Leu 4405</td><td>Leu</td><td>Ual</td><td>Pro</td><td>Phe Thr 4410</td>

<td>Leu</td><td>Asn</td><td>phe</td><td>Thr Ile 4415</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Lys Tyr 4420</td><td>Glu</td><td>Glu</td><td>Asp</td><td>Met His 4425</td>

<td>Cys</td><td>Pro</td><td>Gly</td><td>Ser Arg 4430</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Thr Thr 4435</td><td>Glu</td><td>Arg</td><td>Val</td><td>Leu Gin 4440</td>

<td>Ser</td><td>Leu</td><td>Leu</td><td>Gly Pro 4445</td><td>Met</td><td>Phe</td><td>Lys</td><td>Asn Thr 4450</td><td>Ser</td><td>Val</td><td>Gly</td><td>Pro Leu 4455</td>

<td>Tyr</td><td>Sar</td><td>Gly</td><td>Cys Arg 4460</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Leu Arg 4465</td><td>Ser</td><td>Glu</td><td>Lys</td><td>Asp Gly 4470</td>

<td>Ala</td><td>Ala</td><td>Thr</td><td>Gly Val</td><td>Asp</td><td>Ala</td><td>Ile</td><td>Cya Thr</td><td>His</td><td>Arg</td><td>Leu</td><td>Asp Pro</td>

259

<td></td><td></td><td></td><td> 4475</td><td></td><td></td><td></td><td> 4480</td><td></td><td></td><td></td><td> 4405</td>

<td>Lys</td><td>Ser</td><td>Pro</td><td>Gly Val 4490</td><td>Asp</td><td>Arg</td><td>Glu</td><td>Gin Leu 4495</td><td>Tyr</td><td>Trp</td><td>Glu</td><td>Leu Ser 4500</td>

<td>Gin</td><td>Leu</td><td>Thr</td><td>Asn Gly 4505</td><td>Ile</td><td>Lys</td><td>Glu</td><td>Leu Gly 4510</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Leu Asp 4515</td>

<td>Arg</td><td>Asn</td><td>Ser</td><td>Leu Tyr 4520</td><td>Ual</td><td>Asn</td><td>Gly</td><td>Phe Thr 4525</td><td>His</td><td>Gin</td><td>Thr</td><td>Ser Ala 4530</td>

<td>Pro</td><td>Asn</td><td>Thr</td><td>Ser Thr 4535</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Ser Thr 4540</td><td>Ual</td><td>Asp</td><td>Leu</td><td>Gly Thr 4545</td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>Pro Ser 4550</td><td>Ser</td><td>Leu</td><td>Pro</td><td>Ser Pro 4555</td><td>Thr</td><td>Ser</td><td>Ala</td><td>Gly Pro 4560</td>

<td>Leu</td><td>Leu</td><td>Val</td><td>Pro Phe 4565</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Phe Thr 4570</td><td>Ila</td><td>Thr</td><td>Asn</td><td>Leu Gin 4575</td>

<td>Tyr</td><td>Glu</td><td>Glu</td><td>Asp Met 4580</td><td>His</td><td>His</td><td>Pro</td><td>Gly Ser 4585</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Asn Thr 4590</td>

<td>Thr</td><td>Glu</td><td>Arg</td><td>Val Leu 4595</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Leu Gly 4600</td><td>Pro</td><td>Met</td><td>Phe</td><td>Lys Asn 4605</td>

<td>Thr</td><td>Ser</td><td>Val</td><td>Gly Leu 4610</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Gly Cys 4615</td><td>Arg</td><td>Leu</td><td>Thr</td><td>Leu Leu 4620</td>

<td>Arg</td><td>Pro</td><td>Glu</td><td>Lys Asn 4625</td><td>Gly</td><td>Ala</td><td>Ala</td><td>Thr Gly 4630</td><td>Met</td><td>Asp</td><td>Ala</td><td>He Cys 4635</td>

<td>Ser</td><td>His</td><td>Arg</td><td>Leu Asp 4 64 0</td><td>Pro</td><td>Lys</td><td>Ser</td><td>Pro Gly 4645</td><td>Leu</td><td>Asn</td><td>Arg</td><td>Glu Gin 4650</td>

<td>Leu</td><td>Tyr</td><td>Trp</td><td>Glu Leu 4 655</td><td>Ser</td><td>Gin</td><td>Leu</td><td>Thr His 4660</td><td>Gly</td><td>Ile</td><td>Lys</td><td>Glu Leu 4665</td>

<td>Gly</td><td>Pro</td><td>Tyr</td><td>Thr Leu 4 670</td><td>Asp</td><td>Arg</td><td>Asn</td><td>Ser Leu 4675</td><td>Tyr</td><td>Ual</td><td>Asn</td><td>Gly Phe 4 600</td>

<td>Thr</td><td>His</td><td>Arg</td><td>Ser Ser 4605</td><td>Ual</td><td>Ala</td><td>Pro</td><td>Thr Ser 4 690</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Thr Ser 4695</td>

<td>Thr</td><td>Ual</td><td>Asp</td><td>Leu Gly 4 700</td><td>Thr</td><td>Ser</td><td>Gly</td><td>Thr Pro 4705</td><td>Ser</td><td>Ser</td><td>Leu</td><td>Pro Ser 4710</td>

<td>Pro</td><td>Thr</td><td>Thr</td><td>Ala Vał 4715</td><td>Pro</td><td>Leu</td><td>Leu</td><td>Val Pro 4720</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Asn Phe 4725</td>

<td>Thr</td><td>Ile</td><td>Thr</td><td>Asn Leu 4730</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Glu Asp 4735</td><td>Met</td><td>Arg</td><td>His</td><td>Pro Gly ' 4740</td>

<td>Ser</td><td>Arg</td><td>Lys</td><td>Phe Asn 4745</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Arg Val 4750</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Leu Leu 4755</td>

<td>Gly</td><td>Pro</td><td>Leu</td><td>Phe Lys 4760</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Ual Gly 4755</td><td>Pro</td><td>Leu</td><td>Tyr</td><td>Ser Gly 4 770</td>

<td>Cys</td><td>Arg</td><td>Leu</td><td>Ile Ser 4775</td><td>Leu</td><td>Arg</td><td>Ser</td><td>Glu Lys 47Q0</td><td>Asp</td><td>Gly</td><td>Ala</td><td>Ala Thr 4785</td>

260

<td>Gly</td><td>Val</td><td>Asp</td><td>Ala Ile 4790</td><td>Cys</td><td>Thr</td><td>His</td><td>His Leu 4795</td><td>ASn</td><td>Pro</td><td>Gin</td><td>Ser Pro 4300</td>

<td>Gly</td><td>Leu</td><td>Asp</td><td>Arg Glu 4805</td><td>Gin</td><td>Leu</td><td>Tyr</td><td>Trp Glh 4310</td><td>Leu</td><td>Ser</td><td>Gin</td><td>Met Thr 4315</td>

<td>Asn</td><td>Gly</td><td>Ile</td><td>Lys Glu 4820</td><td>Leu</td><td>Gly</td><td>Pro</td><td>Tyr Thr 4325</td><td>Leu</td><td>Asp</td><td></td><td>Asn Ser 4330</td>

<td>Leu</td><td>Tyr</td><td>val</td><td>Asn Gly 4835</td><td>Phe</td><td>Thr</td><td>His</td><td>Arg Ser 4340</td><td>Ser</td><td>Gly</td><td>Leu</td><td>Thr Thr 4345</td>

<td>Ser</td><td>Thr</td><td>Pro</td><td>Trp Thr 4B50</td><td>Ser</td><td>Thr</td><td>Val</td><td>Asp Leu 4355</td><td>Gly</td><td>Thr</td><td>Ser</td><td>Gly Thr 4860</td>

<td>Pro</td><td>Ser</td><td>Pro</td><td>Val Pro 4865</td><td>Ser</td><td>Pro</td><td>Thr</td><td>Thr Ala 4370</td><td>Gly</td><td>Pro</td><td>Leu</td><td>Leu Val 4375</td>

<td>Pro</td><td>Phe</td><td>Thr</td><td>Leu Asn 4880</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Thr Asn 4eas</td><td>Leu</td><td>Gin</td><td>Tyr</td><td>Glu Glu 4390</td>

<td>Asp</td><td>Met</td><td>His</td><td>Arg Pro 4S95</td><td>Gly</td><td>Ser</td><td>Arg</td><td>Lys Phe 4900</td><td>Asn.</td><td>Ala</td><td>Thr</td><td>Glu Arg 4905</td>

<td>Val</td><td>Leu</td><td>Gin</td><td>Gly Leu 4910</td><td>Leu</td><td>Ser</td><td>Pro</td><td>tle Phe 4915</td><td>Lys</td><td>Asn</td><td>Ser</td><td>Ser Val 4920</td>

<td>Gly</td><td>Pro</td><td>Leu</td><td>Tyr Ser 4925</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Leu Thr 4930</td><td>Ser</td><td>Leu</td><td>Arg</td><td>Pro Glu 4935</td>

<td>Lys</td><td>Asp</td><td>Gly</td><td>Ala Ala 4940</td><td>Thr</td><td>Gly</td><td>Met</td><td>Asp Ala 4945</td><td>Val</td><td>Cys</td><td>Leu</td><td>Tyr His 4950</td>

<td>Pro</td><td>Asn</td><td>Pro</td><td>Lys Arg 4955</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Asp Arg 4960</td><td>Glu</td><td>Gin</td><td>Leu</td><td>Tyr Trp 4965</td>

<td>Glu</td><td>Leu</td><td>Ser</td><td>Gin Leu 4970</td><td>Thr</td><td>His</td><td>Asn</td><td>Ile Thr 4975</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Pro Tyr 4980</td>

<td>Ser</td><td>Leu</td><td>Asp</td><td>Arg Asp 4985</td><td>Ser</td><td>Leu</td><td>Tyr</td><td>Val Asn 4990</td><td>Gly</td><td>Phe</td><td>Thr</td><td>His Gin 4995</td>

<td>Asn</td><td>Ser</td><td>Val</td><td>Pro Thr 5000</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Pro Gly .5005</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Va 1 Tyr 5010</td>

<td>Trp</td><td>Ala</td><td>Thr</td><td>Thr Gly 5015</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Ser Phe 5020</td><td>Pro</td><td>Gly</td><td>His</td><td>Thr Glu 5025</td>

<td>Pro</td><td>Gly</td><td>Pro</td><td>Leu Leu 5030</td><td>Ile</td><td>Pro</td><td>Phe</td><td>Thr Phe 5035</td><td>Asn</td><td>Phe</td><td>Thr</td><td>Ile Thr 5040</td>

<td>Asn</td><td>Leu</td><td>His</td><td>Tyr Glu 5045</td><td>Glu</td><td>Asn</td><td>Met</td><td>Gin His 5050</td><td>Pro</td><td>Gly</td><td>Ser</td><td>Arg Lys 5055</td>

<td>Phe</td><td>Asn</td><td>Thr</td><td>Thr Glu 5060</td><td>Arg</td><td>Val</td><td>Leu</td><td>Gin Gly 5065</td><td>Leu</td><td>Leu</td><td>Lys</td><td>Pro Leu 5070</td>

<td>Phe</td><td>Lys</td><td>Asn</td><td>Thr Ser 5075</td><td>vai</td><td>Gly</td><td>Pro</td><td>Leu Tyr 5030</td><td>Ser</td><td>Gly</td><td>Cys</td><td>Arg Leu 5035</td>

261

<td>Thr</td><td>Leu</td><td>Leu</td><td>Arg Pro 5090</td><td>Glu</td><td>Lys</td><td>Gin</td><td>Glu Ala 5095</td><td>Ala</td><td>Thr</td><td>Gly</td><td>Val Asp 5100</td>

<td>Thr</td><td>Ile</td><td>Cys</td><td>Thr His 5105</td><td>Arg</td><td>Ual</td><td>Asp</td><td>Pro Ile 5110</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Leu Asp 5115</td>

<td>Arg</td><td>Glu</td><td>Arg</td><td>Leu Tyr 5120</td><td>Trp</td><td>Glu</td><td>Leu</td><td>Ser Gin 5125</td><td>LeU</td><td>Thr</td><td>Asn</td><td>Ser Ile 5130</td>

<td>Thr</td><td>Glu</td><td>Leu</td><td>Gly Pro 5135</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Asp Arg 5140</td><td>Asp</td><td>ser</td><td>Leu</td><td>Tyr Ual 5145</td>

<td>Asn</td><td>Gly</td><td>Phe</td><td>Asn Pro 5150</td><td>Trp</td><td>Ser</td><td>Ser</td><td>Ual Pro 5155</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Thr Pro 5160</td>

<td>Gly</td><td>Thr</td><td>Ser</td><td>Thr Ual 5165</td><td>His</td><td>Leu</td><td>Ala</td><td>Ttir Ser 5170</td><td>Gly</td><td>Thr</td><td>Pro</td><td>Ser Ser 5175</td>

<td>Leu</td><td>Pro</td><td>Gly</td><td>His Thr 5180</td><td>%la</td><td>Pro</td><td>Ual</td><td>Pro Leu 5135</td><td>Leu</td><td>Ile</td><td>Pro</td><td>Phe Thr 5190</td>

<td>Leu</td><td>Asn</td><td>Phe</td><td>Thr Ile 5195</td><td>Thr</td><td>Asn</td><td>Leu</td><td>His Tyr 5200</td><td>Glu</td><td>Glu</td><td>Asn</td><td>Met Gin 5205</td>

<td>His</td><td>Pro</td><td>Gly</td><td>Ser Arg S210</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Thr Thr 5215</td><td>Glu</td><td>Arg</td><td>Val</td><td>Leu Gin 5220</td>

<td>Gly</td><td>Leu</td><td>Leu</td><td>Lys Pro 5225</td><td>Leu</td><td>Phe</td><td>Lys</td><td>Ser Thr 5230</td><td>Ser</td><td>Val</td><td>Gly</td><td>Pro Leu 5235</td>

<td>Tyr</td><td>Sar</td><td>Gly</td><td>Cys Arg 5240</td><td>leu</td><td>Thr</td><td>Leu</td><td>Leń Arg 5245</td><td>Pro</td><td>Glu</td><td>Lys</td><td>His Gly 5250</td>

<td>Ala</td><td>Ala</td><td>Thr</td><td>Gly Ual 5255</td><td>Asp</td><td>Ala</td><td>Ile</td><td>Cy3 Thr 5260</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Asp Pro 5265</td>

<td>Thr</td><td>Gly</td><td>Pro</td><td>Gly Leu 5270</td><td>Asp</td><td>Arg</td><td>Glu</td><td>Arg Leu 5275</td><td>Tyr</td><td>Trp</td><td>Glu</td><td>Leu Ser 5280</td>

<td>Gin</td><td>Leu</td><td>Thr</td><td>Asn Ser 5205</td><td>Val</td><td>Thr</td><td>Glu</td><td>Leu Gly 5290</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Leu Asp 5295</td>

<td>Arg</td><td>Asp</td><td>Ser</td><td>Leu Tyr 5300</td><td>Val</td><td>Asn</td><td>Gly</td><td>Phe Thr 5305</td><td>Hi a</td><td>Arg</td><td>Ser</td><td>Ser Ual 5310</td>

<td>Pro</td><td>Ttir</td><td>Thr</td><td>Ser Ile 5315</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Ser Ala 5320</td><td>Ual</td><td>His</td><td>Leu</td><td>Glu Thr 5325</td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>Pro Ala 5330</td><td>Ser</td><td>Leu</td><td>Pro</td><td>Gly His 5335</td><td>Thr</td><td>Ala</td><td>Pro</td><td>Gly Pro S34O</td>

<td>Leu</td><td>Leu</td><td>Val</td><td>Pro Phe 5345</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Phe Thr 5350</td><td>Ile</td><td>Thr</td><td>Asn</td><td>Leu Gin 5355</td>

<td>Tyr</td><td>Glu</td><td>Glu</td><td>Asp Met 5360</td><td>Arg</td><td>His</td><td>Pro</td><td>Gly Ser 5365</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Asn Thr 5370</td>

<td>Thr</td><td>Glu</td><td>Arg</td><td>Val Leu 5375</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Leu Lys 5330</td><td>Pro</td><td>Leu</td><td>Phe</td><td>Lys Ser 5385</td>

<td>Thr</td><td>Ser</td><td>Ual</td><td>Gly pro</td><td>Leu</td><td>Tyr</td><td>Sar</td><td>Gly Cys</td><td>Arg</td><td>Leu</td><td>Thr</td><td>Leu Leu</td>

262

<td colspan="2"></td><td colspan="3"> 5390</td><td colspan="5"> 6395</td><td colspan="2"> 5400</td>

<td>Arg</td><td>Pro</td><td>Glu</td><td>Lys Arg 5405</td><td>Gly</td><td>Ala</td><td>Ala</td><td>Thr Gly 5410</td><td>Val</td><td>Asp</td><td>Thr</td><td>ILe Cys 5415</td>

<td>Thr</td><td>His</td><td>Arg</td><td>Leu Asp 5420</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Pro Gly 5425</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Glu Gin 5430</td>

<td>LeU</td><td>Tyr</td><td>Trp</td><td>Glu Leu 5435</td><td>Ser</td><td>Lys</td><td>Leu</td><td>Thr Arg 5440</td><td>Gly</td><td>Ile</td><td>Ile</td><td>GlU Leu 5445</td>

<td>Gly</td><td>Pro</td><td>Tyr</td><td>Leu Leu 5450</td><td>Asp</td><td>Arg</td><td>Gly</td><td>Ser Leu 5455</td><td>Tyr</td><td>Val</td><td>Asn</td><td>Gly Phe 5460</td>

<td>Thr</td><td>His</td><td>Arg</td><td>Asn Phe 5465</td><td>Vai</td><td>Pro</td><td>Ile</td><td>Thr Ser 5470</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Thr Ser 5475</td>

<td>Thr</td><td>Val</td><td>His</td><td>Leu Gly 5400</td><td>Thr</td><td>Ser</td><td>Glu</td><td>Thr Pro 5485</td><td>Ser</td><td>Ser</td><td>Leu</td><td>Pro Arg 5490</td>

<td>Pro</td><td>Ile</td><td>Val</td><td>Pro Gly 5495</td><td>Pro</td><td>Leu</td><td>Leu</td><td>Val Pro 5500</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Asn Phe 5505</td>

<td>Thr</td><td>Ile</td><td>Thr</td><td>Asn Leu 5510</td><td>Gin</td><td>Tyr</td><td>GlU</td><td>Glu Ala 5515</td><td>Met</td><td>Arg</td><td>His</td><td>Pro Gly 5520</td>

<td>Ser</td><td>Arg</td><td>Lys</td><td>Phe Asn 5525</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Arg Val 5530</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Leu Leu 5535</td>

<td>Arg</td><td>Pro</td><td>Leu</td><td>Phe Lys 5540</td><td>Asn</td><td>Thr</td><td>Ser</td><td>Ile Gly 5545</td><td>Pro</td><td>Leu</td><td>Tyr</td><td>Ser Ser 5550</td>

<td>Cys</td><td>Arg</td><td>Leu</td><td>Thr Leu 5555</td><td>Leu</td><td>Arg</td><td>Pro</td><td>Glu Lys 5560</td><td>Asp</td><td>Lys</td><td>Ala</td><td>Ala Thr 5565</td>

<td>Arg</td><td>Val</td><td>Asp</td><td>Ala Ile 5570</td><td>Cys</td><td>Thr</td><td>Hia</td><td>His Pro 5575</td><td>Asp</td><td>Pro</td><td>Gin</td><td>Ser Pro 5580</td>

<td>Gly</td><td>Leu</td><td>Asn</td><td>Arg Glu 5585</td><td>Gin</td><td>Leu</td><td>Tyr</td><td>Trp Glu 5590</td><td>Leu</td><td>Ser</td><td>Gin</td><td>Leu Thr 5595</td>

<td>His</td><td>Gly</td><td>Ile</td><td>Thr Glu 5600</td><td>Leu</td><td>Gly</td><td>Pro</td><td>Tyr Thr 5605</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Asp Ser 5610</td>

<td>Leu</td><td>Tyr</td><td>Val</td><td>Asp Gly 5615</td><td>Phe</td><td>Thr</td><td>His</td><td>Trp Ser 5620</td><td>Pro</td><td>Ile</td><td>Pro</td><td>Thr Thr 5625</td>

<td>Ser</td><td>Thr</td><td>Pro</td><td>Gly Thr 5630</td><td>Ser</td><td>Ile</td><td>Val</td><td>Asn Leu 5635</td><td>Gly</td><td>Thr</td><td>Ser</td><td>Gly Ile 5640</td>

<td>Pro</td><td>Pro</td><td>Ser</td><td>Leu Pro 5645</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Ala Thr 5650</td><td>Gly</td><td>Pro</td><td>Leu</td><td>Leu Val 5655</td>

<td>Pro</td><td>Phe</td><td>Thr</td><td>Leu Asn 5660</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Thr Asn 5665</td><td>Leu</td><td>Gin</td><td>Tyr</td><td>Glu Glu 5670</td>

<td>Asn</td><td>Met</td><td>Gly</td><td>His Pro 5675</td><td>Gly</td><td>Ser</td><td>Arg</td><td>Lys Phe 5600</td><td>Αξπ</td><td>Ile</td><td>Thr</td><td>Glu Ser 5685</td>

<td>Val</td><td>Leu</td><td>Gin</td><td>Gly Leu 5690</td><td>Leu</td><td>Lys</td><td>Pro</td><td>Leu Phe 5695</td><td>Lys</td><td>Ser</td><td>Thr</td><td>Ser Val 5700</td>

263

<td>Gly</td><td>Pro</td><td>Leu</td><td>Tyr Ser 5705</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Leu Thr 5710</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Pro Glu 5715</td>

<td>Lys</td><td>Asp</td><td>Gly</td><td>Val Ala 5720</td><td>Thr</td><td>Arg</td><td>Val</td><td>Asp Ala 5725</td><td>Ile</td><td>Cys</td><td>Thr</td><td>His Arg . 5730</td>

<td>Pro</td><td>Asp</td><td>Pro</td><td>Lys Ile 5735</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Asp Arg 5740</td><td>Gin</td><td>Giń</td><td>Leu</td><td>Tyr Trp 5745</td>

<td>Glu</td><td>Leu</td><td>Ser</td><td>Gin Leu 5750</td><td>Thr</td><td>His</td><td>Ser</td><td>Ile Thr 5755</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Pro Tyr 5760</td>

<td>Thr</td><td>Leu</td><td>Asp</td><td>Arq Asp 5765</td><td>Ser</td><td>Leu</td><td>Tyr</td><td>Val Asn 5770</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Gin Arg 5775</td>

<td>Ser</td><td>Ser</td><td>Val</td><td>Pro Thr 5780</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Pro Gly 5785</td><td>Thr</td><td>Phe</td><td>Thr</td><td>Val Gin 5790</td>

<td>Pro</td><td>Glu</td><td>Thr</td><td>Ser Glu 5795</td><td>Thr</td><td>Pro</td><td>Ser</td><td>Ser Leu 5800</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Thr Ala 5805</td>

<td>Thr</td><td>Gly</td><td>Pro</td><td>Val Leu 5310</td><td>Leu</td><td>Pro</td><td>Phe</td><td>Thr Leu 5815</td><td>Asn</td><td>Phe</td><td>Thr</td><td>Ile Ile 5820</td>

<td>Asn</td><td>Leu</td><td>Gin</td><td>Tyr Glu 5825</td><td>Glu</td><td>Asp</td><td>Met</td><td>His Arg 5830</td><td>Pro</td><td>Gly</td><td>Ser</td><td>Arg Lys 5835</td>

<td>Phe</td><td>Asn</td><td>Thr</td><td>Thr Glu 5840</td><td>Arg</td><td>vai</td><td>Leu</td><td>Gin Gly 5845</td><td>Leu</td><td>Leu</td><td>Met</td><td>Pro Leu 5850</td>

<td>Phe</td><td>LyS</td><td>Asn</td><td>Thr Ser 5855</td><td>Val</td><td>Ser</td><td>Ser</td><td>Leu Tyr 5860</td><td>Ser</td><td>Gly</td><td>Cys</td><td>Arg Leu 5865</td>

<td>Thr</td><td>Leu</td><td>Leu</td><td>Arg Pro 5870</td><td>Glu</td><td>Lys</td><td>Asp</td><td>Gly Ala 5875</td><td>Ala</td><td>Thr</td><td>Arg</td><td>Val Asp 5880</td>

<td>Ala</td><td>Val</td><td>Cys</td><td>Thr His 58B5</td><td>Arg</td><td>Pro</td><td>Asp</td><td>Pro Lys 5890</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Leu Asp 5895</td>

<td>Arg</td><td>GLu</td><td>Arg</td><td>Leu Tyr 5900</td><td>Trp</td><td>Lys</td><td>Leu</td><td>Ser Gin 5905</td><td>Leu</td><td>Thr</td><td>His</td><td>Gly Ile 5910</td>

<td>Thr</td><td>Glu</td><td>Leu</td><td>Gly Pro 5915</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Asp Arg 5920</td><td>His</td><td>Ser</td><td>Leu</td><td>Tyr vai 5925</td>

<td>Asn</td><td>Gly</td><td>Phe</td><td>Thr His 5930</td><td>Gin</td><td>Ser</td><td>Ser</td><td>Met Thr 5935</td><td>Thr</td><td>Thr</td><td>Arg</td><td>Thr Pro 5940</td>

<td>Asp</td><td>Thr</td><td>Ser</td><td>Thr Met 5945</td><td>His</td><td>Leu</td><td>Ala</td><td>Thr Ser 5950</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Ala Ser 5955</td>

<td>Leu</td><td>Ser</td><td>Gly</td><td>Pro Thr 5960</td><td>Thr</td><td>Ala</td><td>Ser</td><td>Pro Leu 5965</td><td>Leu</td><td>Val</td><td>Leu</td><td>Phe Thr 5970</td>

<td>Ile</td><td>Asn</td><td>Phe</td><td>Thr Ile 5975</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Arg Tvr 5980</td><td>Glu</td><td>Glu</td><td>Asn</td><td>Met His 5985</td>

<td>His</td><td>Pro</td><td>Gly</td><td>Ser Arg 5990</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Thr Thr 5995</td><td>Glu</td><td>Arg</td><td>Val</td><td>Leu Gin 6000</td>

264

<td>Gly</td><td>Leu</td><td>Leu</td><td>Arg Pro 6005</td><td>Val</td><td>Phe</td><td>Lys</td><td>Asn Thr 6010</td><td>Ser</td><td>Val</td><td>Gly</td><td>Pro Leu 6015</td>

<td>Tyr</td><td>Ser</td><td>Gly</td><td>Cys Arg 6020</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Leu Arg 6025</td><td>Pro</td><td>Lys</td><td>Lys</td><td>Asp Gly 6030</td>

<td>Ala</td><td>Ala</td><td>Thr</td><td>Lys Val 6035</td><td>Asp</td><td>Ala</td><td>Ile</td><td>Cys Thr 6040</td><td>Tyr</td><td>Arg</td><td>Pro</td><td>Asp Pro 6045</td>

<td>Lys</td><td>Ser</td><td>Pro</td><td>Gly Leu 6050</td><td>Aap</td><td>Arg</td><td>Glu</td><td>Gin Leu 6055</td><td>Tyr</td><td>Trp</td><td>Glu</td><td>Leu Ser 6060</td>

<td>Gin</td><td>Leu</td><td>Thr</td><td>His Ser 6065</td><td>Ile</td><td>Thr</td><td>Glu</td><td>Leu Gly 6070</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Leu Asp 6075</td>

<td>Arg</td><td>Asp</td><td>Ser</td><td>Leu Tyr 6080</td><td>Val</td><td>Asn</td><td>Gly</td><td>Phe Thr 6085</td><td>Gin</td><td>Arg</td><td>Ser</td><td>Ser Val 6090</td>

<td>Pro</td><td>Thr</td><td>Thr</td><td>Ser Ile 6095</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Pro Thr 6100</td><td>Val</td><td>Asp</td><td>Leu</td><td>Gly Thr 6105</td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>Pro Val 6110</td><td>Ser</td><td>Lys</td><td>Pro</td><td>Gly Pro 6115</td><td>Ser</td><td>Ala</td><td>Ala</td><td>Ser Pro 6120</td>

<td>Leu</td><td>Leu</td><td>Val</td><td>Leu Phe 6125</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Ehe Thr 6130</td><td>Ile</td><td>Thr</td><td>Asn</td><td>Leu Arg 6135</td>

<td>Tyr</td><td>Glu</td><td>Glu</td><td>Asn Met 6140</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly Ser 6145</td><td>Arg</td><td>Lya</td><td>Phe</td><td>Asn Thr 6150</td>

<td>Thr</td><td>Glu</td><td>Arg</td><td>Ual Leu 6155</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Leu Arg 6160</td><td>Ser</td><td>Leu</td><td>Phe</td><td>Lys Ser 6165</td>

<td>Thr</td><td>Ser</td><td>Val</td><td>Gly Pro 6170</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Gly Cys 6175</td><td>Arg</td><td>Leu</td><td>Thr</td><td>Leu Leu 6180</td>

<td>Arg</td><td>Pro</td><td>Glu</td><td>Ly3 Asp 6105</td><td>Gly</td><td>Thr</td><td>Ala</td><td>Thr Gly 6190</td><td>Val</td><td>Asp</td><td>Ala</td><td>Ile Cys 6195</td>

<td>Thr</td><td>His</td><td>His</td><td>Pro Asp 6200</td><td>Pro</td><td>Lys</td><td>Ser</td><td>Pro Arg 6205</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Glu Gin 6210</td>

<td>Leu</td><td>Tyr</td><td>Trp</td><td>Glu Leu 6215</td><td>Ser</td><td>Gin</td><td>Leu</td><td>Thr His 6220</td><td>Asn</td><td>Ile</td><td>Thr</td><td>Glu Leu 6225</td>

<td>Gly</td><td>Pro</td><td>Tyr</td><td>Ala Leu 6230</td><td>Asp</td><td>Asn</td><td>Asp</td><td>Ser Leu 6235</td><td>Phe</td><td>Val</td><td>Asn</td><td>Gly Phe 6240</td>

<td>Thr</td><td>His</td><td>Arg</td><td>Ser Ser 6245</td><td>Val</td><td>Ser</td><td>Thr</td><td>Thr Ser 6250</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Thr Pro 6255</td>

<td>Thr</td><td>Val</td><td>Tyr</td><td>Leu Gly 6260</td><td>Ala</td><td>Ser</td><td>Lys</td><td>Thr Pro 6265</td><td>Ala</td><td>Ser</td><td>Ile</td><td>Phe Gly 6270</td>

<td>Pro</td><td>Ser</td><td>Ala</td><td>Ala Ser 6275</td><td>His</td><td>Leu</td><td>Leu</td><td>Ile Leu 6230</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Asn Phe 6205</td>

<td>Thr</td><td>Ile</td><td>Thr</td><td>Asn Leu 6290</td><td>Arg</td><td>Tyr</td><td>Glu</td><td>Glu Asn 6295</td><td>Met</td><td>Trp</td><td>Pro</td><td>Gly Ser 6300</td>

<td>Arg</td><td>Lys</td><td>Phe</td><td>Asn Thr</td><td>Thr</td><td>Glu</td><td>Arg</td><td>Ual Leu</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Leu Arg</td>

265

6305 6310 6315

<td>Pro</td><td>Leu</td><td>Phe</td><td>Lys Asn 6320</td><td>Thr</td><td>Ser</td><td>Val</td><td>Gly Pro 6325</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Gly Cys 6330</td>

<td>Arg</td><td>Leu</td><td>Thr</td><td>Leu Leu 6335</td><td>Arg</td><td>Pro</td><td>Glu</td><td>Lys Asp 6340</td><td>Gly</td><td>Glu</td><td>Ala</td><td>Thr Gly 634 5</td>

<td>Val</td><td>Asp</td><td>Ala</td><td>Ile Cys 6350</td><td>Thr</td><td>His</td><td>Arg</td><td>Pro Asp 6355</td><td>Pro</td><td>Thr</td><td>Gly</td><td>Pro Gly 6360</td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Glu Gln 6365</td><td>Leu</td><td>Tyr</td><td>Leu</td><td>Glu Leu 6370</td><td>Ser</td><td>Gln</td><td>Leu</td><td>Thr His 6375</td>

<td>Ser</td><td>Ile</td><td>Thr</td><td>Glu Leu 6380</td><td>Gly</td><td>Pro</td><td>Tyr</td><td>Thr Leu 6305</td><td>Asp</td><td>Arg</td><td>Asp</td><td>Ser Leu 6390</td>

<td>Tyr</td><td>Val</td><td>Asn</td><td>Gly Phe 6395</td><td>Thr</td><td>His</td><td>Arg</td><td>Ser Ser 64 00</td><td>Val</td><td>Pro</td><td>Thr</td><td>Thr Ser 6405</td>

<td>Thr</td><td>Gly</td><td>Val</td><td>Val Ser 6410</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Phe Thr 6415</td><td>Leu</td><td>Asn</td><td>Phe</td><td>Thr Ile 5420</td>

<td>Asn</td><td>Asn</td><td>Leu</td><td>Arg Tyr 6425</td><td>Met</td><td>Ala</td><td>Asp</td><td>Met Gly 6430</td><td>Gln</td><td>Pro</td><td>Gly</td><td>Ser Leu 6435</td>

<td>Lys</td><td>Phe</td><td>Asn</td><td>Ile Thr 6440</td><td>Asp</td><td>Asn</td><td>Val</td><td>Met Gln 6445</td><td>His</td><td>Leu</td><td>Leu</td><td>Ser Pro 6450</td>

<td>Leu</td><td>Phe</td><td>Gln</td><td>Arg Ser 6455</td><td>Ser</td><td>Leu</td><td>Gly</td><td>Ala Arg 6460</td><td>Tyr</td><td>Thr</td><td>Gly</td><td>Cys Arg 6465</td>

<td>Val</td><td>Ile</td><td>Ala</td><td>Leu Arg 6470</td><td>Ser</td><td>Val</td><td>Lys</td><td>Asn Gly 6475</td><td>Ala</td><td>Glu</td><td>Thr</td><td>Arg Val 6400</td>

<td>Asp</td><td>Leu</td><td>Leu</td><td>Cys Thr 6485</td><td>Tyr</td><td>Leu</td><td>Gln</td><td>Pro Leu 64 90</td><td>Ser</td><td>Gly</td><td>Pro</td><td>Gly Leu 6495</td>

<td>Pro</td><td>Ile</td><td>Lys</td><td>Gln val 6500</td><td>Phe</td><td>His</td><td>Glu</td><td>Leu Ser 6505</td><td>Gln</td><td>Gln</td><td>Thr</td><td>His Gly 6510</td>

<td>Ile</td><td>Thr</td><td>Arg</td><td>Leu Gly 6515</td><td>Pro</td><td>Tyr</td><td>Ser</td><td>Leu Asp 6520</td><td>Lys</td><td>Asp</td><td>Ser</td><td>Leu Tyr 6525</td>

<td>Leu</td><td>Asn</td><td>Gly</td><td>Tyr Asm 6530</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Pro Asp 6535</td><td>Glu</td><td>Pro</td><td>Pro</td><td>Thr Thr 6540</td>

<td>Pro</td><td>Lys</td><td>Pro</td><td>Ala Thr 6545</td><td>Thr</td><td>Phe</td><td>Leu</td><td>Pro Pro 6550</td><td>Leu</td><td>Ser</td><td>Glu</td><td>Ala Thr 6555</td>

<td>Thr</td><td>Ala</td><td>Met</td><td>Gly Tyr 6560</td><td>His</td><td>Leu</td><td>Lys</td><td>Thr Leu 6565</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Phe Thr 557 0</td>

<td>Ile</td><td>Ser</td><td>Asn</td><td>Leu Gln 6575</td><td>Tyr</td><td>Ser</td><td>Pro</td><td>Asp Met 6530</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Ser Ala 6585</td>

<td>Thr</td><td>Phe</td><td>Asn</td><td>Ser Thr 6590</td><td>Glu</td><td>Gly</td><td>Val</td><td>Leu Gln 6595</td><td>His</td><td>Leu</td><td>Leu</td><td>Arg Pro 6600</td>

<td>Leu</td><td>Phe</td><td>Gln</td><td>Lys Ser</td><td>Ser</td><td>Met</td><td>Gly</td><td>Pm phe</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>Cys Gln</td>

6605 6510 6615

266

<td>Leu</td><td>Ile</td><td>Ser</td><td>Leu Arg 6S20</td><td>Pro</td><td>Glu</td><td>Lys</td><td>Asp Gly 6625</td><td>Ala</td><td>Ala</td><td>Thr</td><td>Gly Val 6630</td>

<td>Asp</td><td>Thr</td><td>Thr</td><td>Cys Thr 6635</td><td>Tyr</td><td>His</td><td>Pro</td><td>Asp Pro 6640</td><td>Val</td><td>Gly</td><td>Pro</td><td>Gly Leu 6645</td>

<td>Asp</td><td>Ile</td><td>Gin</td><td>Gin Leu 6S5O</td><td>Tyr</td><td>Trp</td><td>Glu</td><td>Leu Ser 6655</td><td>Gin</td><td>Leu</td><td>Thr</td><td>His Gly 6660</td>

<td>Val</td><td>Thr</td><td>Gin</td><td>Leu Gly 6665</td><td>Phe</td><td>Tyr</td><td>Val</td><td>Leu Asp 6670</td><td>Arg</td><td>Asp</td><td>Ser</td><td>Leu Phe 6675</td>

<td>Ile</td><td>Asn</td><td>Gly</td><td>Tyr Ala 6630</td><td>Pro</td><td>Gin</td><td>Asn</td><td>Leu Ser 66Θ5</td><td>He</td><td>Arg</td><td>Gly</td><td>Glu Tyr 6690</td>

<td>Gin</td><td>Ile</td><td>Asn</td><td>Phe His 6695</td><td>Ile</td><td>Val</td><td>Asn</td><td>Trp Asn 6700</td><td>Leu</td><td>Ser</td><td>Asn</td><td>Pro Asp 6705</td>

<td>Pro</td><td>Thr</td><td>Ser</td><td>Ser Glu 6710</td><td>Tyr</td><td>Ile</td><td>Thr</td><td>Leu Leu 6715</td><td>Arg</td><td>Asp</td><td>Ile</td><td>Gin Asp 6720</td>

<td>Lys</td><td>Val</td><td>Thr</td><td>Thr Leu 6725</td><td>Tyr</td><td>Lys</td><td>Gly</td><td>Ser Gin 6730</td><td>Leu</td><td>His</td><td>Asp</td><td>Thr Phe 6735</td>

<td>Arg</td><td>Phe</td><td>Cys</td><td>Leu Val 6740</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Thr Met 6745</td><td>Asp</td><td>Ser</td><td>Val</td><td>Leu Val 6750</td>

<td>Thr</td><td>Val</td><td>Lys</td><td>Ala Leu 6755</td><td>Phe</td><td>Ser</td><td>Ser</td><td>Asn Leu 6760</td><td>Asp</td><td>Pro</td><td>Ser</td><td>Leu Va 1 6765</td>