Macropinocytic human anti-CD46 antibodies and targeted cancer therapies

Abstract

The present application relates to macropinocytic human anti-CD46 antibodies and targeted cancer therapies. In various embodiments, human anti-CD46 antibodies that are internalized via the macropinocytic pathway and enter tumor cells, as well as antibody-drug conjugates ADCs developed from these antibodies for diagnostic and / or therapeutic targeting of CD46 overexpressing tumors are provided.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 10, 2015, application number 201580061138.5, and invention name “Macropinocytic human anti-CD46 antibodies and targeted cancer therapies”.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of and priority to USSN 62 / 049,973, filed September 12, 2014, which is incorporated herein by reference in its entirety for all purposes.

[0004] Statement Regarding Rights to Inventions Made Under Federally Sponsored Research and Development

[0005] This research was supported in part by the National Institutes of Health Grant Nos. R01 CA118919, R01 CA129491, and R01 CA171315. The government has certain rights in this invention. Technical Field

[0006] This application generally relates to macropinocytic human anti-CD46 antibodies and targeted cancer therapies. Background Art

[0007] Tumor cell surface antigens are valuable targets for therapeutic development due to their ease of access.The epitope space on the cell surface is highly complex. Relevant antigens may include glycosylated proteins and other post-translational modification products that are not easily predicted from genomic copy number or mRNA expression levels (Liu et al. (2004) Cancer Res. 64:704-710; Kobata and Amano (2005) Immunol. Cell Biol. 83:429-439; Birkle et al. (2003) Biochimie (Paris) 85:455-463; Hakomori (2001) Adv. Exp. Med. Biol. 491:369-402; Hanisch, FG (2001) O-Glycosylation of the mucin-type mucintype.) Biol. Chem. 382, ​​143-149; Ugorski and Laskowska (2002) Acta Biochim. Pol. 49: 303-311).

[0008] Identification of tumor cell surface epitopes allows the production of antibodies that achieve specific binding to neoplastic cells, a capability that can be used, for example, to induce antibody-dependent cellular cytotoxicity (see, e.g., Clynes et al. (2000) Nat. Med. 6:443-446) or inhibit signaling pathways involved in tumor cell migration, growth, and survival (see, e.g., McWhirter et al. (2006) Proc. Natl. Acad. Sci., USA, 103:1041-1046; Fuh et al. (2006) J. Biol. Chem. 281:6625-6631). In addition, antibodies targeted to internalizing tumor epitopes can be used to achieve efficient and specific intracellular delivery of cytotoxins, cytostatics, chemotherapeutic drugs and / or other tumor modulating agents (see, e.g., Liu et al. (2004) Cancer Res. 64:704-710; Nielsen et al. (2002) Biochim. Biophys. Acta 1591:109-118; Pirollo et al. (2006) Hum. Gene Ther. 17:117-124; Song et al. (2005) Nat. Biotechnol. 23:709-717; Liu et al. (2002) J. Mol. Biol. 315:1063-1073).

[0009] Phage antibody display has been widely used to develop cancer-specific antibodies (see, e.g., Liu et al. (2004) Cancer Res. 64:704-710; Liu and Marks (2000) Anal. Biochem. 286:119-128; 15. Marks et al. (1992) Biotechnology (NY) 10:779-783; Marks et al. (1991) J. Mol. Biol. 222:581-597; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Sharon et al. (2005) J. Cell. Biochem. 96:305-313; Silacci et al. (2005) Proteomics 5:2340-2350; Gao et al. (2003) J. Immunol. Methods 274:185-197; Lekkerkerker and Logtenberg (1999) J. Immunol. Meth. 231:53-63; de Kuff ... de Kuff et al. (2003) J. Immunol. Methods 274:185-197; Lekkerkerker and Logtenberg (1999) J. Immunol. Meth. 231:53-63; de Kuff et al. (2003) J. Immunol. Methods 274:185-197; de Kuff et al. (2003) J. Immunol. Methods 274:185-197; de Kuff et al. (2003) J. Im Kruif et al. (1995) Proc. Natl. Acad. Sci., USA, 92: 3938-3942; Pini et al. (1998) J. Biol. Chem. 273: 21769-21776). Combinatorial phage antibody libraries are used as a source of randomly shaped antibody repertoires that can be used to probe neoplastic changes on the surface of cancer cells (see, e.g., Liu et al. (2004) Cancer Res. 64:704-710; Geuijen et al. (2005) Eur. J. Cancer 41:178-187; Poul et al. (2000) J. Mol. Biol. 301:1149-1161; Cai and Garen (1995) Proc. Natl. Acad. Sci., USA, 92:6537-6541).Selection of phage antibody libraries directly on cancer cell lines enables the identification of tumor-targeting antibodies without prior knowledge of the target antigen, see, e.g., (Liu et al. (2004) Cancer Res. 64:704-710; Gao et al. (2003) J. Immunol. Methods 274:185-197; Geuijen et al. (2005) Eur. J. Cancer 41:178-187; Poul et al. (2000) J. Mol. Biol. 301:1149-1161).

[0010] Although many antibodies have been discovered by this approach, the screening process against cell lines does not provide a picture of how specific these antibodies are to the actual cancer cells in the patient population. Nor does it necessarily provide an indication of whether the antibody will be internalized in vivo. Summary of the Invention

[0011] In various embodiments, human anti-CD46 antibodies that are internalized and enter tumor cells via the macropinocytosis pathway are provided, as well as antibody-drug conjugates (ADCs) developed from these antibodies for diagnostic and / or therapeutic targeting of CD46-overexpressing tumors.

[0012] Embodiments encompassed herein may include, but are not limited to, one or more of the following:

[0013] Example 1: An isolated human antibody that specifically binds to CD46 and is internalized into cells that express or overexpress CD46, wherein: the antibody is an antibody that specifically binds to cells that express or overexpress CD46, wherein the antibody specifically binds to an epitope bound by one or more antibodies selected from the group consisting of: YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY, YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ; and the antibody is internalized into the cell by macropinocytosis.

[0014] Embodiment 2: The antibody of embodiment 1, wherein said antibody binds to domain 1 and / or domain 2 of CD46.

[0015] Embodiment 3: The antibody of embodiment 1 or 2, wherein the antibody does not bind to domain 3 and / or domain 4 of CD46.

[0016] Embodiment 4: The antibody according to any one of embodiments 1 to 3, wherein the cell expressing or overexpressing CD46 is a cancer cell.

[0017] Embodiment 5: The antibody according to any one of embodiments 1 to 4, wherein the cell expressing or overexpressing CD46 is a prostate cancer cell.

[0018] Example 6: The antibody of Example 5, wherein the antibody binds to cells of a cell line selected from the group consisting of: DU145 cells, PC3 cells, and LnCaP cells.

[0019] Embodiment 7: The antibody according to any one of embodiments 1 to 6, wherein said antibody binds to prostate tumor cells with an affinity (K of at least about 5-10 nM) when measured by FACS on live prostate tumor cells. D ) binds to prostate tumor cells.

[0020] Example 8: The antibody of Example 7, wherein said antibody binds to prostate tumor cells with an affinity (K) of at least about 3 nM when measured by FACS on live prostate tumor cells. D ) binds to prostate tumor cells.

[0021] Embodiment 9: The antibody of any one of embodiments 1 to 8, wherein the antibody is a substantially intact immunoglobulin.

[0022] Embodiment 10: The antibody of embodiment 9, wherein the antibody comprises IgA, IgE or IgG.

[0023] Example 11: The antibody of Example 9, wherein the antibody comprises IgG1.

[0024] Embodiment 12: The antibody according to any one of embodiments 1 to 8, wherein the antibody is an antibody fragment that specifically binds to cells expressing or overexpressing CD46.

[0025] Embodiment 13: The antibody of embodiment 12, wherein the antibody is an antibody fragment selected from the group consisting of: Fv, Fab, (Fab')2, (Fab')3, IgGΔCH2, and minibody.

[0026] Embodiment 14: The antibody according to any one of embodiments 1 to 8, wherein the antibody is a single-chain antibody.

[0027] Example 15: The antibody of Example 14, wherein the VL region of said antibody is linked to the VH region of said antibody by an amino acid linker, wherein the length of said amino acid linker ranges from about 3 amino acids to about 15 amino acids.

[0028] Example 16: The antibody of Example 14, wherein the VL region of the antibody is connected to the VH region of the antibody via an amino acid linker selected from the group consisting of: GGGGS GGGGS GGGGS (SEQ ID NO:67), GGGGSGGGGS (SEQ ID NO:68), GGGGS (SEQ ID NO:69), GS GGGGS GGGGS GGS GGGGS (SEQ ID NO:70), SGGGGS (SEQ ID NO:71), GGGS (SEQ ID NO:72), VPGV (SEQ ID NO:73), VPGVG (SEQ ID NO:74), GVPGVG (SEQ ID NO:75), GVG VP GVG (SEQ ID NO:76), VP GVG VP GVG (SEQ ID NO:77), GGSSRSS (SEQ ID NO:78), and GGSSRSSSSGGGGSGGGG (SEQ ID NO:79).

[0029] Embodiment 17: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS5 for binding to CD46.

[0030] Embodiment 18: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS5F for binding to CD46.

[0031] Embodiment 19: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS5v1D for binding to CD46.

[0032] Embodiment 20: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with SB1 HGNY for binding to CD46.

[0033] Embodiment 21: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS12 for binding to CD46.

[0034] Embodiment 22: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with 3G7RY for binding to CD46.

[0035] Embodiment 23: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS6 for binding to CD46.

[0036] Embodiment 24: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS1 for binding to CD46.

[0037] Embodiment 25: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS3 for binding to CD46.

[0038] Embodiment 26: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS4 for binding to CD46.

[0039] Embodiment 27: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS8 for binding to CD46.

[0040] Embodiment 28: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS7 for binding to CD46.

[0041] Embodiment 29: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS9 for binding to CD46.

[0042] Embodiment 30: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS10 for binding to CD46.

[0043] Embodiment 31: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with YS11 for binding to CD46.

[0044] Embodiment 32: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with 3G7HY for binding to CD46.

[0045] Embodiment 33: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with 3G7NY for binding to CD46.

[0046] Embodiment 34: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with 3G7 for binding to CD46.

[0047] Embodiment 35: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with SB2 for binding to CD46.

[0048] Embodiment 36: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with 2C8 for binding to CD46.

[0049] Embodiment 37: The antibody according to any one of embodiments 1 to 16, wherein said antibody competes with UA8κ for binding to CD46.

[0050] Embodiment 38: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3, and / or VL CDR1, and / or VL CDR2, and / or VL CDR3 of an antibody selected from the group consisting of: YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY, YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and UA8κ.

[0051] Embodiment 39: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS5 antibody.

[0052] Embodiment 40: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS5 antibody.

[0053] Embodiment 41: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS5 antibody.

[0054] Embodiment 42: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5 antibody.

[0055] Embodiment 43: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS5 antibody.

[0056] Embodiment 44: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5 antibody and the variable heavy (VH) chain of the YS5 antibody.

[0057] Example 45: The antibody of Example 1, wherein the antibody is human YS5 scFv.

[0058] Example 46: The antibody of Example 1, wherein the antibody is human YS5 IgG.

[0059] Embodiment 47: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS5F antibody.

[0060] Embodiment 48: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS5F antibody.

[0061] Embodiment 49: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS5F antibody.

[0062] Embodiment 50: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5F antibody.

[0063] Embodiment 51: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS5F antibody.

[0064] Embodiment 52: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5F antibody and the variable heavy (VH) chain of the YS5F antibody.

[0065] Example 53: The antibody of Example 1, wherein the antibody is human YS5F scFv.

[0066] Example 54: The antibody of Example 1, wherein the antibody is human YS5F IgG.

[0067] Embodiment 55: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS5F antibody.

[0068] Embodiment 56: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS5F antibody.

[0069] Embodiment 57: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS5F antibody.

[0070] Embodiment 58: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5F antibody.

[0071] Embodiment 59: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS5F antibody.

[0072] Embodiment 60: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5F antibody and the variable heavy (VH) chain of the YS5F antibody.

[0073] Example 61. The antibody of Example 1, wherein the antibody is human YS5F scFv.

[0074] Example 62: The antibody of Example 1, wherein the antibody is human YS5F IgG.

[0075] Embodiment 63: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS5V1D antibody.

[0076] Embodiment 64: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS5V1D antibody.

[0077] Embodiment 65: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS5V1D antibody.

[0078] Embodiment 66: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5V1D antibody.

[0079] Embodiment 67: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS5V1D antibody.

[0080] Embodiment 68: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS5V1D antibody and the variable heavy (VH) chain of the YS5V1D antibody.

[0081] Example 69: The antibody of Example 1, wherein the antibody is human YS5V1D scFv.

[0082] Example 70: The antibody of Example 1, wherein the antibody is human YS5V1D IgG.

[0083] Embodiment 71: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the SB1HGNY antibody.

[0084] Embodiment 72: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the SB1HGNY antibody.

[0085] Embodiment 73: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the SB1HGNY antibody.

[0086] Embodiment 74: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the SB1 HGNY antibody.

[0087] Embodiment 75: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the SB1HGNY antibody.

[0088] Embodiment 76: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the SB1HGNY antibody and the variable heavy (VH) chain of the SB1HGNY antibody.

[0089] Example 77: The antibody of Example 1, wherein the antibody is human SB1 HGNY scFv.

[0090] Example 78: The antibody of Example 1, wherein the antibody is human SB1 HGNY IgG.

[0091] Embodiment 79: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS12 antibody.

[0092] Embodiment 80: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS12 antibody.

[0093] Embodiment 81: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS12 antibody.

[0094] Embodiment 82: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS12 antibody.

[0095] Embodiment 83: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS12 antibody.

[0096] Embodiment 84: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS12 antibody and the variable heavy (VH) chain of the YS12 antibody.

[0097] Example 85: The antibody of Example 1, wherein the antibody is human YS12 scFv.

[0098] Example 86: The antibody of Example 1, wherein the antibody is human YS12 IgG.

[0099] Embodiment 87: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the 3G7RY antibody.

[0100] Embodiment 88: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the 3G7RY antibody.

[0101] Embodiment 89: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the 3G7RY antibody.

[0102] Embodiment 90: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7RY antibody.

[0103] Embodiment 91: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the 3G7RY antibody.

[0104] Embodiment 92: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7RY antibody and the variable heavy (VH) chain of the 3G7RY antibody.

[0105] Example 93. The antibody of Example 1, wherein said antibody is human 3G7RY scFv.

[0106] Example 94. The antibody of Example 1, wherein the antibody is human 3G7RY IgG.

[0107] Embodiment 95: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS6 antibody.

[0108] Embodiment 96: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS6 antibody.

[0109] Embodiment 97: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS6 antibody.

[0110] Embodiment 98: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS6 antibody.

[0111] Embodiment 99: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS6 antibody.

[0112] Embodiment 100: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS6 antibody and the variable heavy (VH) chain of the YS6 antibody.

[0113] Example 101: The antibody of Example 1, wherein the antibody is human YS6 scFv.

[0114] Example 102: The antibody of Example 1, wherein the antibody is human YS6 IgG.

[0115] Embodiment 103: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS1 antibody.

[0116] Embodiment 104: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS1 antibody.

[0117] Embodiment 105: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS1 antibody.

[0118] Embodiment 106: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS1 antibody.

[0119] Embodiment 107: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the YS1 antibody.

[0120] Embodiment 108: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS1 antibody and the variable heavy (VH) chain of the YS1 antibody.

[0121] Embodiment 109: The antibody of Embodiment 1, wherein the antibody is human YS1 scFv.

[0122] Example 110: The antibody of Example 1, wherein the antibody is human YS1 IgG.

[0123] Embodiment 111: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS3 antibody.

[0124] Embodiment 112: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS3 antibody.

[0125] Embodiment 113: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS3 antibody.

[0126] Embodiment 114: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS3 antibody.

[0127] Embodiment 115: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS3 antibody.

[0128] Embodiment 116: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS3 antibody and the variable heavy (VH) chain of the YS3 antibody.

[0129] Embodiment 117. The antibody of Embodiment 1, wherein the antibody is human YS3 scFv.

[0130] Example 118. The antibody of Example 1, wherein the antibody is human YS3 IgG.

[0131] Embodiment 119: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS4 antibody.

[0132] Embodiment 120: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS4 antibody.

[0133] Embodiment 121: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS4 antibody.

[0134] Embodiment 122: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS4 antibody.

[0135] Embodiment 123: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS4 antibody.

[0136] Embodiment 124: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS4 antibody and the variable heavy (VH) chain of the YS4 antibody.

[0137] Example 125. The antibody of Example 1, wherein the antibody is human YS4 scFv.

[0138] Example 126. The antibody of Example 1, wherein the antibody is human YS4 IgG.

[0139] Embodiment 127: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS8 antibody.

[0140] Embodiment 128: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS8 antibody.

[0141] Embodiment 129: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS8 antibody.

[0142] Embodiment 130: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the YS8 antibody.

[0143] Embodiment 131: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the YS8 antibody.

[0144] Embodiment 132: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS8 antibody and the variable heavy (VH) chain of the YS8 antibody.

[0145] Example 133: The antibody of Example 1, wherein the antibody is human YS8 scFv.

[0146] Example 134. The antibody of Example 1, wherein the antibody is human YS8 IgG.

[0147] Embodiment 135: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS7 antibody.

[0148] Embodiment 136: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS7 antibody.

[0149] Embodiment 137: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS7 antibody.

[0150] Embodiment 138: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS7 antibody.

[0151] Embodiment 139: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS7 antibody.

[0152] Embodiment 140: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS7 antibody and the variable heavy (VH) chain of the YS7 antibody.

[0153] Example 141. The antibody of Example 1, wherein the antibody is human YS7 scFv.

[0154] Example 142. The antibody of Example 1, wherein the antibody is human YS7 IgG.

[0155] Embodiment 143: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS9 antibody.

[0156] Embodiment 144: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS9 antibody.

[0157] Embodiment 145: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS9 antibody.

[0158] Embodiment 146: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the YS9 antibody.

[0159] Embodiment 147: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the YS9 antibody.

[0160] Embodiment 148: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS9 antibody and the variable heavy (VH) chain of the YS9 antibody.

[0161] Embodiment 149. The antibody of Embodiment 1, wherein said antibody is human YS9 scFv.

[0162] Example 150: The antibody of Example 1, wherein the antibody is human YS9 IgG.

[0163] Embodiment 151: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS10 antibody.

[0164] Embodiment 152: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS10 antibody.

[0165] Embodiment 153: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS10 antibody.

[0166] Embodiment 154: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the YS10 antibody.

[0167] Embodiment 155: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the YS10 antibody.

[0168] Embodiment 156: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS10 antibody and the variable heavy (VH) chain of the YS10 antibody.

[0169] Embodiment 157. The antibody of Embodiment 1, wherein said antibody is human YS10 scFv.

[0170] Example 158: The antibody of Example 1, wherein the antibody is human YS10 IgG.

[0171] Embodiment 159: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the YS11 antibody.

[0172] Embodiment 160: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the YS11 antibody.

[0173] Embodiment 161: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the YS11 antibody.

[0174] Embodiment 162: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the YS11 antibody.

[0175] Embodiment 163: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable heavy (VH) chain of the YS11 antibody.

[0176] Embodiment 164: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the YS11 antibody and the variable heavy (VH) chain of the YS11 antibody.

[0177] Example 165: The antibody of Example 1, wherein the antibody is human YS11 scFv.

[0178] Example 166. The antibody of Example 1, wherein the antibody is human YS11 IgG.

[0179] Embodiment 167: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the 3G7HY antibody.

[0180] Embodiment 168: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the 3G7HY antibody.

[0181] Embodiment 169: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the 3G7HY antibody.

[0182] Embodiment 170: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the 3G7HY antibody.

[0183] Embodiment 171: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the 3G7HY antibody.

[0184] Embodiment 172: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7HY antibody and the variable heavy (VH) chain of the 3G7HY antibody.

[0185] Embodiment 173. The antibody of Embodiment 1, wherein said antibody is human 3G7HY scFv.

[0186] Embodiment 174. The antibody of Embodiment 1, wherein the antibody is human 3G7HY IgG.

[0187] Embodiment 175: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises VH CDR1, and / or VH CDR2, and / or VH CDR3 of the 3G7NY antibody.

[0188] Embodiment 176: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the 3G7NY antibody.

[0189] Embodiment 177: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the 3G7NY antibody.

[0190] Embodiment 178: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7NY antibody.

[0191] Embodiment 179: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the 3G7NY antibody.

[0192] Embodiment 180: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7NY antibody and the variable heavy (VH) chain of the 3G7NY antibody.

[0193] Embodiment 181. The antibody of Embodiment 1, wherein said antibody is human 3G7NY scFv.

[0194] Embodiment 182: The antibody of Embodiment 1, wherein the antibody is human 3G7NY IgG.

[0195] Embodiment 183: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the 3G7 antibody.

[0196] Embodiment 184: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the 3G7 antibody.

[0197] Embodiment 185: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the 3G7 antibody.

[0198] Embodiment 186: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the 3G7 antibody.

[0199] Embodiment 187: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the 3G7 antibody.

[0200] Embodiment 188: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the 3G7 antibody and the variable heavy (VH) chain of the 3G7 antibody.

[0201] Embodiment 189. The antibody of Embodiment 1, wherein said antibody is human 3G7 scFv.

[0202] Embodiment 190. The antibody of Embodiment 1, wherein the antibody is human 3G7 IgG.

[0203] Embodiment 191: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the SB2 antibody.

[0204] Embodiment 192: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the SB2 antibody.

[0205] Embodiment 193: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the SB2 antibody.

[0206] Embodiment 194: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the SB2 antibody.

[0207] Embodiment 195: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the SB2 antibody.

[0208] Embodiment 196: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the SB2 antibody and the variable heavy (VH) chain of the SB2 antibody.

[0209] Embodiment 197. The antibody of Embodiment 1, wherein said antibody is human SB2 scFv.

[0210] Example 198: The antibody of Example 1, wherein the antibody is human SB2 IgG.

[0211] Embodiment 199: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the 2C8 antibody.

[0212] Embodiment 200: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the 2C8 antibody.

[0213] Embodiment 201: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the 2C8 antibody.

[0214] Embodiment 202: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the 2C8 antibody.

[0215] Embodiment 203: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the 2C8 antibody.

[0216] Embodiment 204: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the 2C8 antibody and the variable heavy (VH) chain of the 2C8 antibody.

[0217] Embodiment 205. The antibody of Embodiment 1, wherein said antibody is human 2C8 scFv.

[0218] Embodiment 206. The antibody of Embodiment 1, wherein said antibody is human 2C8 IgG.

[0219] Embodiment 207: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, and / or VH CDR2, and / or VH CDR3 of the UA8κ antibody.

[0220] Embodiment 208: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VL CDR1, and / or VL CDR2, and / or VL CDR3 of the UA8κ antibody.

[0221] Embodiment 209: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the UA8κ antibody.

[0222] Embodiment 210: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable light (VL) chain of the UA8κ antibody.

[0223] Embodiment 211: The antibody according to any one of embodiments 1 to 16, wherein said antibody comprises the variable heavy (VH) chain of the UA8κ antibody.

[0224] Embodiment 212: The antibody according to any one of embodiments 1 to 16, wherein the antibody comprises the variable light (VL) chain of the UA8κ antibody and the variable heavy (VH) chain of the UA8κ antibody.

[0225] Example 213: The antibody of Example 1, wherein the antibody is human UA8κ scFv.

[0226] Example 214: The antibody of Example 1, wherein the antibody is human UA8κ IgG.

[0227] Embodiment 215: An immunoconjugate comprising the antibody of any one of embodiments 1 to 214 linked to an effector, wherein the effector is selected from the group consisting of a second antibody, a detectable label, a cytotoxin or cytostatic agent, a drug-containing liposome, a radionuclide, a drug, a prodrug, a viral particle, a cytokine, and a chelate.

[0228] Example 216: The immunoconjugate of Example 215, wherein the antibody is linked to a cytotoxin.

[0229] Example 217: The immunoconjugate of Example 216, wherein said antibody is linked to a cytotoxin selected from the group consisting of diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, saporin, and thymidine kinase.

[0230] Example 218: The immunoconjugate of Example 215, wherein the antibody is linked to a cytotoxic and / or cytostatic drug.

[0231] Example 219: The immunoconjugate of Example 216, wherein the antibody is linked directly or through a linker to one or more of: the drug; a lipid or liposome containing the drug; a polymeric drug carrier comprising the drug; and a nanoparticle drug carrier comprising the drug.

[0232] Embodiment 220: The immunoconjugate according to any one of embodiments 218 to 219, wherein the drug is an anticancer drug.

[0233] Embodiment 221: The immunoconjugate according to any one of embodiments 218 to 219, wherein the drug is selected from the group consisting of a microtubule inhibitor, a DNA damaging agent, and a polymerase inhibitor.

[0234] Example 222: The immunoconjugate of Example 221, wherein the drug comprises a tubulin inhibitor.

[0235] Example 223: The immunoconjugate of Example 222, wherein the drug comprises a drug selected from the group consisting of auristatin, dolastatin-10, a synthetic derivative of the natural product dolastatin-10, and maytansine or a maytansine derivative.

[0236] Example 224: The immunoconjugate of Example 222, wherein the drug comprises a drug selected from the group consisting of: Monomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF.

[0237] Embodiment 225. The immunoconjugate of Embodiment 222, wherein the drug comprises a maytansine selected from the group consisting of maytansine (DM1), DM3, and DM4.

[0238] Example 226: The immunoconjugate of Example 221, wherein the drug comprises a DNA damaging agent.

[0239] Example 227. The immunoconjugate of Example 226, wherein the drug comprises a drug selected from the group consisting of calicheamicin, duocarmycin, and pyrrolobenzodiazepine.

[0240] Example 228: The immunoconjugate of Example 227, wherein the drug comprises calicheamicin or a calicheamicin analog.

[0241] Example 229: The immunoconjugate of Example 227, wherein the drug comprises duocarmycin.

[0242] Example 230: The immunoconjugate of Example 229, wherein the drug comprises a duocarmycin selected from the group consisting of: duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, cyclopropylbenzindolecarmycin (CC-1065), Centanamycin, Rachelmycin, Adozelesin, Bizelesin, and Carzelesin.

[0243] Example 231: The immunoconjugate of Example 227, wherein the drug comprises a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.

[0244] Example 232: The immunoconjugate of Example 231, wherein the drug comprises a drug selected from the group consisting of: Anthramycin (and its dimer), Mazethramycin (and its dimer), Tomaymycin (and its dimer), Prothracarcin (and its dimer), Chicamycin (and its dimer), Neothramycin A (and its dimer), Neothramycin B (and its dimer), DC-81 (and its dimer), Sibiromycin (and its dimer), Porothramycin A (and its dimer), Porothramycin B (and its dimer), Sibanomycin (and its dimer), Abbeymycin (and its dimer), SG2000 and SG2285.

[0245] Example 233: The immunoconjugate of Example 221, wherein the drug comprises a polymerase inhibitor.

[0246] Example 234: The immunoconjugate of Example 233, wherein the drug comprises a poly (ADP-ribose) polymerase (PARP) inhibitor.

[0247] Example 235: The immunoconjugate of Example 234, wherein the drug comprises a poly (ADP-ribose) polymerase (PARP) inhibitor selected from the group consisting of Iniparib (BSI 201), Talazoparib (BMN-673), Olaparib (AZD-2281), Olaparib, Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, MK4827, BGB-290 and 3-aminobenzamide.

[0248] Embodiment 236: The immunoconjugate according to any one of embodiments 218 to 219, wherein the drug is selected from the group consisting of auristatins, dolastatin, colchicine, combretastatin and mTOR / PI3K inhibitors.

[0249] Embodiment 237: The immunoconjugate according to any one of embodiments 218 to 219, wherein the drug is selected from the group consisting of fluorouracil (5-FU), capecitabine, 5-trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed, pemetrexed, cytosine arabinoside, 6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin, fludarabine phosphate, phosphate), cladribine, floxuridine (5-fluoro-2-nitropropane), ribonucleotide reductase inhibitors (RNR), cyclophosphamide, neosar, ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1,3-bis(2-chloroethyl)-3-cyclohexyl-nitrosourea, methyl (CCNU), hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC), chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin, bendamustine, carmustine, chloromethine, dacarbazine,DTIC), fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine, doxorubicin, daunorubicin citrate, mitoxantrone, actinomycin D), etoposide, topotecan HCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin, belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbinetartrate, vindesine sulfate, paclitaxel, docetaxel, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, and vinflunine.

[0250] Embodiment 238: The immunoconjugate of any one of embodiments 218 to 219, wherein the drug is selected from the group consisting of carboplatin, cisplatin, cyclophosphamide, docetaxel, vinblastine, erlotinib, etoposide, gemcitabine, imatinib mesylate, irinotecan, methotrexate, sorafenib, sunitinib, topotecan, vinblastine and vincristine.

[0251] Embodiment 239: The immunoconjugate of any one of embodiments 218 to 219, wherein the drug is selected from the group consisting of retinoic acid, retinoic acid derivatives, doxirubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, camptothecin derivatives, interferon, tamoxifen and taxol. In certain embodiments, the anticancer compound is selected from the group consisting of albuterol, tadalafil, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, and zoledronic acid.

[0252] Embodiment 240: The immunoconjugate of Embodiment 215, wherein the antibody is linked to a chelate comprising an isotope selected from the group consisting of: 99 Tc, 203 Pb, 67 Ga, 68 Ga, 72 As、 111 In, 113 In, 97 Such as 62 Cu, 64l Cu, 52 Fe, 52 Mn, 51 Cr, 186 Re、 188 Re、 77 As、 90 Y. 67 Cu, 169 Second, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 199 Au, 161 Tb, 109 Pd, 165Dy, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 166 Ho, 172 Tm, 169 Yb, 175 Yb, 177 Lu, 105 Rh and 111 Ag.

[0253] Example 241: The immunoconjugate of Example 215, wherein the antibody is linked to an alpha emitter.

[0254] Example 242: The immunoconjugate of Example 241, wherein the alpha emitter is bismuth 213.

[0255] Example 243: The immunoconjugate of Example 215, wherein the antibody is linked to a lipid or liposome complexed with or containing an anti-cancer drug.

[0256] Example 244: The immunoconjugate of Example 215, wherein the antibody is linked to a detectable label.

[0257] Embodiment 245: The immunoconjugate of Embodiment 244, wherein said antibody is linked to a detectable label selected from the group consisting of a radioactive label, a radiopaque label, an MRI label, and a PET label.

[0258] Embodiment 246: A pharmaceutical formulation comprising: a pharmaceutically acceptable excipient and the antibody according to any one of embodiments 1 to 214; and / or a pharmaceutically acceptable excipient and the immunoconjugate according to any one of embodiments 215 to 245.

[0259] Embodiment 247. The pharmaceutical formulation of Embodiment 246, wherein said formulation is a unit dose formulation.

[0260] Embodiment 248: The formulation of any one of Embodiments 246-247, wherein the formulation is formulated for administration via a route selected from the group consisting of oral administration, nasal administration, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transdermal administration, and intramuscular injection.

[0261] Example 249: A method of inhibiting the growth and / or proliferation of cells that express or overexpress CD46, the method comprising: contacting the cancer cells with an antibody according to any one of Examples 1 to 214; and / or contacting the cancer cells with an immunoconjugate comprising the antibody according to any one of Examples 1 to 214 linked to an effector having cell growth inhibitory and / or cytotoxic activity.

[0262] Embodiment 250: The method of Embodiment 249, wherein the method comprises contacting the cancer cell with the antibody according to any one of Embodiments 1-214.

[0263] Embodiment 251: The method of Embodiment 249, wherein said method comprises contacting said cancer cell with an immunoconjugate comprising an antibody according to any one of Embodiments 1 to 214 linked to an effector having cytostatic and / or cytotoxic activity.

[0264] Example 252: The method of Examples 249-251, wherein the cell is a cancer cell.

[0265] Embodiment 253: The method of Embodiment 252, wherein the cell is a cancer cell that overexpresses CD46.

[0266] Example 254: The method of Example 252, wherein the cancer cells are selected from the group consisting of ovarian cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, mesothelioma, lymphoma, liver cancer, urothelial cancer, gastric cancer, multiple myeloma, glioblastoma multiforme, glioma, neuroblastoma, and cervical cancer.

[0267] Example 255: The method of Example 252, wherein the cancer cells are prostate cancer cells.

[0268] Example 256: The method of Example 255, wherein the cancer cells are castration-resistant prostate cancer cells.

[0269] Example 257: The method of Example 252, wherein the cancer cells are multiple myeloma cells.

[0270] Embodiment 258: The method according to any one of Embodiments 252 to 257, wherein the cell is a metastatic cell.

[0271] Example 259: The method of Example 258, wherein the metastatic cells are bone metastases, liver metastases, bladder metastases and / or lymph node metastases.

[0272] Embodiment 260: The method according to any one of Embodiments 252 to 258, wherein the cell is a solid tumor cell.

[0273] Embodiment 261: The method according to any one of embodiments 249, and 251 to 260, wherein the effector comprises a radionuclide and / or a cytostatic drug.

[0274] Example 262: The method of Example 261, wherein the effector comprises one or more of: a cytotoxic and / or cytostatic drug; a lipid or liposome containing a cytotoxic and / or cytostatic drug; a polymeric drug carrier comprising a cytotoxic and / or cytostatic drug; and a nanoparticle drug carrier comprising a cytotoxic and / or cytostatic drug.

[0275] Embodiment 263. The method of Embodiment 262, wherein the drug is an anticancer drug.

[0276] Embodiment 264: The method of Embodiment 263, wherein the drug is selected from the group consisting of: auristatins, dolastatin, colchicine, combretastatins, and mTOR / PI3K inhibitors.

[0277] Embodiment 265. The method of Embodiment 263, wherein the drug is monomethyl auristatin F.

[0278] Example 266: The method of Example 263, wherein the drug is selected from the group consisting of: fluorouracil (5-FU), capecitabine, 5-trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed, pemetrexed, cytarabine, 6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin, fludarabine phosphate, cladribine, floxuridine (5-fluoro-2), ribonucleotide reductase inhibitors (RNR), cyclophosphamide, carboplatin, ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1,-(2-chloroethyl)-3-cyclohexyl-nitrosourea, methyl (CCNU), hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC), chlorambucil, melphalan, cisplatin, carboplatin, oxadiazine Riboplatin, bendamustine, carmustine, nitrogen mustard, dacarbazine (DTIC), fotemustine, lomustine, mannosufan, nedaplatin, nimustine, prednimustine, ranimustine, seraplatin, semustine, streptozotocin, temozolomide, treosufan, triazoquinone, triethylene melamine, thiotepa, tereplatin tetranitrate, trolofosamide, uramustine, cranberries, daunomycin citrate, mitoxantrone, Actinomycin D, etoposide, topotecan HCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin, belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulfate, paclitaxel, docetaxel, nanoparticle paclitaxel, abelmopan, ixabepilone, larotaxel, ortataxel, tesetaxel, and vinflunine.

[0279] Example 267: The method of Example 263, wherein the drug is selected from the group consisting of carboplatin, cisplatin, cyclophosphamide, docetaxel, vinblastine, erlotinib, etoposide, gemcitabine, imatinib mesylate, irinotecan, methotrexate, sorafenib, sunitinib, topotecan, vinblastine, and vincristine.

[0280] Example 268: The method of Example 263, wherein the drug is selected from the group consisting of retinoic acid, retinoic acid derivatives, doxorubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, camptothecin derivatives, interferon, tamoxifen, and paclitaxel. In certain embodiments, the anticancer compound is selected from the group consisting of abestrol, cypermethrin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol acetate tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, and zoledronic acid.

[0281] Embodiment 269: The method according to any one of Embodiments 262 to 268, wherein said drug is bound to said antibody.

[0282] Embodiment 270: The method according to any one of embodiments 262 to 268, wherein said drug is contained in a lipid or liposome attached to said antibody.

[0283] Embodiment 271: The method according to any one of embodiments 262 to 268, wherein the drug is contained in a polymeric and / or nanoparticle carrier attached to the antibody.

[0284] Example 272: The method of Examples 249, and 251 to 260, wherein the effector comprises a cytotoxin.

[0285] Embodiment 273. The method of Embodiment 272, wherein the cytotoxin is selected from the group consisting of diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, saponin, and thymidine kinase.

[0286] Embodiment 274. The method of Embodiment 249, wherein the effector comprises a radionuclide.

[0287] Embodiment 275: The method according to any one of Embodiments 249 to 274, wherein said immunoconjugate or antibody is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0288] Embodiment 276: The method according to any one of Embodiments 249 to 275, wherein said administering comprises administering to a human.

[0289] Embodiment 277: The method according to any one of Embodiments 249 to 275, wherein said administering comprises administering to a human or non-human mammal.

[0290] Embodiment 278: The method according to any one of Embodiments 249 to 277, wherein said administering comprises parenteral administration.

[0291] Embodiment 279: The method according to any one of Embodiments 249 to 277, wherein said administering comprises administering into a tumor or a surgical site.

[0292] Embodiment 280: The method according to any one of Embodiments 249 to 279, wherein said immunoconjugate is administered as an adjunct therapy to surgery and / or radiation therapy.

[0293] Embodiment 281: The method according to any one of embodiments 249 to 279, wherein the antibody and / or immunoconjugate is administered in conjunction with another anti-cancer drug and / or hormone.

[0294] Embodiment 282: The method of Embodiment 281, wherein the antibody and / or immunoconjugate is administered in conjunction with abiraterone and / or enzalutamide.

[0295] Embodiment 283: The method of Embodiment 282, wherein the cells comprise prostate cancer cells.

[0296] Example 284: The method of Example 283, wherein the prostate cancer cells comprise neuroendocrine prostate cancer (NEPC) cells.

[0297] Example 285: The method of Example 283, wherein the prostate cancer cells comprise metastatic castration-resistant prostate cancer (mCRPC) cells that are resistant to abiraterone (Abi) or enzalutamide (Enz).

[0298] Example 286: A method for detecting cancer cells of a cancer that expresses or overexpresses CD46, the method comprising: contacting the cancer cells with an immunoconjugate comprising an antibody according to any one of Examples 1 to 214 linked to a detectable label; and detecting the presence and / or location of the detectable label, wherein the presence and / or location is indicative of the location and / or presence of the cancer cells.

[0299] Embodiment 287: The method of Embodiment 286, wherein the label comprises a label selected from the group consisting of a radioactive label, a radiopaque label, an MRI label, a PET label, and a SPECT label.

[0300] Embodiment 288: The method of Embodiment 286, wherein the detectable label is selected from the group consisting of a gamma-emitter, a positron emitter, an x-ray emitter, an alpha emitter, and a fluorescent emitter.

[0301] Embodiment 289: The method of any one of Embodiments 286 to 288, wherein the cancer cells are selected from the group consisting of ovarian cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, mesothelioma, lymphoma, liver cancer, urothelial cancer, gastric cancer, multiple myeloma, glioma, neuroblastoma, and cervical cancer.

[0302] Example 290: The method of Example 289, wherein the cancer cells are prostate cancer cells.

[0303] Example 291: The method of Example 290, wherein the cancer cells are castration-resistant prostate cancer cells.

[0304] Example 292: The method of Example 289, wherein the cancer cells are multiple myeloma cells.

[0305] Embodiment 293: The method according to any one of Embodiments 286 to 292, wherein said contacting comprises administering said immunoconjugate to a non-human mammal.

[0306] Embodiment 294: The method according to any one of Embodiments 286 to 292, wherein said contacting comprises administering said immunoconjugate to a human.

[0307] Embodiment 295: The method according to any one of Embodiments 286 to 294, wherein said detecting comprises detecting said marker in vivo.

[0308] Embodiment 296: The method of Embodiment 295, wherein the detecting comprises using a detection method selected from the group consisting of: X-ray, PET, SPECT, MRI, and CAT.

[0309] Embodiment 297: The method according to any one of Embodiments 286 to 294, wherein said detecting comprises detecting said marker ex vivo in a biopsy or in a sample derived from a biopsy.

[0310] Embodiment 298: A nucleic acid encoding the antibody or antibody fragment according to any one of embodiments 1 to 214.

[0311] Example 299: An expression vector comprising the nucleic acid of Example 298.

[0312] Example 300: A cell comprising the expression vector of Example 299.

[0313] definition

[0314] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The terms also include variants of the traditional peptide bonds joining the amino acids that constitute a polypeptide.

[0315] As used herein, the term "nucleic acid" or "oligonucleotide" or grammatical equivalents refers to at least two nucleotides covalently linked together. The nucleic acids of the invention are preferably single-stranded or double-stranded and will generally contain phosphodiester bonds, but in some cases, as outlined below, include nucleic acid analogs that may have alternative backbones, including, for example, phosphoramidites (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res. Res.) 14:3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) Chemica Scripta 26:1419), phosphorothioates (Mag et al. (1991) Nucleic Acids Res. Res. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioates (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, O-methylphosphoramidite bonds (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press). Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114: 1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature 365: 566; Carlsson et al. (1996) Nature 380: 207).Other analog nucleic acids include those with cationic backbones (Dempcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097); nonionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Angewandte Chemie (1991) (English Edition) tl. Ed. English 30:423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Conference Series 580, "Carbohydrate Modifications in Antisense Studies Modifications in Antisense Research, YS Sanghui and P. Dan Cook, eds.; Mesmaeker et al. (1994) Bioorganic & Medicinal Chem. Lett. 4:395; Jeff et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones (including U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Conference Series 580, Carbohydrate Modifications in Antisense Research, YS Sanghui and P. Dan Cook, eds.; Mesmaeker et al. (1994) Bioorganic & Medicinal Chem. Lett. 4:395; Jeff et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones (including U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Conference Series 580, Carbohydrate Modifications in Antisense Research, YS Sanghui and P. Dan Cook, eds. Cook, ed.) are also included within the definition of nucleic acid (see Jenkins et al. (1995), Chem. Soc. Rev., pp. 169-176). Several nucleic acid analogs are described in Rawls, C&E News, June 2, 1997, p. 35. These modifications to the ribose phosphate backbone can be made to facilitate the addition of additional moieties (e.g., labels) or to increase the stability and half-life of such molecules in physiological environments.

[0316] As used herein, the term "residue" refers to a natural, synthetic, or modified amino acid.

[0317] As used herein, "antibody" refers to a protein consisting of one or more polypeptides generally encoded by immunoglobulin genes or immunoglobulin gene fragments. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0318] The typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0319] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds in the hinge region to produce F(ab)' (a fragment which itself is joined to V by a disulfide bond). H -C H 1 light chain Fab dimer). F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. Fab' monomers are essentially Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, edited by WE Paul, Raven Press, New York (NY) (1993)). Although various antibody fragments are defined based on the digestion of intact antibodies, those skilled in the art will appreciate that such Fab' fragments can be synthesized chemically or by utilizing recombinant DNA methods. Therefore, the term antibody as used herein also includes antibody fragments produced by modifying whole antibodies or antibody fragments synthesized recombinantly using recombinant DNA methods. Particularly preferred antibodies include single-chain antibodies (antibodies in the form of a single polypeptide chain), more preferably single-chain Fv antibodies (sFv or scFv), in which the variable heavy chain and the variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. Single-chain Fv antibodies are covalently linked V H -V LHeterodimers, which can be derived from V H and V L Nucleic acid expression of coding sequences. Huston et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Although V H and V L linked to each other in a single polypeptide chain, but V H and V L The domains are associated in a non-covalent manner. The first functional antibody molecule intended to be expressed on the surface of filamentous phage was a single-chain Fv (scFv), however, alternative expression strategies have also been successful. For example, if one chain (heavy or light) is fused to the g3 capsid protein and the complementary chain is exported to the extracellular cytoplasm as a soluble molecule, then the Fab molecule can be displayed on the phage. The two chains can be encoded on the same or different replicons; the key point is that the two antibody chains in each Fab molecule are assembled after translation and the dimer is incorporated into the phage particle via a linkage, for example, to one chain of g3p (see, for example, U.S. Patent No. 5,733,743). Those skilled in the art are aware of scFv antibodies and many other structures that convert naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into molecules that fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site (see, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies include all antibodies displayed on phage (eg, scFv, Fv, Fab, and disulfide-linked Fv (Reiter et al. (1995) Protein Eng. 8: 1323-1331)).

[0320] As used herein, the term "specific binding" when referring to biomolecules (e.g., proteins, nucleic acids, antibodies, etc.) refers to a binding reaction that determines the presence of a biomolecule (e.g., proteins and other biologics) in a heterogeneous population of molecules. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies or stringent hybridization conditions in the case of nucleic acids), a specified ligand or antibody binds to its specific "target" molecule and does not significantly bind to other molecules present in the sample.

[0321] As used herein, the phrase "inhibits proliferation of cells expressing CD46" refers to the ability of an anti-CD46 antibody or immunoconjugate described herein to reduce (preferably statistically significantly) the proliferation of cells expressing CD46 relative to proliferation in the absence of the antibody or immunoconjugate. In one embodiment, when cells are contacted with an antibody, or antigen-binding portion thereof, or immunoconjugate described herein, proliferation of cells expressing CD46 (e.g., cancer cells) can be reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100%, relative to proliferation measured in the absence of the antibody, or antigen-binding portion thereof, or immunoconjugate (control group). Cell proliferation can be analyzed using art-recognized techniques that measure the rate of cell division, the fraction of cells within a cell population that undergo cell division, and / or the rate of cell loss from a cell population due to terminal differentiation or cell death (e.g., using cell titer luminescence assays or thymidine incorporation).

[0322] As used herein, the phrase "inhibits migration of cells expressing CD46" refers to the ability of the anti-CD46 antibodies, or antigen-binding portions thereof, or immunoconjugates described herein to reduce (preferably statistically significantly reduce) the migration of cells expressing CD46 relative to cell migration in the absence of the antibody. In one embodiment, when cells are contacted with the antibody, or antigen-binding portion thereof, or immunoconjugate, the migration of cells expressing CD46 (e.g., cancer cells) can be reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100%, relative to cell migration measured in the absence of the antibody, or antigen-binding portion thereof, or immunoconjugate (control group). Cell migration can be analyzed using techniques recognized in the art. In various embodiments, it is contemplated that the antibodies and / or immunoconjugates described herein can inhibit the migration of cells expressing or overexpressing CD46 (e.g., cancer cells as described herein).

[0323] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody (e.g., CD46 domain 1 and / or domain 2) that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a fragment consisting of V domains, and (ii) a Fab fragment. L 、V H (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of VH and CH1 domains; (iv) Fv fragments, which consist of the V L and V H Domain composition, (v) includes V H and V L (vi) dAb fragments (see, e.g., Ward et al., (1989) Nature 341:544-546) consisting of V H domain; (vii) consists of V H or V L and (viii) an isolated complementarity determining region (CDR), or (ix) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. L and V H ) are encoded by separate genes, but the two domains can be joined using recombinant methods via a synthetic linker that enables their manufacture as a V L and V H The regions pair to form a single protein chain of a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0324] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising this population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be prepared using any art-recognized technique and those described herein, such as the hybridoma method, as described in Kohler et al. (1975) Nature, 256: 495, transgenic animals, for example (see, for example, Lonberg et al. (1994) Nature, 368(6474): 856-859), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), or using phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature, 352: 624-628, and Marks et al. (1991) J. Mol. Biol., 222: 581-597. Monoclonal antibodies include chimeric, human, and humanized antibodies and can occur naturally or be produced recombinantly.

[0325] The term "recombinant antibody" refers to an antibody prepared, expressed, formed or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or fusion tumors prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies, such as from transfectomas, (c) antibodies isolated from recombinant combinatorial antibody libraries (e.g., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, formed or isolated by any other means involving splicing immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis, and thus the V H and V L The amino acid sequence of the region is derived from and related to the human germline V- and V- L sequences, but not sequences that naturally occur in the human antibody germline repertoire in vivo.

[0326] The term "chimeric immunoglobulin" or antibody refers to an immunoglobulin or antibody whose variable region is derived from a first species and whose constant region is derived from a second species. A chimeric immunoglobulin or antibody can be constructed, for example, by genetic engineering from immunoglobulin gene segments belonging to different species.

[0327] As used herein, the term "human antibody" is intended to include antibodies in which both the framework and CDR regions in the variable region are derived from human germline immunoglobulin sequences, as described, for example, by Kabat et al. (see Kabat et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) are grafted onto human framework sequences.

[0328] At least one or more amino acids of a human antibody can be replaced with an amino acid residue, e.g., an activity-enhancing amino acid residue that is not encoded by a human germline immunoglobulin sequence. Typically, a human antibody can have up to twenty positions replaced with an amino acid residue that is not part of a human germline immunoglobulin sequence. In a specific embodiment, as described in detail below, these replacements are within the CDR regions.

[0329] The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that comprises at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light chain or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., a light chain or heavy chain, respectively) that comprises a variable framework region substantially derived from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially derived from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further comprises a constant region (e.g., in the case of a light chain, at least one constant region or portion thereof, and in the case of a heavy chain, preferably three constant regions). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and a complementarity determining region (CDR) substantially derived from a non-human immunoglobulin or antibody.

[0330] As used herein, "heterologous antibodies" are defined with respect to transgenic non-human organisms or plants that produce such antibodies.

[0331] As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CD46 is substantially free of antibodies that specifically bind to antigens other than CD46). Additionally, isolated antibodies are typically substantially free of other cellular material and / or chemicals. In one embodiment, a combination of "isolated" monoclonal antibodies having different CD46 binding specificities are combined in a well-defined composition.

[0332] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. In one embodiment, the antibody or its antigen-binding portion has an isotype selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE antibody isotypes. In some embodiments, the monoclonal antibodies of the invention have an IgG1 isotype. In other embodiments, the monoclonal antibodies of the invention have an IgG2 isotype.

[0333] An "antigen" is an entity (eg, a protein entity or a peptide) to which an antibody or antigen-binding portion thereof binds. In various embodiments of the present invention, the antigen is, for example, CD46 presented on a cell (eg, a CD46-positive cancer cell).

[0334] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by continuous amino acids or discontinuous amino acids connected by tertiary folding of a protein. Epitopes formed by continuous amino acids are generally retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are generally lost when treated with denaturing solvents. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an epitope include techniques in the art and those described herein, such as x-ray crystallography and 2D nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0335] Also contemplated herein are antibodies that bind to the same or overlapping epitopes as the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ antibodies described herein. Antibodies that recognize the same epitope can be identified using conventional techniques (e.g., immunoassays), for example, by demonstrating the ability of one antibody to block binding of another antibody to the target antigen (i.e., competitive binding assays). Competitive binding is determined in an assay where the immunoglobulin being tested inhibits specific binding of a reference antibody to a common antigen (e.g., CD46 domain 1 and / or domain 2). Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, for example, Stahli et al. (1983) Meth. Enzymol., 9:242); solid phase direct biotin-avidin EIA (see, for example, Kirkland et al. (1986) J. Immunol. 137:3614); solid phase direct label assays, solid phase direct label sandwich assays (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); for example, using 125I-labeled solid phase direct label RIA (see, e.g., Morel et al. (1988) Mol. Immunol. 25(1):7); solid phase direct biotin-avidin EIA (Cheung et al. (1990) Virology 176:546); and direct labeled RIA (Moldenhauer et al. (1990) Scand J. Immunol. 32:77). Typically, such assays involve the use of purified antigen (e.g., CD46 domain 1 and / or domain 2) bound to a solid surface or cell carrying either an unlabeled test immunoglobulin or a statically labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. The test immunoglobulin is typically present in excess. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

[0336] As used herein, the terms "specifically bind" and "selectively bind" mean that an antibody or antigen-binding portion thereof exhibits significant affinity for a particular antigen or epitope and generally does not exhibit significant cross-reactivity with other antigens and epitopes. "Significant" or preferential binding includes binding with a KD of at least (equal to or less than) 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 The affinity binding of M is greater than 10 -9 M, preferably greater than 10 -10 Affinities of M are more preferred. Values ​​between the values ​​described herein are also intended to be within the scope of the invention, and preferably the binding affinity can be expressed as an affinity range, for example 10 -6 M to 10 -11 M, preferably 10 -7 M or 10 -8 M to 10 -10M. An antibody that "does not exhibit significant cross-reactivity" is one that will not significantly bind to undesirable entities (e.g., undesirable protein entities). For example, in one embodiment, an antibody, or antigen-binding portion thereof, that specifically binds to a CD46 (e.g., domain 1 and / or domain 2) protein will not significantly react with other molecules and non-CD46 proteins or peptides. Specific or selective binding can be determined according to any art-recognized means for determining such binding, including, for example, Scatchard analysis and / or competitive binding assays.

[0337] As used herein, the term "K D "is intended to refer to the dissociation equilibrium constant for a particular antibody-antigen interaction or the affinity of the antibody for the antigen. In one embodiment, an antibody, or antigen-binding portion thereof, according to the present invention binds to an antigen with an affinity (K) of 5 nM or better (i.e., 10 nM or less) (e.g., 40 nM or 30 nM or 20 nM or 10 nM or less) as measured using a surface plasmon resonance assay or a cell binding assay. D ) binds to an antigen (e.g., CD46 domain 1 and / or domain 2). In a specific embodiment, an antibody, or antigen-binding portion thereof, according to the present invention binds to an antigen (e.g., CD46 domain 1 and / or domain 2) with an affinity (K) of 5 nM or better (e.g., 4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1 nM or less) as measured using a surface plasmon resonance assay or a cell binding assay. D ) binds to CD46. In other embodiments, antibodies or antigen-binding portions thereof are detected by FACS using live prostate tumor cells at approximately 10 -10 M, or 100×10 -11 M, or 10×10 -11 M, or even lower affinity (K D ) binds to an antigen (e.g., CD46).

[0338] As used herein, the term "K off ” is intended to refer to the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0339] As used herein, the term "EC50" refers to the concentration of an antibody or antigen-binding portion thereof or immunoconjugate described herein that induces 50% of the maximal response (i.e., halfway between the maximal response and the baseline value) in an in vitro or in vivo assay.

[0340] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0341] As used herein, the term "modification" is intended to refer to altering one or more amino acids in an antibody or antigen-binding portion thereof. The alteration can be produced by adding, substituting, or deleting amino acids at one or more positions. The alteration can be produced using known techniques (e.g., PCR mutagenesis). For example, in some embodiments, an antibody or antigen-binding portion thereof identified using the methods of the present invention can be modified to alter the binding affinity of the antibody or antigen-binding portion thereof for CD46.

[0342] In certain embodiments, "conservative amino acid substitutions" in the sequences of the anti-CD46 antibodies described herein are contemplated, i.e., nucleotide and amino acid sequence modifications that do not abolish binding of the antibody encoded by the nucleotide sequence or the antibody comprising the amino acid sequence to the antigen (e.g., CD46). Conservative amino acid substitutions include substitutions of an amino acid of one class with an amino acid of the same class, where the classes are defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature (as determined, for example, by the standard Dayhoff frequency exchange matrix or BLOSUM matrix). Six general classes of amino acid side chains have been classified and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of Asp for another Class III residue (e.g., Asn, Gln, or Glu) is a conservative substitution. Thus, a predicted nonessential amino acid residue in an anti-CD46 antibody is preferably replaced with another amino acid residue from the same class. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al. (1993) Biochem. 32: 1180-1187; Kobayashi et al. (1999) Protein Eng. 12(10): 879-884; and Burks et al. (1997) Proc. Natl. Acad. Sci. USA 94: 412-417).

[0343] The term "non-conservative amino acid substitution" refers to the substitution of an amino acid from one class with an amino acid from another class; for example, substitution of a Class II residue Ala with a Class III residue (eg, Asp, Asn, Glu, or Gin).

[0344] In another example, mutations (conservative or non-conservative) can be introduced randomly along all or part of an anti-CD46 antibody coding sequence, for example, by saturation mutagenesis, and the resultant modified antibodies can be screened for binding activity.

[0345] A "consensus sequence" is a sequence formed by the most commonly occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). Within a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either one may be included in the consensus sequence. The "consensus framework" of an immunoglobulin refers to the framework region in the consensus immunoglobulin sequence.

[0346] Similarly, consensus sequences for the CDRs can be generated by optimally aligning the CDR amino acid sequences of the anti-CD46 antibodies described herein.

[0347] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, typically at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides, with appropriate insertions or deletions of nucleotides. Alternatively, substantial homology exists when a segment will hybridize under selective hybridization conditions to the complement of a strand.

[0348] The percent identity between the two sequences varies with the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions multiplied by 100), taking into account the number of gaps required to introduce for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[0349] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of Meyers and Miller (1989) CABIOS, 4: 11-17, incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm, which has been incorporated into the GAP program in the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0350] The nucleic acid and protein sequences encompassed herein can further be used as "query sequences" to search against public databases, e.g., to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) as described by Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described by Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the individual programs (e.g., XBLAST and NBLAST) can be used.

[0351] Although the nucleic acid compositions described herein (e.g., nucleic acids encoding all or a portion of an anti-CD46 antibody or immunoconjugate) are generally native sequences (except for modified restriction sites, etc.), cDNA, genomic, or mixtures thereof can be mutated according to standard techniques to provide variant sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. Specifically, DNA sequences described herein that are substantially homologous to or derived from native V, D, J, constant sequences, switch sequences, and other such sequences are contemplated (where "derived" indicates that a sequence is identical to or modified from another sequence).

[0352] The term "operably linked" refers to a nucleic acid sequence that is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is operably linked to a coding sequence; or if a ribosome binding site is positioned to promote translation, it is operably linked to a coding sequence. Generally speaking, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linkage is achieved by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide attachments or linkers are used according to conventional practice. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of the sequence, they are operably linked to a coding sequence. With respect to transcriptional regulatory sequences, "operably linked" means that the linked DNA sequences are contiguous and, if necessary, join two protein coding regions that are contiguous and in reading frame. For switch sequences, operably linked indicates that the sequence is capable of effecting switch recombination.

[0353] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA that can be joined to other DNA segments. Another type of vector is a viral vector, in which additional DNA segments can be joined to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome when introduced into the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors used in recombinant DNA technology are typically in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably. However, the present invention is intended to include expression vectors of this type, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), that provide equivalent functions.

[0354] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific host cell, but also to the progeny of such cells. Because certain modifications can occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0355] As used herein, the term "treat" refers to therapeutic or preventive measures as described herein. "Treatment" methods employ administering to a subject (e.g., a subject in need thereof) an anti-CD46 antibody or antigen-binding portion as described herein, or an immunoconjugate comprising such an antibody or antigen-binding portion. In certain embodiments, the subject is diagnosed with a CD46-positive cancer (e.g., prostate cancer) and / or is being treated for a CD46-positive cancer (e.g., prostate cancer) to prevent the disease or condition or recurring disease or condition, cure the disease or condition or recurring disease or condition, delay the disease or condition or recurring disease or condition, lessen the severity of the disease or condition or recurring disease or condition, or ameliorate one or more symptoms of the disease or condition or recurring disease or condition, or prolong the subject's survival beyond the expected survival rate in the absence of such treatment.

[0356] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. CD46-positive cancer refers to cancers characterized by cells that express or overexpress CD46. Illustrative CD46 cancers include, but are not limited to, ovarian, breast, lung, prostate, colon, kidney, and pancreatic cancers.

[0357] As used herein, the term "effective amount" refers to an amount of an anti-CD46 antibody, or antigen-binding portion thereof, and / or immunoconjugate thereof, as described herein, sufficient to achieve treatment, prognosis, or diagnosis of a disease associated with CD46-positive cell growth and / or proliferation (e.g., a CD46-positive cancer) when administered to a subject. The therapeutically effective amount will vary depending on the subject and disease condition to be treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. The dosage can be, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 50 0 ng to about 4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg, about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg, about 30 μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50 μg to about 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg to about 2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175 mg, about 50 0 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875 mg, about 10 mg

[0014] The effective amount is preferably from about 100 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg of an anti-CD46 antibody and / or antigen-binding portion thereof described herein, and / or an immunoconjugate thereof as described herein. The dosage regimen can be adjusted to provide the optimal therapeutic response. An effective amount is also an amount in which any toxic or adverse effects (e.g., side effects) of the antibody or antigen-binding portion thereof are minimized and / or outweighed by the therapeutically beneficial effects.

[0358] The term "patient" includes human and other mammalian individuals receiving prophylactic or therapeutic treatment.

[0359] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject suffering from cancer. In a specific embodiment, the subject is a human. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0360] "Effector" refers to any molecule or combination of molecules whose activity is required for delivery into and / or localization in cells. Effectors include, but are not limited to, markers, cytotoxins, enzymes, growth factors, transcription factors, antibodies, drugs, and the like.

[0361] Phrases such as "inhibiting the growth and / or proliferation" of cancer cells include, inter alia, inducing apoptosis or other cell killing mechanisms, reducing the invasiveness of cells, arresting cells at a point in the cell cycle, and the like.

[0362] The term "immunoconjugate" refers to an antibody or multiple antibodies linked to one or more effectors. The term "immunoconjugate" is intended to include effectors chemically bound to antibodies as well as antibodies expressed as fusion proteins in which the antibody (or a portion thereof) is directly linked or linked through a linker to a peptide effector or an effector comprising a peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0363] Figure 1A Shown are YS5 (SEQ ID NO:1), YS5F (SEQ ID NO:2), YS5vlD (SEQ ID NO:3), SB1HGNY (SEQ ID NO:4), YS12 (SEQ ID NO:5), 3G7RY (also known as 3G8) (SEQ ID NO:6), YS6 (SEQ ID NO:7), YS1 (SEQ ID NO:7) NO:8), YS3 (SEQ ID NO:9), YS4 (SEQ ID NO:10), YS8 (SEQ ID NO:11), YS7 (SEQ ID NO:12), YS9 (SEQ ID NO:13), YS10 (SEQ ID NO:14), YS11 (SEQ ID NO:15), 3G7HY (SEQ ID NO:16), 3G7NY (SEQ ID NO:17), 3G7 (SEQ ID NO:18), SB2 (SEQ ID NO:19), 2C8 (SEQ ID NO:20) and UA8κ (SEQ ID NO:21) VH framework and CDR regions. Figure 1BShown are YS5 (SEQ ID NO:22), YS5F (SEQ ID NO:23), YS5vlD (SEQ ID NO:24), SB1HGNY (SEQ ID NO:25), YS12 (SEQ ID NO:26), 3G7RY (also known as 3G8) (SEQ ID NO:27), YS6 (SEQ ID NO:28), YS1 (SEQ ID NO:29), YS3 (SEQ ID NO:30), YS4 (SEQ ID NO:31), YS8 (SEQ ID NO:32), YS7 (SEQ ID NO:33), YS9 (SEQ ID NO:34), YS10 (SEQ ID NO:35), YS11 (SEQ ID NO:36), 3G7HY (SEQ ID NO:36) NO:37), 3G7NY (SEQ ID NO:38), 3G7 (SEQ ID NO:39), SB2 (SEQ ID NO:40), 2C8 (SEQ ID NO:41) and UA8κ (SEQ ID NO:41) NO:42) of the VL framework and CDR regions.

[0364] Figure 2 YS5 IgG1 KD measurement on Du-145 cells. YS5 was incubated with Du-145 cells overnight at 4°C and binding was analyzed by FACS. Mean fluorescence intensity (MFI) values ​​were curve-fitted using Prism (GraphPad), yielding an estimated KD value of 2.19 + / - 0.73 nM.

[0365] Figure 3 YS12 IgG1 KD measurement on Du-145 cells. YS12 was incubated with Du-145 cells overnight at 4°C and binding analyzed by FACS. MFI values ​​were curve-fitted using Prism (GraphPad), yielding an estimated KD value of 0.043 + / - 0.019 nM.

[0366] Figure 4 Our anti-CD46 antibodies bind to both human and cynomolgus macaque CD46. FACS analysis of CHO cells transfected with human CD46 (left) and cynomolgus macaque CD46 (right). Results for YS5 are shown, but all our antibodies bind to both human and macaque CD46. Ctr IgG: No binding to human IgG1. Ctr: CHO cells stained with secondary antibody only.

[0367] Figure 5YS5 KD measurements on CHO cells transfected with human CD46. YS5 was incubated with CHO-huCD46 cells overnight at 4°C and binding analyzed by FACS. MFI values ​​were curve-fitted using Prism (GraphPad), yielding an estimated K of 0.867 + / - 0.299 nM. D value.

[0368] Figure 6 YS5 KD measurements on CHO cells transfected with cynomolgus macaque CD46. YS5 was incubated with CHO-cynoCD46 cells overnight at 4°C and binding analyzed by FACS. MFI values ​​were curve-fitted using Prism (GraphPad), yielding an estimated KD value of 1.952 + / - 0.508 nM.

[0369] Figure 7 Epitope mapping by competition assay. FACS binding on Du145 cells with or without UA20Fc as a competitor. UA20Fc versus UA20Fc served as a control for complete competition. YS5, YS12, and 3G8 (i.e., 3G7, also known as 3G8) showed distinct competition patterns with SB1HGNY, thus defining two groups with non-overlapping epitopes.

[0370] Figure 8 Competition with the Edmonton strain measles virus H protein. Antibody binding to Du-145 cells in the presence or absence of excess recombinant H protein-Fc fusion was measured by FACS. MFI values ​​were normalized to H protein-Fc versus H protein-Fc as a positive control (total competition) to generate a normalized competition index. Non-CD46-binding, Du-145 cell-binding antibody served as a negative control (lack of competition).

[0371] Figure 9 Internalization by macropinocytosis. YS5 IgG1 was incubated with the metastatic castration-resistant prostate cancer cell line Du-145 and the macropinocytosis indicator ND70-TRITC (Life Technologies) for 4 and 24 hours, respectively. Colocalization was analyzed by Olympus fluorescence confocal microscopy.

[0372] Figure 10 Immunohistochemistry study of an anti-CD46 antibody on an FDA-standard panel of frozen tissue for therapeutic antibody evaluation. Shading indicates the strongest level of positive staining with placental trophoblasts. Signals in non-shaded areas are weak or undetectable.

[0373] Figure 11HPLC analysis of an anti-CD46 antibody-drug conjugate. YS5 IgG1 was conjugated to monomethyl auristatin (MMAF) via an mc-vc-pab linker and analyzed by HIC. The number of peaks above the peak indicates the number of drug molecules. On average, approximately three drug molecules were bound to each IgG molecule.

[0374] Figure 12 Killing curve of prostate cancer cell line LNCaP-C4-2b.

[0375] Figure 13 Killing curve of anti-CD46 ADC (YS5) against metastatic castration-resistant prostate cancer cell line Du-145.

[0376] Figure 14 In vivo tumor killing by an anti-CD46 (YS5) ADC was performed using a subcutaneous prostate cancer xenograft model. SCID mice were subcutaneously implanted with LNCaP-C4-2B cells. Injections were initiated on day -12 at 5 mg / kg ADC and four injections were administered (one every 4-5 days). Mice in the anti-CD46 ADC group were monitored for a longer period after treatment. N = 6.

[0377] Figure 15 CD46 is highly expressed on the multiple myeloma cell line RPMI8226. Anti-CD46 antibodies bind to both prostate cancer (LNCaP) and multiple myeloma cells, while anti-PSMA antibody J591 binds only to prostate cancer cells.

[0378] Figure 16 Anti-CD46 ADC (YS5) kills RPMI8226 cells in vitro. Ctr ADC: unconjugated human IgG1 bound to MMAF.

[0379] Figure 17 In vivo anti-CD46 (YS5) ADC activity. RPMI8226-Luc cells were injected intravenously into NSG mice to form disseminated tumor xenografts. CD46-MMAF: YS5 ADC; IgG-MMAF: control ADC. Injections were given every 4 days for a total of 4 injections. Treatment began on day 10. Three mice in the bortezomib-treated group did not survive past day 31.

[0380] Figure 18 Kaplan-Meier analysis of survival data of RPMI8226 xenograft-bearing mice following YS5 ADC treatment.

[0381] Figure 19 Killing curve of anti-CD46 (YS5) ADC against colorectal cancer cell line HT29.

[0382] Figure 20 .Anti-CD46 ADC (YS5) on MM1S xenografts.

[0383] Figure 21 Kaplan-Meier analysis of mice bearing orthotopic metastatic MM1.S xenografts after ADC treatment. 100% of mice treated with 4 mg / kg anti-CD46 ADC survived until the end of the experiment (day -212). Injections began on day -10 post-implantation. Anti-CD46 was injected as a single dose of 4 mg / kg or four times at two dosing levels (0.8 mg / kg and 4 mg / kg). A control ADC (Ctr ADC) was injected four times at 4 mg / kg.

[0384] Figure 22 Killing curve of anti-CD46 (YS12) ADC against colorectal cancer cell line HT29.

[0385] Figure 23 Killing curve of anti-CD46 (SB1HGNY) ADC against colorectal cancer cell line HT29.

[0386] Figure 24 Killing curve of anti-CD46 YS5 ADC against pancreatic cancer cell line MiaCaPa2.

[0387] Figure 25 Killing curve of anti-CD46 YS5 ADC against mesothelioma cell line M28.

[0388] Figure 26 Killing curve of anti-CD46 YS5 ADC against ovarian cancer cell line OVCAR3.

[0389] Figure 27 Anti-CD46 ADC had no effect on BPH-1 cells. BPH-1 cells express very low levels of CD46 and were unaffected by the YS5 ADC.

[0390] Figure 28 Anti-CD46 (YS5) ADC on HS27 cells. HS27 cells were seeded at 3,000 cells per well.

[0391] Figure 29 The anti-CD46 ADC (YS5) showed minimal toxicity against standard CD3+ T cells. 10,000 CD3+ T cells were seeded in a 96-well culture plate and incubated with various concentrations of YS5 ADC at 37°C for 96 hours. Cell viability was assessed using CCK-8 (Dojindo) assay.

[0392] Figure 30Anti-CD46 ADC had no effect on standard CD14-depleted PBMCs. 10,000 cells were seeded in 96-well culture plates and incubated with various concentrations of ADC at 37°C for 98 hours. Cell viability was determined using a CCK8 counting kit.

[0393] Figure 31 Toxicity assessment of anti-CD46 ADCs in transgenic mice expressing human CD46 Following intravenous injection of 6 mg / kg anti-CD46 and control ADCs, mice were followed for 14 days, sacrificed, and major organs harvested for histological examination.

[0394] Figure 32 CD46 ADCs demonstrate efficacy in an intrafemoral prostate cancer xenograft model. Mice were injected into the femurs of the mCRPC cell line LnCaP-C4-2B harboring a firefly luciferase reporter. On day 7, CD46 ADCs (YS5-mcvcpab-MMAF) were injected every four days for a total of four injections. Mice were monitored post-treatment until day 65. Ctr ADC: unbound IgG1 bound to MMAF (ctr IgG-mcvcpab-MMAF).

[0395] Figure 33 CD46 expression in castration-resistant prostate cancer (CRPC) is shown. Prostate tissue samples are taken from patients with hormonal obstruction and are difficult to cure. Arrows indicate tumor cells (selective tumor area only). H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) is used for staining, followed by detection using the Envision+ system (Dako North America).

[0396] Figure 34 Figure 2 shows bone metastasis of mCRPC. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and subsequently detected using the Envision+ system (North American Dakota).

[0397] Figure 35 Lymph node metastasis of mCRPC is shown. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and subsequently detected using the Envision+ system (North American Dakota).

[0398] Figure 36Bladder metastasis of mCRPC is shown. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and subsequently detected using the Envision+ system (North American Dakota).

[0399] Figure 37 This figure shows that CD46 is highly expressed in the prostate cancer neuroendocrine cell line H660. Left: FACS analysis. Ctr: unbound mAb. Right: Western blot analysis confirms CD46 expression and expression of the neuroendocrine marker NSE in H660 cells.

[0400] Figure 38 Shown is the internalization of anti-CD46 antibodies by the prostate cancer neuroendocrine cell line H660. YS5 IgG1 was incubated with H660 cells overnight. The cells were fixed, permeabilized, stained, and imaged by confocal microscopy. A single confocal slice is shown. Anti-CD46 antibodies are internalized and colocalized with the lysosomal marker LAMP1.

[0401] Figure 39 Demonstrating CD46 ADC killing of H660 cells. Neuroendocrine cell line H660 was incubated with various concentrations of CD46 ADC (YS5-mcvcpab-MMAF) at 37°C for 7 days. Viability was assessed using a Calcein AM assay. Ctr ADC: Unbound IgG1-mcvcpab-MMAF.

[0402] Figure 40 Shown that treatment of the metastatic castration-resistant prostate cancer cell line LNCaP-C4-2B with 10 μM abiraterone (abi) for 7 days results in upregulation of cell surface CD46 expression as measured by FACS. MFI: mean fluorescence intensity.

[0403] Figure 41 Figure 2 shows that LNCaP C4-2B cells treated with abiraterone (Abi) are more sensitive to CD46 ADC. LNCaP-C4-2B cells were cultured with abiraterone for 7 days, washed, and cultured with CD46 ADC for an additional 96 hours in abiraterone-free medium. C4-2B_CD46 ADC: LNCaP C4-2B cells were cultured with CD46 ADC (YS5-mcvcpab-MMAF) without prior exposure to abiraterone (EC50 = 169 pM). C4-2BABI_CD46 ADC: LNCaP C4-2B cells were previously exposed to abiraterone and cultured with CD46 ADC (EC50 = 21 pM). Ctr ADC: Unbound IgG1 bound to MMAF.

[0404] Figure 42 Shown is upregulation of cell surface CD46 on the neuroendocrine cell line H660 following enzalutamide (ENZ) treatment. H660 cells were treated with 10 μM enzalutamide for 7 days, and cell surface antigen expression was analyzed by FACS. CD46 is highly expressed by H660 cells, while prostate-specific membrane antigen (PSMA) is barely detectable. In addition, exposure to enzalutamide upregulates CD46 expression on the tumor cell surface. MFI: median fluorescence intensity.

[0405] Figure 43 CD46 is highly expressed in primary colorectal cancer. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and then detected using the Envision+ system (North American Dakota). Images were acquired using a digital microscope at two magnification levels (4× and 20×).

[0406] Figure 44 CD46 is highly expressed in colorectal cancer that metastasizes to the liver. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and detection was performed using the Envision+ system (North American Dakota). Images were acquired using a digital microscope at two magnification levels (4× and 20×).

[0407] Figure 45 CD46 is highly expressed in colorectal cancer that has metastasized to lymph nodes. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and then detected using the Envision+ system (North American Dakota). Images were acquired using a digital microscope at two magnification levels (4× and 20×).

[0408] Figure 46 CD46 is highly expressed in colorectal cancer that metastasizes to the bladder. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and then detected using the Envision+ system (North American Dakota). Images were acquired using a digital microscope at two magnification levels (4× and 20×).

[0409] Figure 47CD46 is highly expressed in mesothelioma as shown by immunohistochemistry of CD46 staining in mesothelioma. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and subsequently detected using the Envision+ system (North American Dakota).

[0410] Figure 48 Showing that CD46 is highly expressed in pancreatic cancer. Arrows indicate tumor cells (selective tumor areas only). Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and then detected using the Envision+ system (North American Dakota).

[0411] Figure 49 CD46 is overexpressed by glioblastoma multiforme (GBM) as shown by immunohistochemical analysis of CD46 staining in GBM and standard brain. No CD46 staining was observed in normal human brain, but strong staining was observed in GBM specimens. Staining was performed using H-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) and then detected using the Envision+ system (North American Dakota).

[0412] Figure 50 In vivo inhibition of mesothelioma (M28) xenograft growth by CD46 ADC is shown. YS5-mcvcpab-MMAF was injected once every 3-4 days at 5 mg / kg for a total of 5 times (indicated by arrows). Tumor volume was measured by caliper. YSC10 is a control that does not bind human IgG1. DETAILED DESCRIPTION

[0413] In various embodiments, a number of novel anti-CD46 antibodies are provided. The prototype antibodies described herein were identified by a combination of selection on the cell surface and recombinant CD46 using phage and yeast display technologies. These antibodies are internalized via a tumor-selective macropinocytosis pathway without the need for cross-linking and localize to lysosomes, making them well-suited for the development of antibody-drug conjugates (ADCs) and other targeted therapies utilizing intracellular payload release.

[0414] The antibodies described herein are antibodies that bind to domains 1 and 2 of the CD46 molecule rather than the primary complement binding domains 3 and 4, and therefore do not directly block the standard complement sequence cascade.

[0415] Fine epitope mapping also revealed differences in the antigenic contact sites between the anti-CD46 antibodies described herein and the UA20 and 2B10 antibodies described in PCT Application No. PCT / US2008 / 076704 and co-pending U.S. Application No. 14 / 205,101, respectively. Testing on CHO cells transfected with cynomolgus macaque CD46 cDNA revealed that our anti-CD46 antibodies bind to an epitope that is conserved between humans and cynomolgus macaques, thereby identifying species suitable for regulatory toxicology studies.

[0416] Evaluation of therapeutic antibodies using an FDA-approved frozen human tissue panel revealed that the CD46 epitope bound by the antibodies (described herein) is actually expressed at low levels in tissues other than placental trophoblasts and, to a lesser extent, in prostate epithelial cells. In contrast, CD46 has been found to be overexpressed in a variety of tumors, including, but not limited to, prostate cancer, multiple myeloma, colorectal cancer, pancreatic cancer, mesothelioma, lung cancer, breast cancer, ovarian cancer, liver cancer, glioma, neuroblastoma, and the like.

[0417] Because CD46 is located on chromosome 1q32.2 and 1q gain has been observed in a wide range of human cancers, it may be an excellent target for the development of antibody therapies for a variety of malignancies. Antibody-drug conjugates (ADCs) utilizing the anti-CD46 antibodies described herein were constructed and found to kill CD46-overexpressing cancer cell lines in vitro, including (but not limited to) metastatic castration-resistant prostate cancer, multiple myeloma, colorectal cancer, mesothelioma, ovarian cancer, and others. Most importantly, the anti-CD46 ADCs described herein exhibited strong in vivo anti-tumor activity, significantly reducing tumor burden in xenograft models of castration-resistant prostate cancer and multiple myeloma. This potent anti-tumor activity is also believed to be applicable to other CD46-overexpressing tumor models. The studies described herein therefore validate CD46 as a tumor cell surface antigen suitable for the development of targeted therapies. In addition, the anti-CD46 antibodies described herein may be used as companion diagnostics for patient stratification and monitoring treatment outcomes.

[0418] In view of these findings, it is believed that the anti-CD46 antibodies described herein specifically bind to and internalize into cells that express or overexpress CD 46. Because CD46 is expressed / overexpressed by many cancers, including but not limited to ovarian, breast, lung, prostate, colon, kidney, pancreatic, mesothelioma, lymphoma, liver, urothelial, gastric, and cervical cancers, these antibodies can be used to specifically target and internalize into these and other CD46-positive cancer cells.

[0419] In certain embodiments, these antibodies can be used without an attached effector on cells (specifically cancer cells) for their inherent cytotoxic and / or cytostatic and / or anti-proliferative activity. In certain embodiments, these antibodies can be attached to one or more effectors (e.g., a second antibody, a detectable label, a cytotoxin, a drug-containing liposome, a radionuclide, a drug, a prodrug, a virion, a cytokine, a chelate, etc.), thereby forming an immunoconjugate that will specifically bind and internalize into cancer cells that express or overexpress CD46. In certain embodiments, multiple effectors will be attached to a single antibody, or in certain embodiments, multiple antibodies will be attached to a single effector, or in certain embodiments, a single antibody will be attached to a single antibody.

[0420] In various embodiments, methods of using these antibodies and / or immunoconjugates are provided. In certain embodiments, the methods involve contacting cells that express or overexpress CD46 (e.g., cancer cells, such as ovarian cancer cells, breast cancer cells, lung cancer cells, prostate cancer cells, colon cancer cells, renal cancer cells, pancreatic cancer cells, etc.) with a construct, resulting in internalization of the construct (or portion thereof) into the cell and thereby delivering the effector to the target cell. In certain embodiments, "contacting" comprises administering the antibody or construct to a subject in need thereof (e.g., a human or non-human mammal).

[0421] Antibodies that bind to CD46

[0422] For example, the antibodies were found to specifically bind to CD46 (specifically domains 1 and / or 2) and to be internalized on-site by prostate (and other CD46-positive cancer cells) when the cancer cells are in the tissue microenvironment. As noted above, such antibodies are useful for targeting cancer when used alone or when linked to an effector to form a "targeted effector."

[0423] Thus, in certain embodiments, isolated antibodies are provided that specifically bind to CD46 and are internalized via macropinocytosis into cells expressing or overexpressing CD46 (e.g., prostate cancer cells). In various embodiments, the antibodies bind to domain 1 and / or domain 2 of CD46. In various embodiments, the antibodies do not bind to domain 3 and / or domain 4 of CD46.

[0424] The antibodies designated herein as YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also referred to as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8κ (see, e.g., Table 1) are illustrative prototype antibodies. In certain embodiments, antibodies comprising the VL CDR1 and / or VLCDR2, and / or VL CDR3, and / or VH CDR1 and / or VH CDR2, and / or VH CDR3 of one or more of these antibodies are contemplated. In certain embodiments, antibodies comprising the VH domain and / or VL domain of one or more of the Zhexi antibodies are contemplated. Also contemplated are antibodies that compete for binding to CD46 with one or more of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ.

[0425] The amino acid sequences of the VH and VL domains of the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8 kappa antibodies are shown in Table 1 (see Example 1).

[0426] Table 1. Novel human anti-CD46 antibody sequences. YS5 and YS5F differ by one amino acid in the VH CDR1 (L vs. F). YS5 and YS5v1D have the same VH but one amino acid difference in the VL CDR2 (N vs. D). 3G7HY, 3G7NY, 3G7RY (also known as 3G8), and 3G7 differ by one residue in the VH CDR3 but have completely different VLs. YS6 and 3G7 have the same VH but different VLs.

[0427]

[0428]

[0429]

[0430]

[0431] In various embodiments, the antibodies encompassed herein specifically exclude antibodies that are prepared from three VH CDRs and / or three VL CDRs of antibodies 3051.1, G12FC3, M6c42b, 4F3YW, M40pr146, UA20, UA8, 585II41, 585II41.1, 585II56, 3076, 3051, M49R, RCI-14, II79_4, II79_3, T5II-4B.1, T5II-4B.2, RCI-11, RCI-20, CI-11A, CI-14A, S95-2 and / or mPA7 antibodies described in PCT / US2008 / 076704 (WO2009 / 039192). The amino acid sequences of the VH and VL chains of these antibodies and the CDRs comprising these domains are shown in PCT / US2008 / 076704, and the amino acid sequences of these domains are reproduced in Table 2 below.

[0432] Table 2. Excluded antibodies. The antibodies shown below are scFv antibodies (VL and VH regions joined by a (Gly4Ser)3 (SEQ ID NO: 43)) linker, however, it will be appreciated that other antibody formats comprising CDRs (or VH and / or VL domains) are similarly excluded.

[0433]

[0434]

[0435]

[0436]

[0437] Using the amino acid sequences provided for YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8κ antibodies, a variety of antibody formats can be prepared, for example, as described below. Such formats include, but are not limited to, substantially intact (e.g., full-length) immunoglobulins (e.g., IgA, IgE, IgG, etc.), antibody fragments (e.g., Fv, Fab, (Fab')2, (Fab')3, IgGD, CH2, minibodies, etc.), single-chain antibodies (e.g., scFv), diabodies, monobodies, affibodies, etc.

[0438] It will be appreciated that when the antibody is a single-chain antibody, the VH and VL domains comprising such an antibody may be joined together directly or through a peptide linker. Illustrative peptide linkers include, but are not limited to, GGGGS GGGGS GGGGS (SEQ ID NO: 67), GGGGS GGGGS (SEQ ID NO: 68), GGGGS (SEQ ID NO: 69), GS GGGGS GGGGS GGS GGGGS (SEQ ID NO: 70), SGGGGS (SEQ ID NO: 71), GGGS (SEQ ID NO: 72), VPGV (SEQ ID NO: 73), VPGVG (SEQ ID NO: 74), GVPGVG (SEQ ID NO: 75), GVG VP GVG (SEQ ID NO: 76), VP GVG VPGVG (SEQ ID NO: 77), GGSSRSS (SEQ ID NO: 78), and GGSSRSSSSGGGGSGGGG (SEQ ID NO: 79), among others.

[0439] As indicated above, in various embodiments, the antibodies bind (e.g., specifically bind) to CD46 (e.g., domains 1 and / or 2). Typically, the antibodies contemplated herein will specifically bind to prostate cancer cells, including but not limited to, cell lines selected from the group consisting of: DU145 cells, PC3 cells, and LnCaP cells. In certain embodiments, the antibodies bind to prostate cancer cells at a concentration greater than (K) when measured by FACS on live prostate tumor cells. D In certain embodiments, the affinity of the antibody is greater than (K D less than) about 1 nm, or about 100 pM, or about 50 pM, or about 10 pM, or about 1 pM.

[0440] Using the sequence information provided herein, antibodies comprising one or more of the CDRs of, for example, YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8κ, or antibodies comprising the VH and / or VL domains of these antibodies, are readily prepared using standard methods well known to those skilled in the art (e.g., as described below), such as chemical synthesis methods and / or recombinant expression methods.

[0441] Additionally, other "related" prostate cancer-specific antibodies can be screened for binding to the same epitope (e.g., an epitope that competes with one or more of the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ antibodies for binding to CD446 and / or cells expressing or overexpressing CD46 (e.g., prostate cancer cells)). Antibodies identified herein and / or by modifying the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ antibodies identified herein to generate modified antibody libraries and then rescreening the antibodies in the libraries for improved binding to and / or internalization into cells that express or overexpress CD46, e.g., prostate cancer cells.

[0442] Identify and compare with YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ bind to the same CD46 epitope. It antibodies.

[0443] Having identified CD46 (particularly domains one and / or two) as a useful antibody target and YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8κ antibodies as useful prototype antibodies, other "related" antibodies that bind to CD46 and are preferably internalized via macropinocytosis can be readily identified, for example, by raising antibodies (e.g., monoclonal antibodies) that specifically bind to domains 1 and / or 2 of CD46 and by screening for antibodies that bind to domains 1 and / or 2 of CD46. Additionally or alternatively, other antibodies that bind to CD46 and are internalized by macropinocytosis can be identified by screening, for example, YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or Antibodies that cross-react with the epitope bound by UA8κ and one or more of antibodies YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ; Antibodies that cross-react with one or more of YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ to bind to prostate cancer cells (e.g., CaP cells, PC3 cells, etc.); and / or antibodies that cross-react with idiotypic antibodies raised against YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ antibodies.

[0444] Monoclonal antibodies.

[0445] Monoclonal antibodies that bind to the epitope bound by one or more of CD46 domains 1 and / or 2, preferably YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ, can be generated using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein (1975) Nature 256:495, viral or oncogenic transformation of B lymphocytes, or phage display techniques using human antibody gene repertoires. In specific embodiments, the antibody is a fully human monoclonal antibody.

[0446] Thus, in one embodiment, antibodies that bind to CD46, preferably domain 1 and / or domain 2 of CD46, are produced using a hybridoma approach. In this approach, mice or other appropriate host animals can be immunized with a suitable antigen to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes can then be fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells (Goding (1986) Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press)). Culture medium containing the hybridoma cells is analyzed for the production of monoclonal antibodies against the antigen. After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Id.). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the fusion tumor cells can be grown into ascites tumors in animals in vivo. The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites or serum by conventional immunoglobulin purification procedures (e.g., protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography).

[0447] In another embodiment, antibodies and antibody portions that bind to domains 1 and / or 2 of CD46 can be used with, for example, McCafferty et al. (1990) Nature, 348:552-554, Clackson et al. (1991) Nature, 352:624-628, Marks et al. (1991) J. Mol. Biol., 222:581-597, Hoet et al. (2005) Nature Biotechnol., 23:344-348; Ladner et al. (2006) Nature Biotechnol., 23:350-351; and Nos. 5,223,409; 5,403,484; and 5,571,698 to ) et al.; 5,427,908 and 5,580,717 to Dower et al.; 5,969,108 and 6,172,197 to McCafferty et al.; and 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081 to Griffiths et al. Additionally, high affinity (nM range) human antibodies can be made by chain shuffling (Marks et al. (1992) Bio / Technology, 10:779-783) and combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al. (1993) Nucl. Acids. Res., 21:2265-2266).

[0448] In a specific embodiment, monoclonal antibodies or antigen-binding portions thereof that bind to an epitope bound by one or more of CD46, preferably YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ, are produced using the phage display technology described in Hoet et al., supra. This technology involves the generation of human Fab libraries having a unique combination of immunoglobulin sequences isolated from human donors and having synthetic diversity in the heavy chain CDRs. The library is then screened for Fabs that bind to CD46, preferably competing for binding to one or more of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ.

[0449] In another embodiment, human monoclonal antibodies to CD46, preferably comprising an epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ, can be generated using transgenic or transchromosomic mice that carry parts of the human immune system rather than the mouse system (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859; Lonberg and Huszar, (1995) Int. Rev. Immunol. 114). 3:65-93, Harding and Lonberg (1995) Ann. NY. Acad. Sci. 764:536-546, and Lonberg and Kay, U.S. Patents Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425 No. 5,789,650; No. 5,877,397; No. 5,661,016; No. 5,814,318; No. 5,874,299; and No. 5,770,429; U.S. Patent No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962 to Lonberg and Kay; and PCT Publication No. WO 01 / 14424 to Korman et al.).

[0450] In another embodiment, human antibodies to CD46 that preferably bind to an epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ can be raised using mice that carry human immunoglobulin sequences on a transgene and a transchromosome, e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome (see, e.g., PCT Publication WO 02 / 43478 to Ishida et al.).

[0451] Alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CD46 antibodies of the present invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and 6,162,963 to Kucherlapati et al.

[0452] Alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CD46 antibodies encompassed herein. For example, mice carrying human heavy chain transchromosomes and human light chain transchromosomes can be used; as described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. In addition, cattle carrying human heavy and light chain transchromosomes have been described in the art (e.g., see Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti-CD46 CCP1 antibodies.

[0453] In another embodiment, antibodies that specifically bind to CD46, preferably to an epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ, can be prepared using transgenic plants and / or cultured plant cells that produce such antibodies (e.g., tobacco, corn, and duckweed). For example, transgenic tobacco leaves expressing an antibody, or antigen-binding portion thereof, can be used to produce such antibodies, for example, by using an inducible promoter (see, e.g., Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118). In addition, transgenic corn can be used to express such antibodies and antigen-binding portions thereof (see, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464: 127-147). Antibodies can also be produced in large quantities from transgenic plant seeds comprising antibody portions, such as single-chain antibodies (scFv), using, for example, tobacco seeds and potato tubers (see, e.g., Conrad et al. (1998) Plant Mol. Biol. 38: 101-109). Methods for making antibodies or antigen-binding portions in plants can also be found, for example, in Fischer et al. (1999) Biotechnol. Appl. Biochem. 30:99-108, Ma et al. (1995) Trends Biotechnol. 13:522-527, Ma et al. (1995) Plant Physiol. 109:341-346; Whitelam et al. (1994) Biochem. Soc. Trans. 22:940-944 and U.S. Pat. Nos. 6,040,498 and 6,815,184.

[0454] The binding specificity of a monoclonal antibody or portion thereof that binds to CD46, preferably comprising an epitope that preferentially binds to one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ, prepared using any of the techniques disclosed herein, can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody or portion thereof can also be determined by Scatchard analysis of Munson et al. (1980) Anal. Biochem., 107:220.

[0455] Cross-reactivity with anti-idiotypic antibodies.

[0456] The idiotype represents the highly variable antigen binding site of the antibody and is itself immunogenic. During the generation of an antibody-mediated immune response, an individual will produce antibodies against the antigen as well as anti-idiotype antibodies whose immunogenic binding site (idiotype) mimics the antigen.

[0457] Anti-idiotypic antibodies can be raised against the variable regions of the antibodies identified herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) using standard methods well known to those skilled in the art. Briefly, anti-idiotypic antibodies can be made by injecting an antibody of the invention, or fragments thereof (e.g., CDRs), into an animal, thereby eliciting antisera against various antigenic determinants on the antibody, including those in the idiotypic region.

[0458] Methods for producing anti-analyte antibodies are well known in the art. Large molecular weight antigens (greater than about 5000 Daltons) can be injected directly into animals, while small molecular weight compounds (less than about 5000 Daltons) are preferably coupled to a high molecular weight immunogenic carrier (usually a protein) to render them immunogenic. Antibodies produced in response to immunization can be used in serum, ascites, immunoglobulin (Ig) fractions, IgG fractions, or affinity-purified monospecific materials.

[0459] Polyclonal anti-idiotypic antibodies can be prepared by immunizing an animal with the antibodies of the present invention, prepared as described above. Generally, it is desirable to immunize an animal that is of the same species as the animal producing the antibodies (e.g., a phage display library) and is allotypicly matched to it. This minimizes the production of antibodies against non-idiotypic determinants. The antiserum thus obtained is then typically fully absorbed against standard serum from the same species that produced the phage display library, thereby eliminating antibodies against non-idiotypic determinants. Absorption can be achieved by passing the antiserum through a gel formed by cross-linking standard (non-immune) serum proteins with glutaraldehyde. Antibodies with anti-idiotypic specificity will pass directly through the gel, while those with specificity for non-idiotypic determinants will bind to the gel. Immobilizing the non-immune serum proteins on an insoluble polysaccharide support (e.g., agarose gel) also provides a matrix suitable for absorption.

[0460] Monoclonal anti-idiotypic antibodies can be made using the method of Kohler et al. (1975) Nature 256:495. Specifically, monoclonal anti-idiotypic antibodies can be made using the hybridoma technique, which comprises fusing (1) splenocytes from mice immunized with an antigen or hapten-carrier conjugate of interest (i.e., an antibody of the invention or a subsequence thereof) with (2) a mouse myeloma cell line selected for resistance to a drug (e.g., 8-azaguanine). Generally, it is desirable to use a myeloma cell line that does not secrete immunoglobulins. Several such lines are known in the art. A generally preferred cell line is P3X63Ag8.653. This cell line is deposited with the American Type Culture Collection as CRL-1580.

[0461] Fusion can be performed in the presence of polyethylene glycol according to existing methods (see, for example, Monoclonal Antibodies, R. Kennett, J. McKearn, and K. Bechtol, eds., New York, NY, Plenum Press, 1980, and Current Topics in Microbiology & Immunology, Vol. 81, F. Melchers, M. Potter, and NL Warner, eds., New York, NY, Springer-Verlag, 1978). The resulting mixture of fused and unfused cells is plated in hypoxanthine-aminopterin-thymidine (HAT) selective medium. Under these conditions, only hybrid cells will grow.

[0462] When the cells are fully grown (usually 10-14 days after fusion), the culture medium is collected and screened for the presence of monoclonal idiotypic anti-analyte antibodies by any of a number of methods including solid phase RIA and enzyme-linked immunosorbent assay. Cells from culture wells containing antibodies with the desired specificity are then expanded and recloned. Cells from those cultures that are positive for the antibody of interest are then typically passaged into ascites tumors in susceptible histocompatible pristane-primed mice.

[0463] Ascites fluid is collected by tapping the peritoneal cavity, retested for antibodies, and purified as described above. If a non-secreting myeloma line is used in the fusion, affinity purification of the monoclonal antibody is generally not necessary because the antigen-binding characteristics of the antibody are already homogeneous. It is only necessary to separate it from contaminating proteins in the ascites fluid, i.e., to generate the immunoglobulin fraction.

[0464] Alternatively, the hybrid cell strain of being paid close attention to can be grown in serum-free tissue culture and collect antibodies from culture medium. Generally speaking, this is not the ideal method for obtaining a large amount of antibodies, because output is low. It is also possible to transmit cells intravenously in mice and collect antibodies from serum. This method is not preferred usually, because a small amount of serum can be obtained when bleeding and because large-scale purification from other serum components is needed. However, some hybridomas will not grow into ascites tumors, and therefore must use a kind of in these alternative methods of obtaining antibodies.

[0465] Compatible with YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, Cross-reactivity with YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ.

[0466] In another approach, antibodies that bind to CD46 can be identified by binding to the same epitope as a "prototype" antibody of the invention (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ). Isolation of the target epitope is not necessary to identify such antibodies. In certain embodiments, antibody libraries can be screened for antibodies that compete with the prototype antibodies of the invention for binding to prostate cancer cells (e.g., CaP cells, PC3 cells, etc.) and / or are internalized by such cells, and / or compete for binding to CD46.

[0467] Methods of screening libraries for epitope binding and / or cell binding and / or internalization are well known to those skilled in the art. In certain embodiments, the cross-reactive prostate antibodies show at least 60%, preferably 80%, more preferably 90%, and most preferably at least 95% or at least 99% cross-reactivity with one or more of the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8 kappa antibodies described herein.

[0468] Select other "related" anti-CD46 antibodies for phage display.

[0469] Various phage display (or yeast display) methods can be used to display the known sequences of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ and / or other prostate-specific antibodies. Additional antibodies are generated that specifically bind to CD46 with the same or even higher affinity, preferably binding to the epitope to which YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ bind.

[0470] Chain shuffling method.

[0471] One method for generating antibody variants has been to replace the original VH or VL gene with an antibody repertoire of V genes to generate new partners in a phage display or yeast display library (chain shuffling) (Clackson et al. (1991) Nature 352: 624-628). Using chain shuffling and phage display, the affinity of human scFv antibody fragments binding to the hapten phenyloxazolone (phOx) was increased from 300 nM to 1 nM (300-fold) (Marks et al. (1992) Bio / Technology 10: 779-783).

[0472] Thus, for example, to alter the affinity of an anti-CD46 antibody described herein, a library of mutant scFv genes can be generated containing the V sequences of the prototype YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8 kappa antibodies. H Genes (e.g. Figure 2 shown) and human V L Genetic antibody library (light chain shuffling). The scFv gene antibody library can be cloned into a phage display vector, such as pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137) or other vectors, and after transformation, a library of transformants is obtained.

[0473] Similarly, for heavy chain shuffling, a library of mutant scFv genes can be generated containing the V sequences of the prototype YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8 kappa antibodies. L Genes (e.g. Figure 2 shown) and human V H Genetic antibody library (heavy chain shuffling). The scFv gene antibody library can be cloned into a phage display vector, such as pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137) or other vectors, and after transformation, a library of transformants is obtained.

[0474] The resulting libraries can be screened for related targets (e.g., CD46) and / or cross-reactivity with YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ.

[0475] Site-directed mutagenesis to improve binding affinity.

[0476] The majority of antigen-contacting amino acid side chains are typically located in the complementarity determining regions (CDRs), with three in the V H(CDR1, CDR2 and CDR3) and three in V L The CDR1, CDR2, and CDR3 residues are located in the CDR2 region of the CDR3 region (Chothia et al. (1987) J. Mol. Biol., 196:901-917; Chothia et al. (1986) Science, 233:755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues are responsible for most of the binding energetics of the antibody's affinity for the antigen. In other molecules, mutating amino acids that contact the ligand has been shown to be an effective way to increase the affinity of a protein molecule for its binding partner (Lowman et al. (1993) J. Mol. Biol., 234:564-578; Wells (1990) Biochemistry, 29:8509-8516). Site-directed mutagenesis of the CDRs and screening against prostate cancer cells (particularly for binding at CD46, such as described herein in the Examples) can generate antibodies with improved binding affinity.

[0477] CDR randomization to generate higher affinity human scFv.

[0478] In a simple site-directed mutagenesis extension, a library of mutant antibodies can be generated in which some or all of the CDRs are randomized (VL CDR1 CDR2 and / or CDR3 and / or VH CDR1, CDR2 and / or CDR3). In one embodiment, each CDR is randomized in a separate library using a known antibody (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ) as a template. Additive increases in affinity are achieved by combining the CDR sequences of the highest affinity mutants from each CDR library. A similar approach has been used to randomize the affinity of human growth hormone (hGH) for the growth hormone receptor from 3.4×10 -10 to 9.0×10 -13 M increased more than 1500-fold (Lowman et al. (1993) J. Mol. Biol., 234: 564-578).

[0479] V HCDR3 typically occupies the center of the binding pocket, and therefore mutations in this region may result in improved affinity (Clackson et al. (1995) Science, 267:383-386). In one embodiment, V H CDR3 residues were randomized (see, e.g., Schier et al. (1996) Gene, 169: 147-155; Schier and Marks (1996) Human Antibodies and Hybridomas, 7: 97-105, 1996; and Schier et al. (1996) J. Mol. Biol. 263: 551-567).

[0480] Other Antibody Modifications.

[0481] In one embodiment, partial antibody sequences derived from YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8 kappa antibodies can be used to make structurally and functionally related antibodies. For example, antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than between sequences outside of the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors comprising CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al. (1998) Nature 332: 323-327; Jones et al. (1986) Nature 321: 522-525; and Queen et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences.

[0482] Thus, one or more structural features (e.g., CDRs) of the anti-CD46 antibodies of the invention can be used to form structurally related anti-CD46 antibodies that retain at least one functional property of, for example, the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ antibodies, which antibodies bind to and internalize into prostate cancer cells, for example.

[0483] In a specific embodiment, one or more YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8 kappa CDR regions (e.g., VH CDR1, and / or CDR2, and / or CDR3, and / or VL CDR1, and / or CDR2, and / or CDR3) are recombinantly combined with human framework regions and CDRs to form additional recombinantly engineered anti-CD46 antibodies. The heavy and light chain variable framework regions can be derived from the same or different antibody sequences.

[0484] It is well known in the art that the antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of the antibody for the antigen (see, e.g., Hall et al. (1992) J. Immunol., 149: 1605-1612; Polymenis et al. (1994) J. Immunol., 152: 5318-5329; Jahn et al. (1995) Immunobiol., 193: 400-419; Klimka et al. (2000) Brit. J. Cancer. =Cell Biology, 83: 252-260; Beiboer et al. (2000) J. Mol. Biol., 296: 833-849; Rader et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 8910-8915; Barbas et al. (1994) J. Am. Chem. Soc., 116: 2161-2162; Ditzel et al. (1996) J. Immunol., 157: 739-749). Thus, in certain embodiments, antibodies are generated that comprise the heavy and / or light chain CDR3s of specific antibodies described herein (e.g., YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ). Thus, in certain embodiments, antibodies are generated that comprise the heavy and / or light chain CDR1s of specific antibodies described herein (e.g., YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ). The antibody can further include other heavy and / or light chain CDRs of an antibody of the invention (e.g., YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ).

[0485] In certain embodiments, the CDR1, 2, and / or 3 regions of the engineered antibodies described above may comprise the precise amino acid sequences disclosed herein (e.g., CDRs of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ). However, one of ordinary skill in the art will appreciate that some deviations from the precise CDR sequences may exist while still retaining the ability of the antibody to effectively bind CD46 (e.g., conservative amino acid substitutions). Thus, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98%, 99% or 99.5% identical to one or more CDRs of the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ antibodies.

[0486] In another embodiment, one or more residues of a CDR can be altered to modify binding, resulting in a more favorable binding. Using this strategy, one can achieve a CDR having, for example, 10 10 M -1 Antibodies with ultra-high binding affinity or higher. Affinity maturation techniques well known in the art and described herein can be used to alter CDR regions, and the resulting binding molecules can then be screened for the desired changes in binding. Thus, as CDRs are altered, changes in binding affinity and immunogenicity can be monitored and scored, allowing for the achievement of antibodies optimized for binding and low immunogenicity for optimal combinations.

[0487] In addition to or as an alternative to modifications within the CDRs, modifications may be made within one or more of the framework regions FR1, FR2, FR3, and FR4 of the heavy and / or light chain variable regions of the antibody, as long as these modifications do not abolish the binding affinity of the antibody.

[0488] In another embodiment, the antibody is further modified with respect to effector function, for example, to improve the utility of the antibody in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby forming interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complementary sequence-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Caron et al. (1992) J. Exp Med. 176: 1191-1195; Shopes (1992) J. Immunol. 148: 2918-2922). Homodimeric antibodies with improved anti-tumor activity can also be prepared using heterobifunctional cross-linkers (see, e.g., Wolff et al. (1993) Cancer Res. 53: 2560-2565). Alternatively, antibodies can be engineered that have dual Fc regions and may thereby have improved complementary sequence lysis and ADCC capabilities (see, e.g., Stevenson et al. (1989) Anti-Cancer Drug Design 3:219-230).

[0489] Antibody production.

[0490] In various embodiments, the antibodies described herein can be produced by chemical synthesis or can be recombinantly expressed.

[0491] Chemical synthesis.

[0492] Using the sequence information provided herein, the CD46-specific antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ) or variants thereof can be chemically synthesized using well-known peptide synthesis methods. Solid-phase synthesis, in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence, is a preferred method for chemically synthesizing single-chain antibodies. Solid phase synthesis techniques are described by Barany and Merrifield, Solid Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A, Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156; pp. 3-284 and Stewart et al., (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111.

[0493] Recombinant expression of prostate cancer-specific antibodies.

[0494] In certain embodiments, the CD46-specific antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ), or variants thereof, are recombinantly expressed using methods well known to those skilled in the art. For example, using the YS5, S5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ sequence information provided herein, nucleic acids encoding the desired antibodies can be prepared according to a variety of standard methods known to those skilled in the art. The nucleic acids are transfected into host cells, which then express the desired antibody or its chain.

[0495] Molecular cloning techniques for achieving these goals are known in the art. A variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Examples of these techniques and instructions sufficient to guide one skilled in the art through many cloning exercises can be found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (Sambrook); and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates and John Wiley & Sons. Associates, Inc. and John Wiley & Sons, Inc.), (1994 Supplement) (Ausubel). Methods for producing recombinant immunoglobulins are also known in the art. See Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86:10029-10033. Specific protocols for expressing antibodies are also provided by Liu et al. (2004) Cancer Res. 64:704-710 and Poul et al. (2000) J. Mol. Biol. 301:1149-1161.

[0496] Formation of other antibody forms.

[0497] Using the known and / or identified sequences of the single chain antibodies provided herein (e.g., V H and / or V Lsequences), can form other antibody formats. Such formats include (but are not limited to) multivalent antibodies, complete antibodies, scFv, (scFv')2, Fab, (Fab')2, chimeric antibodies, etc.

[0498] Forming homodimers.

[0499] For example, to form a (scFv')2 antibody, two anti-CD46 antibodies are joined by a linker (e.g., a carbon linker, a peptide, etc.) or by a disulfide bond, for example, between two cysteines. Thus, for example, to form a disulfide-linked scFv, a cysteine ​​residue can be introduced by site-directed mutagenesis at the carboxyl termini of the antibodies described herein.

[0500] scFv can be expressed from this construct, purified by IMAC, and analyzed by gel filtration. To make (scFv')2 dimers, cysteine ​​is reduced by incubation with 1mM 3-mercaptoethanol, and half of the scFv is blocked by adding DTNB. Blocked and unblocked scFv are incubated together to form (scFv')2, and the resulting material can be analyzed by gel filtration. The affinity of the resulting dimer can be determined using standard methods, such as by BIAcore.

[0501] In one illustrative embodiment, (scFv')2 dimers are formed by joining scFv fragments via a linker, such as a peptide linker. This can be achieved in a variety of ways well known to those skilled in the art. For example, one method is described by Holliger et al. (1993) in Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94 / 13804).

[0502] It should be noted that the V H and / or V L Sequence, but also easy to prepare Fab and (Fab')2 dimer. Fab is connected to V by disulfide bond H -C H 1 light chain and is readily formed using standard methods known to those skilled in the art. For example, as described above for (scFv')2 dimers, F(ab)'2 can be produced by dimerizing Fab.

[0503] Chimeric antibodies.

[0504] The antibodies encompassed herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855).

[0505] Although the prototype antibodies provided herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) are fully human antibodies, chimeric antibodies are encompassed, particularly when such antibodies are used in species other than humans (e.g., veterinary applications). A chimeric antibody is an antibody that comprises portions from two different species (e.g., human and non-human portions). Typically, the antigen-combining regions (or variable regions) of the chimeric antibody are derived from one species of origin, and the constant regions (which confer biological effector function to the immunoglobulin) of the chimeric antibody are derived from another source. Many methods for producing chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369, and PCT application WO 91 / 0996).

[0506] In general, the process for making chimeric antibodies consists of the following steps (the order of some steps can be interchanged): (a) identifying and cloning the correct gene segment encoding the antigen-binding portion of the antibody molecule; this gene segment (called the VDJ variable diversity and joining region of the heavy chain, or the VJ variable joining region of the light chain, or simply V or variable region or V H and V LThe present invention relates to a method for producing a chimeric antibody comprising: (a) cloning a gene segment encoding a human constant region or a desired portion thereof; (b) cloning a gene segment encoding a human constant region or a desired portion thereof; (c) joining the variable region to the constant region to encode the entire chimeric antibody in a transcribable and translatable form; (d) joining this construct to a vector containing a selectable marker and gene control regions (e.g., a promoter, enhancer, and poly(A) addition signal); (e) amplifying this construct in a host cell (e.g., bacteria); (f) introducing the DNA into eukaryotic cells (transfection), typically mammalian lymphocytes; and culturing the host cells under conditions suitable for expression of the chimeric antibody.

[0507] Several antibodies with unique antigen binding specificities have been manipulated by these approaches to generate chimeric proteins (e.g., anti-TNP: Boulianne et al. (1984) Nature, 312: 643) and anti-tumor antigens (see, e.g., Sahagan et al. (1986) J. Immunol., 137: 1066). Similarly, several different effector functions have been achieved by linking new sequences to those encoding the antigen binding region. Some of these include enzymes (Neuberger et al. (1984) Nature, 312: 604), immunoglobulin constant regions from another species, and the constant region of another immunoglobulin chain (Sharon et al. (1984) Nature, 309: 364; Tan et al. (1985) J. Immunol., 135: 3565-3567).

[0508] In certain embodiments, a cell line producing an anti-CD46 (e.g., prostate cancer-specific) antibody is transfected with a recombinant DNA vector. The novel recombinant DNA vector contains a "replacement gene" that replaces all or a portion of a gene encoding an immunoglobulin constant region in the cell line (e.g., a replacement gene can encode all or a portion of the constant region of a human immunoglobulin, a specific immunoglobulin class, or an enzyme, toxin, biologically active peptide, growth factor, inhibitor, or linker peptide to facilitate binding to a drug, toxin, or other molecule), and a "target sequence" that allows for targeted homologous recombination with the immunoglobulin sequence within the antibody-producing cell.

[0509] In another embodiment, a cell line is transfected with a recombinant DNA vector to produce an antibody with desired effector function (e.g., a constant region of a human immunoglobulin), in which case the replacement gene contained in the recombinant vector can encode the prostate cancer-specific antibody of the present invention and all or part of the region of the target sequence contained in the recombinant vector to allow homologous recombination and targeted gene modification in the antibody-producing cell. In another embodiment, when only a portion of the variable region or constant region is replaced, the resulting chimeric antibody can be altered or improved while still defining the same antigen and / or having the same effector function, such that the chimeric antibody can demonstrate higher antigen specificity, higher affinity binding constant, increased effector function or increased secretion, and be manufactured by a transfected antibody-producing cell line, etc.

[0510] Regardless of the example performed, methods of selecting for integrated DNA (via a selectable marker), screening for chimeric antibody production, and cell cloning can be used to obtain clones of cells that produce chimeric antibodies.

[0511] Therefore, the modified DNA fragment encoding the monoclonal antibody can directly target the site of the immunoglobulin gene expressed in the B cell or hybridoma cell line. DNA constructs can be prepared for any specific modification to change the protein product of any monoclonal cell line or hybridoma. When the gene is in its natural chromosomal position rather than a random position, the expression level of the chimeric antibody should be higher. Detailed methods for preparing chimeric (humanized) antibodies can be found in U.S. Patent No. 5,482,856.

[0512] Fully human antibodies.

[0513] In another embodiment, the present invention provides complete fully human anti-CD46 (e.g., prostate cancer specific) antibodies. Such antibodies are readily produced using a method similar to that used to prepare chimeric human antibodies. In this example, instead of using, for example, a recognition function derived from a murine species, the fully human recognition function of the antibodies described herein (e.g., V H and V L ).

[0514] Bifunctional antibodies.

[0515] In certain embodiments, it is contemplated that the V H and V L The term "bifunctional antibody" refers to an antibody fragment that typically has two antigen-binding sites. The fragment typically comprises the same polypeptide chain (V H -V L ) is connected to the light chain variable domain (V L ) of the heavy chain variable domain (V HBy using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and form two antigen-binding sites. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 93 / 11161 and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0516] Monoclonal antibody.

[0517] In certain embodiments, using the sequence information provided herein, anti-CD46 antibodies can be constructed as monoclonal antibodies. Monoclonal antibodies are an antibody technology that produces stable smaller antibody formats with a therapeutic window longer than expected for some small antibody formats. In certain embodiments, monoclonal antibodies are made from IgG4 antibodies by eliminating the hinge region of the antibody. Unlike full-sized IgG4 antibodies, half-molecule fragments are very stable and are called monoclonal antibodies. The IgG4 molecule is cut in half, leaving only one area on the monoclonal antibody that can bind to the target. Methods for making monoclonal antibodies are described in detail in PCT Publication WO2007 / 059782, which is incorporated herein by reference in its entirety (see also Kolfschoten et al. (2007) Science 317: 1554-1557).

[0518] Affinity antibodies.

[0519] In certain embodiments, the sequence information provided herein is used to construct an affibody molecule that binds to CD46. Affibody molecules are a class of affibodies based on a 58 amino acid residue protein domain, derived from one of the IgG binding domains of Staphylococcal protein A. These three helical bundle domains have been used as a backbone for the construction of combinatorial phagemid libraries, from which affibody variants targeting the desired molecule can be selected using phage display technology (see, e.g., Nord et al. (1997) Nat. Biotechnol. 15:772-777; Ronmark et al. (2002) Eur. J. Biochem., 269:2647-2655). Details of affibodies and methods of manufacture are known to those skilled in the art (see, e.g., U.S. Patent No. 5,831,012, which is incorporated herein by reference in its entirety).

[0520] It will be appreciated that the antibodies described above can be provided as whole intact antibodies (e.g., IgG), antibody fragments, or single-chain antibodies using methods well known to those skilled in the art. Additionally, although antibodies can be derived from essentially any mammalian species to reduce immunogenicity, it is desirable to use antibodies from the species in which the antibody and / or immunoconjugate is intended to be utilized. In other words, for use in humans, it is desirable to use human, humanized, or chimeric human antibodies.

[0521] Measuring antibody / peptide binding affinity.

[0522] As explained above, selection for improved avidity may involve measuring the affinity of the antibody for the target antigen (e.g., CD46, particularly the epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ). Methods for performing such measurements are well known to those skilled in the art. Briefly, for example, the K of an antibody is determined from the kinetics of binding to, for example, a target cell in a BIAcore (surface plasmon resonance-based biosensor). d For this technique, antigens or cells are coupled to a derivatized sensor chip that can detect mass changes. As the antibody passes over the sensor chip, it binds to the antigen, resulting in a quantifiable mass increase. The association rate constant (k) can be calculated using measurements of the association rate as a function of antibody concentration. on After the association phase, the buffer was passed through the chip and the antibody dissociation rate (k off ). K on Usually measured at 1.0×10 2 to 5.0×10 6 In the range and k off In 1.0×10 -1 to 1.0×10 -6 The equilibrium constant K d Usually calculated as k off / k on and is therefore usually measured at 10 -5 to 10 -12 The affinities measured in this manner correlate well with those measured in solution by fluorescence quenching titration.

[0523] Including YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, Immunization with YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8 and / or UA8κ or other anti-CD46 antibodies Combined.

[0524] The prototype anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) specifically bind to prostate cancer cells and other CD46-positive cancer cells and are internalized by the cells. The antibodies can be used alone as a therapy (e.g., to inhibit prostate cancer cell growth and / or proliferation) or they can be coupled to effectors to form immunoconjugates that provide efficient and specific delivery of effectors (e.g., cytotoxins, labels, radionuclides, ligands, antibodies, drugs, liposomes, nanoparticles, virions, cytokines, etc.) to a variety of cancer cells expressing CD46 (e.g., isolated cells, metastatic cells, solid tumor cells, etc.).

[0525] Anti-CD46 immunoconjugates can be formed by conjugating an antibody described herein, or an antigen-binding portion thereof, to an effector (e.g., a detectable label, another therapeutic agent, etc.). Suitable agents include, for example, cytotoxic or cytostatic agents (e.g., chemotherapeutic agents), toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and / or radioactive isotopes (i.e., radioconjugates).

[0526] In certain embodiments, the effector comprises a detectable label. Suitable detectable labels include, but are not limited to, radiopaque labels, nanoparticles, PET labels, MRI labels, radiolabels, and the like. Among the radionuclides and applicability of various embodiments of the present invention, gamma-emitters, positron emitters, x-ray emitters, and fluorescent emitters are suitable for localization, diagnosis, and / or classification, and / or therapy, while beta and alpha-emitters and electron and neutron capture agents (e.g., boron and uranium) may also be used for therapy.

[0527] Detectable labels can be used in conjunction with external and / or internal detection agents and provide a means of effectively locating and / or observing prostate cancer cells. Such detection / observation can be applicable to a variety of situations, including but not limited to pre-operative and intra-operative scenarios. Thus, in one embodiment, the present invention relates to a method for intra-operative detection of prostate cancer in a mammal. These methods generally involve administering to a mammal a composition comprising a sufficient amount of the following to be detected by a detector (e.g., a gamma detection detector): a prostate cancer-specific antibody (e.g., a prostate cancer-specific antibody) labeled with a detectable label; 161 Tb, 123 I. 125I etc.), for example, a radioisotope-labeled antibody of the present invention), and after the active substance is taken up by the target tissue, and preferably after the label is cleared from the blood, a radioimmunoassay technique is performed on the relevant area of ​​the mammal's body, for example using a gamma detection probe.

[0528] In certain embodiments, labeled antibodies can be used in radiation-guided surgery techniques, in which relevant tissues within an individual's body can be detected and located during surgery with the aid of a detector (e.g., a gamma-detection detector). Surgeons can use this detector during surgery to find tissue that has taken up a compound labeled with a radioactive isotope (e.g., a low-energy gamma photon emitter). In certain embodiments, such methods are particularly useful for locating and removing secondary cancers arising from metastatic cells of a primary tumor.

[0529] In addition to detectable labels, certain preferred effectors include, but are not limited to, cytotoxins (e.g., Pseudomonas aeruginosa exotoxin, ricin, abrin, diphtheria toxin, etc.), or cytotoxic drugs or prodrugs, in which case the chimeric molecule can be used as a potent cell-killing agent that specifically targets the cytotoxin to prostate cancer cells.

[0530] In other embodiments, the effector may include liposomes that encapsulate a drug (e.g., an anticancer drug such as albuterol, ciprofloxacin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), antigens that stimulate bound cells to be recognized by components of the immune system, antibodies that specifically bind to components of the immune system and direct them to prostate cancer, etc.

[0531] Illustrative effectors.

[0532] Imaging compositions.

[0533] In certain embodiments, anti-CD46 immunoconjugates can be used to direct detectable markers to the tumor site. This can facilitate tumor detection and / or localization. This can effectively detect primary tumors, or in certain embodiments, secondary tumors produced by, for example, prostate metastatic cells. In certain embodiments, the effector component of the immunoconjugate comprises a "radiopaque" marker, such as a marker that is easily observed using x-rays. Radiopaque substances are well known to those skilled in the art. The most common radiopaque substances include iodides, bromides, or barium salts. Other radiopaque substances are also known and include, but are not limited to, organobismuth derivatives (see, for example, U.S. Patent No. 5,939,045), radiopaque polyurethanes (see, for example, U.S. Patent No. 5,346,981), organobismuth complexes (see, for example, U.S. Patent No. 5,256,334), radiopaque barium polymer complexes (see, for example, U.S. Patent No. 4,866,132), etc.

[0534] The anti-CD46 antibodies described herein can be coupled directly to a radiopaque moiety, such as described below, or they can be attached to a "package" (e.g., a chelate, liposome, polymer microbeads, nanoparticles, etc.) that carries, contains, or comprises a radiopaque substance.

[0535] In addition to radiopaque labels, other labels are also suitable. Detectable labels suitable for immunoconjugates include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include magnetic beads (e.g., DYNABEADS TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g. 3 H. 125 I. 35 S. 14 C or 32 P), enzymes (such as horseradish peroxidase and alkaline phosphatase commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads, nanoparticles, quantum dots, etc.

[0536] In certain embodiments, suitable radiolabels include, but are not limited to, 99 Tc, 203 Pb, 67 Ga, 68 Ga, 72 As、 111 In, 113m In, 97 Such as 62 Cu, 64lCu, 52 Fe, 52m Mn, 51Cr, 186 Re、 188 Re、 77 As、 90 Y. 67 Cu, 169 Second, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 199 Au, 161 Tb, 109 Pd, 165 Dy, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 166 Ho, 172 Tm, 169 Yb, 175 Yb, 177 Lu, 105 Rh and 111 Ag.

[0537] Means for detecting such labels are well known to those skilled in the art. Thus, for example, specific radioactive labels can be detected using photographic film, scintillation detectors, PET imaging, MRI, and the like. Fluorescent labels can be detected using a photodetector to detect luminescence. Enzymatic labels are typically detected by providing an enzyme to a substrate and detecting the reaction product produced by the enzyme acting on the substrate, and colorimetric labels are detected by simple visual inspection of the colored label.

[0538] Radiation sensitizers.

[0539] In another embodiment, the effector may comprise an agent that increases the sensitivity of the cell to ionizing radiation (e.g., 60 Many radiation sensitizers are known and include, but are not limited to, benzoporphyrin derivatives (see, e.g., U.S. Pat. No. 5,945,439), 1,2,4-benzotriazine oxide (see, e.g., U.S. Pat. No. 5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat. No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107), radiation-sensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S. Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S. Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No. 4,921,963), and the like.

[0540] Alpha emitter.

[0541] In certain embodiments, the effector may comprise an alpha emitter, i.e., a radioisotope that emits alpha particles. Alpha-emitters have recently been shown to be effective in treating cancer (see, e.g., McDevitt et al. (2001) Science 294:1537-1540; Ballangrud et al. (2001) Cancer Res. 61:2008-2014; Borchardt et al. (2003) Cancer Res. 63:5084-50). Suitable alpha emitters include, but are not limited to, Bi, 213 Bi, 211 At et al.

[0542] Chelate

[0543] Many of the agents and / or radiolabels described herein can be provided as chelates. The chelator molecule is typically coupled to a molecule (e.g., biotin, avidin, streptavidin, etc.) that specifically binds to an epitope tag attached to an anti-CD46 antibody described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ).

[0544] Chelating groups are well known to those skilled in the art. In certain embodiments, the chelating group is derived from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl 1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis(-2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraaminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N'-,N",N'"-tetraacetic acid (DOTA), hydroxyethylenediaminetriacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), substituted DTPA, substituted EDTA, and the like.

[0545] Examples of specific preferred chelating agents include unsubstituted or substituted 2-iminothiolanes and 2-iminothiolanes, specifically 2-imino-4-mercaptomethylsulfane.

[0546] One chelator, 1,4,7,10-tetraazacyclododecane-N,N,N",N'"-tetraacetic acid (DOTA), has received particular attention due to its ability to chelate many diagnostically and therapeutically important metals, such as radionuclides and radiolabels.

[0547] Conjugates of DOTA to proteins such as antibodies have been described. For example, U.S. Patent No. 5,428,156 teaches a method for conjugating DOTA to antibodies and antibody fragments. To prepare these conjugates, one of the carboxyl groups of DOTA is converted to an active ester, which can react with an amine or sulfhydryl group on the antibody or antibody fragment. Lewis et al. (1994) Bioconjugate Chem. 5:565-576 describe a similar method in which one of the carboxyl groups of DOTA is converted to an active ester, and the activated DOTA is mixed with an antibody, which is then linked to DOTA via the epsilon-amino group of a lysine residue of the antibody, thereby converting one of the carboxyl groups of DOTA to an amide moiety.

[0548] In certain embodiments, the chelator may be coupled to the epitope tag or to a moiety that binds the epitope tag, directly or via a linker. Conjugates of DOTA and biotin have been described (see, for example, Su (1995) J. Nucl. Med., 36(5 Suppl): 154P, which discloses that DOTA is linked to biotin via an amino side chain biotin derivative (e.g., DOTA-LC-biotin or DOTA-benzyl-4-(6-amino-hexanamide)-biotin). Yau et al., WO 95 / 15335 disclose a method for making a nitro-benzyl-DOTA compound that can be conjugated to biotin. The method comprises: a cyclization reaction via temporary protection of a hydroxyl group; tosylation of an amine; deprotection of the temporarily protected hydroxyl group; tosylation of the deprotected hydroxyl group; and intramolecular tosylate cyclization. Wu et al., (1992) Nucl. Med. Biol, 19(2): 239-244 discloses a method for preparing a nitro-benzyl-DOTA compound that can be conjugated to biotin. The method comprises: a cyclization reaction via temporary protection of a hydroxyl group; tosylation of an amine; deprotection of the temporarily protected hydroxyl group; tosylation of the deprotected hydroxyl group; and intramolecular tosylate cyclization. Wu et al., (1992) Nucl. Med. Biol, 19(2): 239-244 discloses a method for preparing a nitro-benzyl-DOTA compound that can be conjugated to biotin. 111 IN and 90 Synthesis of a macrocyclic chelator for gamma-radiolabeled proteins. Wu et al. prepared labeled DOTA-biotin conjugates to study the stability and biodistribution of avidin, a model protein used in their studies. This conjugate was prepared using a biotin hydrazide containing a free amino group to react with an activated DOTA derivative generated in situ.

[0549] Cytotoxins / cytostatics.

[0550] The anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) can be used to deliver a variety of cytotoxic and / or cytostatic drugs, including therapeutic drugs, radiation-emitting compounds, cytotoxic molecules of plant, fungal, or bacterial origin, biological proteins, and mixtures thereof. In certain embodiments, the cytotoxic drug can include a cytotoxic drug that acts intracellularly, such as a small organic molecule, a cytotoxic protein or peptide, a radiation emitter, including, for example, the short-range, high-energy alpha emitters described above, and the like.

[0551] Thus, in certain embodiments, anti-CD46 antibodies are linked to cytotoxic / cytostatic drugs. In various embodiments, drugs used to construct ADCs include, but are not limited to, microtubule inhibitors and DNA damaging agents, polymerase inhibitors (e.g., polymerase II inhibitors, α-amanitin), and the like. In certain embodiments, the antibody is conjugated to the drug directly or via a linker, while in other embodiments, the antibody is conjugated to a drug carrier (e.g., drug-containing liposomes, polymeric drug carriers, nanoparticle drug carriers, lipid drug carriers, dendrimer drug carriers, etc.).

[0552] In certain embodiments, the drug comprises a tubulin inhibitor, including but not limited to auristatins, dolastatin-10, synthetic derivatives of the natural product dolastatin-10, and maytansine or a maytansine derivative.

[0553] In certain embodiments, the drug comprises an auristatin. In certain embodiments, the auristatin is selected from the group consisting of auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), vcMMAE, and vcMMAF.

[0554] In certain embodiments, the drug comprises a maytansine. Illustrative maytansines include, but are not limited to, maytansine (DM1); and analogs of maytansine, such as DM3 or DM4.

[0555] In certain embodiments, the drug comprises a DNA-interactive agent. In certain embodiments, DNA-interactive agents include, but are not limited to, calicheamicin, duocarmycin, pyrrolobenzodiazepine (PBD), and the like.

[0556] In one illustrative but non-limiting embodiment, the drug comprises calicheamicin. Calicheamicin targets DNA and causes strand breaks. In certain embodiments, the drug comprises calicheamicin or a calicheamicin analog. Calicheamicin analogs are described in U.S. Patent No. 5,264,586, which is incorporated herein by reference.

[0557] In another illustrative but non-limiting embodiment, the drug comprises a duocarmycin. Duocarmycins are DNA damaging agents that can exert their mode of action at any stage in the cell cycle. Agents that are part of this class of duocarmycins typically have potency in the low picomolar range. Illustrative duocarmycins (e.g., duocarmycin analogs) that can be used as effectors in the chimeric constructs contemplated herein include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, cyclopropylbenzindoleduocarmycin (CC-1065), sentanycin, rachelmycin, adolesin, bisezolexin, carzelesin, and the like.

[0558] In another illustrative but non-limiting embodiment, the drug comprises a pyrrolobenzodiazepine. In certain embodiments, the drug comprises a synthetic derivative of two pyrrolobenzodiazepines linked by a flexible polymethylene tether. Pyrrolobenzodiazepines (PBDs) and PBD dimers are described in U.S. Patent No. 7,528,126 B2, which is incorporated herein by reference. In certain embodiments, the pyrrolobenzodiazepine is selected from the group consisting of anthramycin (and its dimer), methylanthramycin (and its dimer), tofusanmycin (and its dimer), pracacosin (and its dimer), cheikamycin (and its dimer), neosclathromycin A (and its dimer), neosclathromycin B (and its dimer), DC-81 (and its dimer), sibiricomycin (and its dimer), pilomycin A (and its dimer), pilomycin B (and its dimer), sibamycin (and its dimer), gibberellin (and its dimer), SG2000, and SG2285.

[0559] In certain embodiments, the drug comprises a polymerase inhibitor, including but not limited to polymerase II inhibitors such as α-amanitin and poly (ADP-ribose) polymerase (PARP) inhibitors. Illustrative PARP inhibitors include but are not limited to enaparib (BSI 201), talaraparib (BMN-673), olaparib (AZD-2281), olaparib, caprarib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, and the like.

[0560] In certain embodiments, the cytotoxic / cytostatic agent comprises a protein or peptide toxin or a fragment thereof. Examples of enzymatically active toxins and fragments thereof are, for example, diphtheria toxin A fragments, non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modicin A chain, α-sarcin, specific Aleurites fordii proteins, specific Dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotonin, Sapaonaria officinalis inhibitor, gelonin, mitogens, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides are suitable for making radioconjugated antibodies. Examples include, but are not limited to 212 Bi, 131 I. 131 In, 90 Y. 186 Re et al.

[0561] In certain embodiments, cytotoxins may include, but are not limited to, Pseudomonas exotoxin, diphtheria toxin, ricin, abrin, and derivatives thereof. Pseudomonas exotoxin A (PE) is an extremely active monomeric protein (molecular weight 66 kD) secreted by Pseudomonas aeruginosa that inhibits protein synthesis in eukaryotic cells by catalyzing ADP-ribosylation to inactivate elongation factor 2 (EF-2) (catalyzing the transfer of the ADP-ribosyl moiety of oxidized NAD to EF-2).

[0562] The toxin contains three domains that work together to cause cytotoxicity. Domain Ia (amino acids 1 to 252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol, and domain III (amino acids 400-613) mediates ADP-ribosylation of elongation factor 2, which does not activate the protein and causes cell death. The function of domain Ib (amino acids 365-399) remains uncertain, but a large portion of it (amino acids 365-380) can be deleted without loss of cytotoxicity. See Siegall et al. (1989) J. Biol. Chem. 264:14256-14261.

[0563] In certain embodiments, the antibody is linked to a preferred molecule in which domain Ia (amino acids 1 to 252) is deleted and amino acids 365 to 380 have been deleted from domain Ib. In certain embodiments, all of domain Ib and a portion of domain II (amino acids 350 to 394) may be deleted, particularly when the deleted sequence is replaced with a connecting peptide.

[0564] Additionally, PE and other cytotoxic proteins can be further modified using site-directed mutagenesis or other techniques known in the art to tailor the molecules for specific desired applications. For example, means of altering PE molecules in a manner that does not substantially affect the functional advantages provided by the PE molecules described herein can also be used and such resulting molecules are intended to be encompassed herein.

[0565] Methods for cloning genes encoding PE fused to various ligands are well known to those skilled in the art (see, e.g., Siegall et al. (1989) FASEB J., 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 4538-4542).

[0566] Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation factor 2, thereby inhibiting protein synthesis. However, diphtheria toxin is divided into two chains, A and B, linked by a disulfide bridge. In contrast to PE, chain B of DT at the carboxyl terminus is responsible for receptor binding, while chain A, present at the amino terminus, contains enzymatic activity (Uchida et al. (1972) Science, 175:901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

[0567] In certain embodiments, the native receptor binding domain of the antibody-diphtheria toxin immunoconjugates of the invention is removed by truncation of the diphtheria toxin B chain. An illustrative modified diphtheria toxin is DT388, a DT with the carboxyl-terminal sequence removed starting at residue 389 (see, e.g., Chaudhary et al. (1991) Bioch. Biophys. Res. Comm., 180:545-551). As with the PE chimeric cytotoxin, the DT molecule can be chemically conjugated to a prostate cancer-specific antibody, but in certain preferred embodiments, the antibody will be recombinantly fused to the diphtheria toxin (see, e.g., Williams et al. (1990) J. Biol. Chem. 265:11885-11889).

[0568] Virus particles.

[0569] In certain embodiments, the effector comprises a viral particle (e.g., a filamentous phage, adeno-associated virus (AAV), a lentivirus, etc.). The antibody can be bound to the viral particle and / or can be expressed on the surface of the viral particle (e.g., a filamentous phage). The viral particle can additionally include a nucleic acid to be delivered to a target (e.g., prostate cancer) cell. The use of viral particles to deliver nucleic acids to cells is described in detail in WO 99 / 55720, US 6,670,188, US 6,642,051, and US 6,669,936.

[0570] Other therapeutic components.

[0571] Other suitable effector molecules include pharmacological agents or encapsulation systems containing multiple pharmacological agents.Thus, in various embodiments, it will be appreciated that a targeting molecule (eg, a targeting antibody) can be linked directly or via a linker to a drug intended for direct delivery to a tumor.

[0572] Such drugs are well known to those skilled in the art and include, but are not limited to, anti-cancer antibodies (e.g. ), antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, somatostatin analogs, glucocorticoids, aromatase inhibitors, mTOR inhibitors, protein kinase B (PKB) inhibitors, phosphatidylinositol, 3-kinase (PI3K) inhibitors, cyclin-dependent kinase inhibitors, anti-TRAIL molecules, MEK inhibitors, etc. In certain embodiments, anticancer compounds include, but are not limited to, fluorouracil (5-FU), capecitabine / XELODA, 5-trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed / Tomudex, pemetrexed / Cytarabine (Ara-C) / thioguanine, 6-mercaptopurine (6-MP), azathioprine / Azasan, 6-thioguanine (6-TG) / Purinethol (TEVA), pentostatin / Nipent, fludarabine phosphate / Cladribine (2-CdA, 2-chlorodeoxyadenosine) / Leustatin, floxuridine (5-fluoro-2) / FUDR (Hospira, Inc.), ribonucleotide reductase inhibitors (RNR), cyclophosphamide / Cytoxan (BMS), carcinosan, ifosfamide / Mitoxana, thiotepa, BCNU-1,3-bis(2-chloroethyl)-1-nitrosourea, 1,-(2-chloroethyl)-3-cyclohexyl-nitrosourea, methyl CCNU, hexamethylmelamine, busulfan / Myleran, procarbazine HCL / Matulane, dacarbazine (DTIC), chlorambucil / Melphalan / Alkeran, Cisplatinum (CDDP) / Platinol, Carboplatin / Paraplatin, Oxaliplatin / Eloxitan, Bendamustine, Carmustine, Nitrogen mustard, Dacarbazine (DTIC), Fotemustine, Lomustine, Mannosufan, Nedaplatin, Nimustine, Prednimustine, Ranimustine, Satraplatin, Semustine, Streptozotocin, Temozolomide, Trioxosulfan, Triethylenetriamine, Thiotepa, Teraplatin tetranitrate, Trolofosamide, Uramustine, Doxil HCL, Daunorubicin Citrate / Mitoxantrone HCL / Novantrone, Actinomycin D, Etoposide / Vepesid, Topotecan HCL / Hycamtin, Teniposide (VM-26), Irinotecan HCL (CPT-11) / Camptothecin, belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulfate, paclitaxel / Taxol, docetaxel / Taxotere, nanoparticle paclitaxel, abeltaxel, ixabepilone, larotaxel, ortataxel, tesetaxel, vinflunine, etc. In certain embodiments, the anticancer drug comprises one or more drugs selected from the group consisting of: carboplatin (e.g., ), cisplatin (e.g. PLATINOL Novo- ), cyclophosphamide (e.g. ), docetaxel (e.g. ), cranberries (e.g. ), erlotinib (e.g. ), etoposide (e.g. ), fluorouracil (e.g. ), gemcitabine (e.g. ), imatinib mesylate (e.g. ), irinotecan (e.g. ), methotrexate (e.g. ), paclitaxel (e.g. ), Sorafenib (e.g. ), sunitinib (e.g. ), topotecan (e.g. ), vinblastine (e.g. ), vincristine (e.g. ). In certain embodiments, the anticancer drug comprises one or more drugs selected from the group consisting of retinoic acid, retinoic acid derivatives, doxorubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, camptothecin derivatives, interferon, tamoxifen and paclitaxel. In certain embodiments, the anticancer compound is selected from the group consisting of abestrol, raspberry, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol acetate tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), antisense molecules, siRNA, etc.

[0573] Alternatively, the effector molecule may comprise an encapsulation system, such as a viral capsid, liposome, or micelle containing a therapeutic composition (e.g., a drug), a nucleic acid (e.g., an antisense nucleic acid or another nucleic acid delivered to the cell), or another therapeutic moiety, which preferably can avoid direct exposure to the circulatory system. Methods for preparing liposomes linked to antibodies are well known to those skilled in the art (see, for example, U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28: 341-365, etc.).

[0574] B) Linking of antibodies to effectors.

[0575] Those skilled in the art will appreciate that the anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) and effector molecules can be conjugated together in any order. Thus, when the antibody is a single-chain polypeptide, the effector molecule can be conjugated to the amino or carboxyl terminus of the targeting molecule. The antibody can also be conjugated to an internal region of the effector molecule, or, relatively speaking, the effector molecule can be conjugated to an internal location of the antibody, as long as the linkage does not interfere with the respective activities of the molecules.

[0576] The antibody and effector can be linked by any of a number of means well known to those skilled in the art. Typically, the effector is bound to the antibody directly or through a linker (spacer). However, in certain embodiments, when the two effector molecules are or comprise polypeptides, it is preferred to express the chimeric molecule as a single-chain fusion protein in a recombinant manner.

[0577] Binding of effector molecules to antibodies.

[0578] In one embodiment, the CD46-specific antibody is chemically conjugated to an effector molecule (eg, a cytotoxin, a label, a ligand, a drug, a liposome, etc.) Methods for chemically conjugating molecules are well known to those skilled in the art.

[0579] The procedures used to link effectors to antibodies vary depending on the chemical structure of the effector and / or antibody. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH) groups or free amine (-NH2) groups, which can be used to react with appropriate functional groups on the effector molecule to bind the effector.

[0580] Alternatively, the antibody and / or effector can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve attachment of any of a number of linker molecules, such as those commercially available from Pierce Chemical Company, Rockford Illinois.

[0581] As used herein, a "linker" is a molecule used to join a targeting molecule to an effector molecule. The linker is capable of forming a covalent bond to the targeting molecule and to the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the targeting molecule and the effector molecule are polypeptides, the linker can be joined to the component amino acids via their side groups (e.g., disulfide bonds to cysteine). However, in a preferred embodiment, the linker will be joined to the alpha carbon amino group or carboxyl group of the terminal amino acid.

[0582] Immunoconjugates can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imino esters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolylene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 3-methyldiethylenetriaminepentaacetic acid 1-benzyl isothiocyanate (MX-DTPA) is an illustrative, non-limiting, chelating agent used to bind, for example, radionucleotides to antibodies (see, for example, WO 1994 / 011026 (PCT / US1993 / 010953)).

[0583] In certain embodiments, the effector (e.g., a drug, liposome, etc.) or a linker attached to the effector is conjugated to the antibody at a solvent-accessible reactive amino acid (e.g., a lysine or cysteine ​​generated by reduction of interchain disulfide bonds in the antibody). In certain embodiments, cysteine ​​conjugation can be performed after reduction of the four interchain disulfide bonds.

[0584] In certain embodiments, site-specific conjugation can be performed to generate highly homogeneous constructs in which a known number of linker-drugs are consistently bound to a defined site in the antibody. The drug-to-antibody ratio (DAR) can be precisely controlled and can be tailored to a variety of linker-drugs, generating, for example, 2- or 4-DAR site-directed ADCs.

[0585] Many methods are known to achieve site-specific binding. For example, the amino acid cysteine ​​contains a reactive thiol group, which plays a fundamental role in the structure and function of many proteins. Attachment of sulfhydryl-reactive probes to proteins via cysteine ​​residues has long been a protein labeling method and is also suitable for the generation of antibody-drug conjugates (ADCs). In certain illustrative but non-limiting embodiments, this approach involves partial reduction of existing disulfide bonds (e.g., interchain disulfide bonds).

[0586] In certain embodiments, cysteine ​​residues can be engineered into proteins to maintain disulfide bonds. Successful use of introduced cysteine ​​residues for site-specific binding relies on the ability to select appropriate sites where cysteine ​​substitutions do not alter protein structure or function. To achieve this, a phage Elisa (PHESELECTOR) for selecting reactive thiols was developed by introducing reactive cysteine ​​residues at multiple sites into an antibody-Fab (trastuzumab-Fab 4D5), displaying the Fab on phage, and screening to identify reactive cysteines that do not interfere with antigen binding (see, e.g., Junutula et al. (2008) J. Immunol. Meth. 332: 41-52).

[0587] The PHESELECTOR method has been shown to be highly efficient and specific, especially when compared to conventional cysteine ​​conjugation. Optimal cysteine ​​sites found using, for example, antibody fragments (e.g., Fab) and the PHESELECTOR method have been shown to be applicable to full-length antibodies, and data indicate that these sites are effective for site-specific conjugation with other mAbs (see, e.g., Boswell et al. (2011) Bioconjug. Chem. 22: 1994-2004; Boswell et al. (2012) Soc. Nuclear Med. 53: 1454-1461; Shen et al. (2012) Nat. Biotechnol. 30: 184-189).

[0588] Another illustrative but non-limiting strategy for site-specific binding focuses on inserting amino acids (such as amino acid selenocysteine ​​and non-natural amino acid acetylphenylalanine (pAcPhe)) with bioorthogonal reactive handles. Two methods of using these amino acids have been developed and both methods utilize stop codons. However, one method incorporates selenocysteine ​​(Sec) into the sequence by pairing the opal stop codon UGA and the Sec insertion sequence, and another method uses tRNA / aminoacyl-tRNA synthetase to incorporate acetylphenylalanine into the amber stop codon UAG. The selenocysteine ​​used in the first method is very similar to the amino acid cysteine, but contains a selenium atom instead of a sulfur atom. Selenol radical is a nucleophile that is more reactive than the thiol radical corresponding part, which enables it to be combined with electrophilic compounds under the conditions of selective activation of selenocysteine. There are about 25 known selenoproteins in mammals, including proteins such as glutathione peroxidase and sulfhydryl reductase (Kryukov et al. 2003 Science, 300: 1439-1443). Under normal conditions, UGA encodes transcription termination; however, in the presence of a Sec insertion sequence (SECIS) located in the 3'UTR of a Sec-containing protein, termination is prevented by forming an mRNA secondary structure, and Sec is inserted at the UGA codon (Caban and Copeland (2006) Cell Mol. Life Sci. 63: 73-81). Sec insertion can be engineered into non-Sec-encoding genes by inserting a UGA codon and SECIS at the 3' end of the gene. This technique has been used, inter alia, for Sec-labeling of mAbs and subsequent site-specific conjugation (see, eg, Hofer et al. (2009) Biochem. 48: 12047-12057).

[0589] Another illustrative method for site-specific conjugation utilizes the unnatural amino acid p-acetylphenylalanine (pAcPhe). pAcPhe contains a ketone group that can selectively bind to alkoxy-amine-containing drugs via oxime conjugation. To incorporate pAcPhe into an antibody, an amber stop codon is substituted into the antibody at the desired position. The antibody cDNA is then co-expressed with an amber suppressor tRNA and an appropriately paired mutant tRNA synthetase. tRNA synthetase charges pAcPhe onto the amber tRNA and thereby incorporates pAcPhe into the antibody at the amber site UAG (see, e.g., Liu et al. 2007 Nat. Meth. 4:239-244; Wang et al. (2003) Proc. Natl. Acad. Sci. USA, 100:56-61; Axup (2012) Proc. Natl. Acad. Sci. USA, 109:16101-16116).

[0590] In addition to pAcPhe, other unnatural amino acids were employed for site-specific incorporation using a similar process involving matching tRNA / aminoacyl-tRNA synthetase pairs (see, e.g., Young (2002) J. Mol. Biol. 395:361-374; Kiick et al. (2002) Proc. Natl. Acad. Sci. USA; 99:19-24).

[0591] In various embodiments, enzyme-catalyzed bond formation can be used for site-specific conjugation. For example, a glycosyltransferase platform uses mutant transferases to attach chemically active sugar moieties to glycosylation sites on antibodies. The selected molecule can then be conjugated to the chemical handle on the sugar moiety. In another illustrative but non-limiting approach, transglutaminase is used to form a bond between an amine group on the linker / drug and an engineered glutamine residue on the antibody.

[0592] Glycosyltransferases are a large family of proteins involved in oligosaccharide synthesis and are responsible for transferring sugar residues from activated sugar nucleotides to sugar acceptors or glycoproteins / lipids. The structures of several glycosyltransferases are known, and it has been revealed that sugar donor specificity is determined by several amino acids in the catalytic pocket (Qasba et al. (2005) Trends Biochem. Sci. 30: 53-62). Using this knowledge, residues in the glycosyltransferase pocket (e.g., B4Gal-T1) are mutated to broaden donor specificity and allow transfer of chemically reactive sugar residues, 2-keto-Gal (see, e.g., Ramakrishnan et al. (2002) J. Biol. Chem. 277: 20833-20839). This technology provides the ability to transfer chemically reactive sugars to any lipid or protein containing a glycosylation site. Human IgG antibodies contain an N-glycosylation site at the conserved Asn-297 of the Fc fragment. Glycans attached to this site are typically complex but can be degalactosylated to G0, to which mutant glycosyltransferases can efficiently transfer C2-keto-Gal (see, e.g., Boeggeman et al. (2009) Bioconjug. Chem. 20: 1228-1236). The reactive chemical handle of C2-keto-Gal can then be coupled to biomolecules using orthogonal reactive groups. This approach has been successfully used to site-specifically conjugate the anti-Her2 antibody trastuzumab with Alexa Fluor 488 aminooxyacetamide and is a viable technique for site-specific ADC generation (Id.).

[0593] The second platform utilizes transglutaminase to catalyze the formation of a covalent bond between a free amine group and a glutamine side chain. Transglutaminase (mTG) from Streptoverticillum mobara is commercially available and has been widely used as a protein cross-linking agent (see, e.g., Yokoyama et al. (2004) Appl. Microbiol. Biotechnol. 64: 447-454). mTG does not recognize any of the naturally occurring glutamine residues in the Fc region of glycosylated antibodies, but rather recognizes a "glutamine tag" that can be engineered into antibodies (see, e.g., Jeger et al. (2010) Angew Chem. Int. Ed. Engl. 49: 9995-9997). As an illustration, the glutamine tag LLQG has been engineered into different sites of the constant domain of an antibody targeting the epidermal growth factor receptor. mTG was then used to conjugate these sites with fluorophores or monomethyl dolastatin 10 (MMAD), and several sites were found to have excellent biophysical properties and high binding. mTG was also able to conjugate glutamine tags to anti-Her2 and anti-M1S1 antibodies. The anti-M1S1-vc-MMAD conjugate exhibited strong in vitro and in vivo activity, indicating that conjugation using this approach does not alter antibody binding or affinity and demonstrating the utility of this approach for site-specific conjugation of ADCs (see, e.g., Strop et al. (2013) Chem. Biol. 20: 161-167).

[0594] In addition to glycosyltransferases and transglutaminases, other enzymes have been developed for protein labeling (Sunbul and Yin (2009) Org. Biomol. Chem. 7: 3361-3371). One such enzyme, a formylglycine generator, recognizes the sequence CxPxR and oxidizes cysteine ​​residues to form formylglycine, thereby generating a protein with an aldehyde tag. The aldehyde group can then be attached to a molecule of choice using, for example, hydrozino-Pictet-Spengler chemistry.

[0595] Many other procedures and linker molecules for conjugating a variety of compounds, including radionuclide metal chelates, toxins, and drugs, to proteins, such as antibodies, are known (see, e.g., European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789, and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47:4071-4075). In particular, the manufacture of various immunotoxins is well known in the art and can be found in, for example, "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S. Pat. Nos. 4,545,985 and 4,894,443.

[0596] In some cases, it is desirable to release the effector from the antibody when the immunoconjugate reaches its target site. Thus, when releasing the effector at the target site, an immunoconjugate comprising a bond that can be broken near the target site can be used. Bond cleavage can be promoted to release the reagent from the antibody by enzymatic activity or the conditions to which the immunoconjugate is subjected in the target cell or near the target site. When the target site is a tumor, a linker that is cleavable under conditions present at the tumor site (e.g., when exposed to tumor-associated enzymes or acidic pH) can be used.

[0597] Many different cleavable linkers are known to those skilled in the art. See U.S. Patent Nos. 4,618,492; 4,542,225; and 4,625,014. Illustrative cleavable linkers include, but are not limited to, acid-labile linkers, protease-cleavable linkers, disulfide linkers, and the like. Acid-labile linkers are designed to be stable at pH levels encountered in blood, but become unstable and degrade when exposed to the low pH environment of lysosomes. Protease-cleavable linkers are also designed to be stable in blood / plasma, but when cleaved by lysosomal enzymes, rapidly release the free drug inside lysosomes in cancer cells. They exploit the high levels of protease activity inside lysosomes and typically include peptide sequences that are recognized and cleaved by these proteases, such as the case with the dipeptide Val-Cit bond, which is rapidly hydrolyzed by cathepsins. Disulfide linkers utilize high levels of intracellular reduced glutathione to release the free drug inside the cell.

[0598] In view of the numerous methods reported for linking a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine an appropriate method for linking a given agent to an antibody or other polypeptide.

[0599] Chelate binding.

[0600] In certain embodiments, the effector comprises a chelate linked to an antibody or epitope tag. The anti-CD46 antibody carries the corresponding epitope tag or antibody such that simple contact of the antibody with the chelate results in binding of the antibody to the effector. A combination step can be performed prior to use of the moiety (targeting strategy) or the target tissue can be bound to the antibody prior to delivery of the chelate. Methods for making chelates suitable for coupling to a variety of targeting moieties are well known to those skilled in the art (see, e.g., U.S. Pat. Nos. 6,190,923, 6,187,285, 6,183,721, 6,177,562, 6,159,445, 6,153,775, 6,149,890, 6,143,276, 6,18 ... No. 6,143,274, No. 6,139,819, No. 6,132,764, No. 6,123,923, No. 6,123,921, No. 6,120,768, No. 6,120,751, No. 6,117,412, No. 6,106,866, No. 6,096,290, No. 6,093,382, No. 6,090,80 0, No. 6,090,408, No. 6,088,613, No. 6,077,499, No. 6,075,010, No. 6,071,494, No. 6,071,490, No. 6,060,040, No. 6,056,939, No. 6,051,207, No. 6,048,979, No. 6,045,821, No. 6,04 No. 5,775, No. 6,030,840, No. 6,028,066, No. 6,022,966, No. 6,022,523, No. 6,022,522, No. 6,017,522, No. 6,015,897, No. 6,010,682, No. 6,010,681, No. 6,004,533 and No. 6,001,329).

[0601] Production of fusion proteins.

[0602] If the antibody and / or effector are relatively short (e.g., less than about 50 amino acids), they can be synthesized using standard chemical peptide synthesis techniques. When both molecules are relatively short, the chimeric molecule can be synthesized as a single continuous polypeptide. Alternatively, the targeting molecule and the effector molecule can be synthesized separately and then fused by condensing the amino terminus of one molecule to the carboxyl terminus of the other molecule, thereby forming a peptide bond. Alternatively, the targeting and effector molecules can each be fused to one end of a peptide spacer molecule, thereby forming a continuous fusion protein.

[0603] Solid-phase synthesis, in which the C-terminal amino acid of a sequence is attached to an insoluble support followed by the sequential addition of the remaining amino acids in the sequence, is a preferred method for chemically synthesizing the polypeptides of the present invention. Solid-phase synthesis techniques are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A, Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156; pp. 3-284 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111. (1984).

[0604] In certain embodiments, the chimeric fusion proteins of the present invention are synthesized using recombinant DNA methods. Generally, this involves generating a DNA sequence encoding the fusion protein, placing the DNA into an expression cassette under the control of a specific promoter, expressing the protein in a host, isolating the expressed protein, and, if desired, restoring the protein to its original properties.

[0605] DNA encoding the fusion protein of the present invention can be prepared by any suitable method, including, for example, cloning and restricting appropriate sequences, or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylaminophosphonate method of Beaucage et al. (1981) Tetra. Lett., 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

[0606] Chemical synthesis produces single-stranded oligonucleotides. This can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using a single strand as a template. Those skilled in the art will recognize that although chemical synthesis of DNA is limited to sequences of approximately 100 bases, longer sequences can be obtained by joining shorter sequences.

[0607] Alternatively, in certain embodiments, subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be joined to generate the desired DNA sequence.

[0608] In certain embodiments, DNA encoding the fusion protein of the present invention can be cloned using PCR cloning methods.

[0609] Although in certain embodiments, the antibody and effector are essentially directly joined together, those skilled in the art will appreciate that the molecules can be separated by a spacer, such as a peptide spacer consisting of one or more amino acids (e.g., (Gly4Ser)3, SEQ ID NO: 80). Generally, the spacer will not have specific biological activity other than binding to the protein or maintaining a certain minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer can be selected to affect certain properties of the molecule, such as folding, net charge, or hydrophobicity.

[0610] The nucleic acid sequence encoding the fusion protein can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and a variety of higher eukaryotic cells, such as COS, CHO, and HeLa cell lines and myeloma cell lines. The recombinant protein gene will be operably linked to the appropriate expression control sequences of each host.

[0611] The plasmids of the present invention can be transferred into selected host cells by well-known methods, such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Plasmid-transformed cells can be selected by antibiotic resistance conferred by genes contained on the plasmid, such as amp, gpt, neo, and hyg genes.

[0612] Once expressed, the recombinant fusion protein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like (see generally R. Scopes (1982) Protein Purification, Springer-Verlag, New York (NY); Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc., New York (NY)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or greater homogeneity is most preferred for pharmaceutical uses. Once partially purified or purified to the desired homogeneity, the polypeptide can then be used for therapeutic purposes.

[0613] Those skilled in the art will recognize that, after chemical synthesis, biological expression, or purification, the fusion protein may have a configuration that is substantially different from the native configuration of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptides, followed by refolding of the polypeptides into the preferred configuration. Methods for reducing and denaturing proteins and inducing refolding are well known to those skilled in the art (see, for example, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner et al. (1992) Anal. Biochem., 205: 263-270).

[0614] Those skilled in the art will recognize that modifications can be made to the fusion protein without reducing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into the fusion protein. Such modifications are well known to those skilled in the art and include, for example, the addition of a methionine to the amino terminus to provide a priming site, or additional amino acids placed at either terminus to form appropriately positioned restriction sites or stop codons.

[0615] Pharmaceutical compositions.

[0616] The anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (also known as 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8κ) and / or immunoconjugates thereof are suitable for parenteral, topical, oral, or local administration (e.g., injection into a tumor site), aerosol administration, or transdermal administration for prophylactic, but primarily therapeutic, treatment. Pharmaceutical compositions can be administered using a variety of unit dosage forms, depending on the method of administration. For example, unit dosage forms suitable for oral administration include powders, tablets, pills, capsules, and lozenges. It will be appreciated that the antibodies and / or immunoconjugates thereof described herein, and pharmaceutical compositions comprising the antibodies and / or immunoconjugates thereof, when administered orally, are preferably protected from digestion. This can be achieved in a variety of ways known to those of ordinary skill in the art, such as by complexing the protein with a composition to render it resistant to acid and enzymatic hydrolysis or by encapsulating the protein in an appropriate drug-resistant carrier, such as a liposome. Means for protecting proteins from digestion are well known in the art.

[0617] In various embodiments, compositions (eg, pharmaceutical compositions) are provided that contain one or a combination of anti-CD46 antibodies, or antigen-binding portions thereof, or immunoconjugates thereof, formulated together with a pharmaceutically acceptable carrier.

[0618] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody, immunoconjugate) may be coated in a material that protects the compound from the action of acids and other natural conditions that may not activate the compound.

[0619] In certain embodiments, the antibodies and / or immunoconjugates can be administered in "native" form, or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, as long as the salts, esters, amides, prodrugs, or derivatives are pharmacologically suitable, i.e., effective in the methods of the invention. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and, for example, as described by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th ed. Wiley-Interscience, New York, NY, and as described above.

[0620] As an illustration, pharmaceutically acceptable salts can be prepared for any of the antibodies and / or immunoconjugates described herein that have functional groups capable of forming salts. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not impart any harmful or undue effects to the individual to which it is administered and to the context in which it is administered.

[0621] In various embodiments, pharmaceutically acceptable salts can be derived from organic or inorganic bases. Salts can be monovalent or multivalent ions. Of particular interest are inorganic ions, lithium, sodium, potassium, calcium, and magnesium. Organic salts can be prepared using amines, specifically ammonium salts, such as monoalkyl, dialkyl, and trialkylamines, or ethanolamine. Salts can also be formed using caffeine, tromethamine, and similar molecules.

[0622] Methods for formulating pharmaceutically active agents into salts, esters, amides, prodrugs, and the like are well known to those skilled in the art. For example, salts can be prepared from free bases using conventional methods that typically involve reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent (e.g., methanol or ethanol) and an acid is added thereto. The resulting salt precipitates or can be precipitated from the solution by adding a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to, organic acids (e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.) and inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.). Acid addition salts can be converted back to the free base by treatment with a suitable base. Particularly preferred acid addition salts of the active agents herein include halide salts, such as those prepared using hydrochloric acid or hydrobromic acid. In contrast, the preparation of basic salts of the active agents of the present invention is prepared in a similar manner using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc. Particularly preferred basic salts include alkali metal salts such as sodium salts and copper salts.

[0623] For the formulation of a salt form of a basic drug, the pKa of the counterion is preferably at least about 2 pH units lower than the pKa of the drug. Similarly, for the formulation of a salt form of an acidic drug, the pKa of the counterion is preferably at least about 2 pH units higher than the pKa of the drug. This allows the counterion to bring the pH of the solution to a pH lower than that at which the salt plateaus. max The solubility of the salt exceeds that of the free acid or base at low levels. The general rule that the difference in the pKa units of the ionizable groups in the active pharmaceutical ingredient (API) and the acid or base is intended to make proton transfer energetically favorable. When the pKa of the API and the counterion are not significantly different, a solid complex can form but rapidly disproportionates (i.e., breaks down into the individual entities of the drug and the counterion) in an aqueous environment.

[0624] Preferably, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to, acetate, benzoate, benzyl, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, glucoheptonate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylsulfate, galactarate, napsylate, nitrate, pamoate (enbolate), phosphate and diphosphate, salicylate and hydrogen salicylate, stearate, succinate, sulfate, tartrate, toluenesulfonate, triethyl iodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to, aluminum, benzathine penicillin, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.

[0625] The preparation of esters generally involves functionalization of hydroxyl and / or carboxyl groups present within the molecular structure of the antibody and / or immunoconjugate. In certain embodiments, esters are generally acyl-substituted derivatives of free alcohol groups, i.e., moieties derived from carboxylic acids of the formula RCOOH, wherein R is an alkyl group, and preferably a lower alkyl group. If desired, esters can be converted back to the free acid using conventional hydrogenolysis or hydrolysis procedures.

[0626] Amides can also be prepared using techniques known to those skilled in the art or described in the relevant literature. For example, amides can be prepared from esters using suitable amine reactants, or they can be prepared from anhydrides or acid chlorides by reaction with ammonia or lower alkylamines.

[0627] Pharmaceutical compositions comprising the antibodies and / or immunoconjugates described herein can be administered alone or in combination therapy (i.e., in combination with other agents). For example, a combination therapy can include an antibody or immunoconjugate with at least one or more other therapeutic agents, such as an anticancer agent described below. Pharmaceutical compositions can also be administered in combination with radiation therapy and / or surgery.

[0628] Compositions comprising antibodies and / or immunoconjugates described herein can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary depending on the desired outcome. The active compound can be prepared using a carrier that protects the compound from rapid release, such as a controlled release formulation, including inserts, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations have been patented and are generally known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978).

[0629] In certain embodiments, administration of an anti-CD46 antibody or immunoconjugate can be facilitated by coating the antibody or immunoconjugate composition, or by co-administering the antibody or immunoconjugate with a material that prevents its inactivation. For example, the compound can be administered to a patient in an appropriate carrier, such as a liposome, or a diluent. Pharmaceutically acceptable diluents include, but are not limited to, saline and aqueous buffer solutions. Liposomes include, but are not limited to, water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol, 7:27).

[0630] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, they are contemplated for use in pharmaceutical compositions. Supplementary active compounds may also be incorporated into the compositions.

[0631] In a number of embodiments, therapeutic compositions are usually sterile and stable under manufacturing and storage conditions. Compositions can be formulated into solutions, microemulsions in lipids or liposomes or other ordered structures suitable for containing high drug concentrations. In certain embodiments, the carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), in the case of a dispersion by maintaining the required particle size and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyols (such as mannitol, sorbitol) or sodium chloride. The extended absorption of injectable compositions can be achieved by including a medicament (such as monostearate and gelatin) that delays absorption in the composition.

[0632] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound (e.g., antibody and / or immunoconjugate as described herein) into a suitable solvent together with one or a combination of the ingredients listed above, followed by sterilization microfiltration if necessary. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and the desired other ingredients from those ingredients listed above. Where sterile powders are used to prepare sterile injectable solutions, illustrative preparation methods include vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0633] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be given, several divided doses can be given over time, or the dose can be proportionally reduced or increased, as indicated by the exigencies of the therapeutic situation. For example, in certain embodiments, the antibodies and / or immunoconjugates described herein can be administered by subcutaneous injection once or twice daily, or once or twice weekly, or once or twice monthly.

[0634] It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit that is suitable for use as a single dose for the individual to be treated. Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, and the required pharmaceutical carrier. The specifications of the unit dosage form are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of mixing such active compounds for treating individuals.

[0635] In certain embodiments, the formulation comprises a pharmaceutical antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0636] For therapeutic compositions, formulations of antibodies and / or immunoconjugates described herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. These formulations can be suitably presented as unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual and specific mode of administration to be treated. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. In general, in one hundred percent, this amount will be in the range of about 0.001% to about 90% active ingredient, preferably about 0.005% to about 70% active ingredient, and most preferably about 0.01% to about 30% active ingredient.

[0637] Formulations of the antibodies and / or immunoconjugates described herein suitable for vaginal administration also include vaginal suppositories, tampons, creams, gels, pastes, foams, or spray formulations containing, for example, suitable carriers known in the art. Dosage forms for topical or transdermal administration of the antibodies and / or immunoconjugates described herein include powders, sprays, ointments, pastes, creams, emulsions, gels, solutions, patches, and inhalants. In certain embodiments, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0638] As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, generally by injection and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0639] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions containing the antibodies and / or immunoconjugates described herein include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, etc. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0640] In various embodiments, these compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Specific examples of adjuvants well known in the art include, for example, inorganic adjuvants (e.g., aluminum salts, such as aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g., squalene), oil-based adjuvants, and virus particles (e.g., virus particles containing membrane-bound hemagglutinin and neuraminidase derived from influenza virus).

[0641] Prevention of the presence of microorganisms in the formulation can be ensured by sterilization procedures, and / or by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenols, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0642] When the antibodies and / or immunoconjugates described herein are administered to humans and animals as pharmaceutical agents, they can be administered alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably 0.005 to 70%, such as 0.01 to 30%) active ingredient and a pharmaceutically acceptable carrier.

[0643] Regardless of the route of administration chosen, the antibodies and / or immunoconjugates described herein, which may be used in suitable hydrated form and / or pharmaceutical compositions, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of ordinary skill in the art.

[0644] The actual dosage level of the active ingredient (e.g., the antibody and / or immunoconjugate described herein) in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention or its ester, salt, or amide, the route of administration, the time of administration, the secretion rate of the specific compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field. A physician or veterinarian generally skilled in the art can easily determine and prescribe the desired effective amount of the pharmaceutical composition. For example, a physician or veterinarian can begin administering the compound of the present invention employed in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, the suitable daily dose of the antibody and / or immunoconjugate described herein will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect. The effective dose will generally depend on the factors described above. In certain embodiments, intravenous, intramuscular, intraperitoneal or subcutaneous administration is preferred, preferably near the target site. If desired, the effective daily dose of the therapeutic composition can be administered in a single dose, or optionally in a unit dosage form as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. Although the antibodies and / or immunoconjugates described herein can be administered alone, it is generally preferred to administer the compound in the form of a pharmaceutical formulation (composition).

[0645] In certain embodiments, therapeutic compositions can be administered using medical devices known in the art. For example, in one illustrative embodiment, the antibodies and / or immunoconjugates described herein can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of suitable well-known implants and modules are described, for example, in U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for administering drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering a drug through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump that delivers drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable fluid implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such inserts, delivery systems, and modules are known to those skilled in the art.

[0646] In certain embodiments, the anti-CD46 antibodies and / or immunoconjugates described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. Methods for making liposomes are described, for example, in U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, Ranade (1989) J. Clin. Pharmacol. 29:685). Illustrative targeting moieties include, but are not limited to, folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman et al. (1995) FEBS Lett. 357: 140; Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).

[0647] Reagent test kit.

[0648] If radioactive or other effectors are used as diagnostic and / or therapeutic agents, it is often not possible for the user to use a ready-to-use composition because the shelf life of radiolabeled compounds is often poor and / or the half-life of the radionuclides used is short. In such cases, the user can perform a radionuclide labeling reaction in a clinical setting, a physician's office, or a laboratory. For this and other purposes, the various reaction components can then be provided to the user in the form of a so-called "kit." The kit is preferably designed so that the manipulations required to perform the desired reaction are as simple as possible, allowing the user to prepare the desired composition from the kit using the facilities at their disposal. Therefore, the present invention also relates to a kit for preparing the composition according to the present invention.

[0649] In certain embodiments, such kits comprise one or more antibodies or immunoconjugates described herein. The antibodies or immunoconjugates may be provided with, if desired, an inert pharmaceutically acceptable carrier and / or formulation and / or adjuvant. In addition, the kits optionally include a solution of a salt or chelate of a suitable radionuclide (or other active agent), and (iii) instructions for use with a prescription for administering and / or reacting the components present in the kit.

[0650] The kit intended for supply to the user may also comprise the components defined above, together with instructions for use, while the solution of the salt or chelate of the radionuclide defined in sub-item (ii) above may be disposed of separately by the user, said solution having a limited shelf life.

[0651] The kit may optionally further comprise a reducing agent and / or a chelating agent as required, and / or instructions for use of the composition and / or a recipe for reacting the components of the kit to form the desired product. If desired, the components of the kit may be combined as long as they are compatible.

[0652] In certain embodiments, immunoconjugates can be produced simply by combining the components in a neutral medium and allowing them to react. For such purposes, the effector can be presented to the antibody, for example, in the form of a chelate.

[0653] When the kit components are used as components for drug administration (e.g., as an injectable liquid form), they are preferably sterile. When the components are provided in a dry state, the user should preferably use a sterile physiological saline solution as a solvent. If necessary, the components can be stabilized in a conventional manner using suitable stabilizers, such as ascorbic acid, gentisic acid, or salts of these acids, or they can contain other adjuvants, such as fillers, for example, glucose, lactose, mannitol, etc.

[0654] While instructional materials, if any, typically comprise written or printed materials, they are not limited to such. The present invention encompasses any medium capable of storing such instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, sleeves, chips), optical media (e.g., CD ROMs), and the like. Such media may include the URL of an Internet website that provides such instructional materials.

[0655] Examples

[0656] The following examples are provided to illustrate, but not to limit, the present invention.

[0657] Example 1

[0658] Novel anti-CD46 antibodies and uses thereof

[0659] To identify novel anti-human CD46 antibodies, a recombinant Fc fusion protein consisting of Sushi domains 1 and 2 of human CD46 was generated. Because complementary sequence elements primarily bind to domains 3 and 4, the selection of domains 1 and 2 minimizes the selection of antibodies that might strongly interfere with the function of the canonical complementary sequence. This CD46-Fc fusion was produced and purified from transfected HEK293 cells by protein A affinity chromatography. For human antibody selection, 5×10 cells were generated using cDNA pooled from peripheral blood mononuclear cells of 426 healthy human donors. 9 A phagemid display library containing 10 members was constructed, and the library was selected for recombinant CD46-Fc fusion protein. After three rounds of selection, binding phagemids were screened by FACS and sequencing. Simultaneously, an alternative strategy was employed, which involved first selecting the library on live tumor cells, followed by transfer of the results of the first round of phagemid selection to a yeast surface display vector. FACS-based selection using low concentrations of ligand was then used to enrich for high-affinity binders to recombinant CD46-Fc fusion protein. The resulting antibodies bound with high affinity to both live tumor cells and recombinant human CD46 protein. All binding clones were sequenced, and the unique sequences are listed in Table 1.

[0660] The scFv was converted to fully human IgG1 and the binding affinity was measured on live tumor cells. The FACS binding data were curve fitted to generate KD values ​​( Figure 2 For YS5 vs. Du-145, and Figure 3 For YS12 versus Du-145). Affinities were in the low nanomolar to subnanomolar (nM) range for the antibodies studied.

[0661] In addition to human CD46, the antibodies described herein bind with similar affinities ( Figure 5 YS5 against CHO-huCD46 and Figure 6 YS5 binds to CHO-cynoCD46) and cynomolgus macaque CD46 ( Figure 4 ), thus identifying the appropriate species for regulatory toxicology studies.

[0662] All anti-CD46 antibodies, as well as the previously identified UA20 and 2B10, bind to CD46 Sushi domains 1 and 2. This region was further analyzed to reveal epitope differences. We first performed FACS-based competition experiments on live tumor cells and found that our antibodies either competed or did not compete with UA20-Fc: Group 1 consisted of YS5 and others, and Group 2 consisted of SB1HGNY ( Figure 7For the antibodies in Group 1, additional epitope differences were demonstrated by the selective binding of the antibodies to various CD46 mutants (Table 3). For example, YS5 differs from the other antibodies in that a mutation at position 39 uniquely affects its binding (Table 3), indicating that position 39, along with position 40, which affects binding of all antibodies, is part of the epitope of YS5 that binds to CD46.

[0663] Table 3. Alanine scanning reveals epitope differences. CD46 residue changes are as indicated (top row, E13A, position 13 changed from E to A, etc.). Binding to CHO cells transfected with the various constructed mutants was quantified by FACS using MFI normalized to cells transfected with wild-type CD46. Grey shading indicates significant loss of binding. R40A reduces binding of all antibodies, indicating that position 40 is a critical contact site for all. D39A uniquely affects YS5 binding, indicating that position 39 is the only contact site that contributes to YS5 (and YS6) binding but not the other antibodies listed in the table. YS6 differs from YS5 in that its selectivity is affected by the mutation at position 31 (P31A).

[0664]

[0665] All antibodies competed with the laboratory strain (Edmonston) measles virus H protein. Measles virus enters target cells via macropinocytosis (Crimeen-Irwin et al. (2003) J. Biol. Chem. 278:46927-46937). The H protein responds to binding and crosslinking of cell surface CD46, which is required for viral entry (Id.). To determine if our antibodies compete with H protein binding to CD46, recombinant H protein-Fc fusions were generated and tested for competition with anti-CD46 antibodies by FACS. The antibodies tested were found to compete with H protein ( Figure 8 ), indicating overlapping binding sites.

[0666] As measles virus enters target cells via macropinocytosis, it was then determined whether anti-CD46 antibodies were also internalized by tumor cells via macropinocytosis. Using 70 kDa neutral density polydextrose (ND70) as an indicator of macropinocytosis (Ha et al. (2014) Mol. Cell Proteomics. 13(12): 3320-3331), it was found that anti-CD46 antibodies were actually internalized via the macropinocytosis pathway ( Figure 9Macropinocytosis is inherently tumor-selective (Commisso et al. (2013) Nature, 497: 633-637; Ha et al. (2014) Mol. Cell Proteomics. 13(12): 3320-3331; Reyes-Reyes et al. (2010) Cancer Res. 70: 8617-8629), thus conferring additional selectivity to anti-CD46 antibodies against tumor cells.

[0667] To validate CD46 as a target for targeted therapeutic development, the tissue specificity of the CD46 epitope was determined by immunohistochemistry. CD46 epitope expression in tumors was first investigated. Immunohistochemistry studies were performed on frozen and formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue. For frozen tissue, 18 / 18 cases (100%) showed strong staining signals, indicating overexpression in all cases. For FFPE tissue, strong CD46 staining was found in 63 / 87, moderate staining was found in 23 / 87, and weak staining was found in 1 / 87 cases, supporting the conclusion that CD46 is overexpressed by the majority of prostate tumors. An overview of the immunohistochemistry results is shown in Tables 4 and Figure 10 middle.

[0668] Table 4. Immunohistochemistry results of CD46 expression in prostate cancer tissues. All cases with positive control studies are indicated. FFPE: formalin-fixed paraffin-embedded (tissue). Biotinylated anti-human CD46 antibody was used in this study.

[0669] Tissue type powerful medium weak Negative freezing 18 / 18 0 0 0 FFPE 63 / 87 23 / 87 1 / 87 0

[0670] The above studies strongly support the development of CD46-targeted monoclonal antibodies for the treatment of human cancers. To this end, a novel panel of anti-CD46 antibodies was used to develop antibody-drug conjugates (ADCs). Monomethyl auristatin F (MMAF) was conjugated to YS5 IgG1 via a MC-vc-PAB linker (McDonagh et al. (2008) Mol. Canc. Therap. 7:2913-2923; Sutherland et al. (2006) J. Biol. Chem. 281:10540-10547). By hydrophobic interaction chromatography (HIC), an average of approximately 3 drug molecules ( Figure 11 ). A panel of metastatic castration-resistant prostate cancer cell lines (LNCaP-C4-2B and Du145) were then used to test the in vitro tumor killing activity. Potent tumor cell killing was observed with EC50 values ​​as low as the sub-nM range ( Figure 12 and 13 ).

[0671] The in vivo anti-tumor activity of the anti-CD46 ADC was tested using the LNCaP-C4-2B subcutaneous xenograft model. Potent inhibition of tumor growth and survival was observed, with tumor volume reduced to undetectable levels after five doses of 5 mg / kg ( Figure 14 There was no tumor recurrence during the designated period after ADC injection ( Figure 14 ).

[0672] In addition to prostate cancer, anti-CD46 ADCs were found to potently kill a variety of other cancers that express CD46. For example, in addition to prostate cancer, CD46 was found to be highly expressed on the surface of multiple myeloma cells ( Figure 15 In addition, anti-CD46 ADC potently killed the multiple myeloma cell line RPMI8226 with an EC50 in the sub-nM range ( Figure 16 Finally, it has been shown that anti-CD46 ADCs significantly reduce multiple myeloma burden in vivo. A reporter expression line (RPMI8226-Luc expressing the firefly luciferase gene) was injected into immunodeficient mice via the tail vein and disseminated tumor cells were formed in the bones and joints. Four doses of anti-CD46 ADC were then injected at 5 mg / kg (once every 4 days) and tumor status was monitored by bioluminescence. It was found that anti-CD46 ADCs were highly effective in reducing tumor burden in vivo ( Figure 17 Survival data were collected after ADC treatment ( Figure 18 ), showed that 60% of the treated mice were "cured" and the remaining 40% had a greatly delayed onset of death during the duration of the experiment.

[0673] To broaden the applicability, the anti-CD46 ADC was studied using a secondary multiple myeloma cell line, MM1.S, expressing a luciferase reporter. Potent tumor-killing activity of the anti-CD46 ADC was observed in vivo. Four doses of 4 mg / kg completely eliminated MM1.S tumor cells ( Figure 20 In contrast, the control ADC (MMAF bound to a non-binding antibody) had no effect at all at the same dose and dosing frequency. In addition, even a single dose of the anti-CD46 ADC at 4 mg / kg greatly reduced tumor burden, causing a profound inhibition of tumor development ( Figure 20 Four doses of anti-CD46 ADC at 0.8 mg / kg also caused sustained inhibition of MM1.S xenograft development ( Figure 20 Finally, even naked antibody (YS5 IgG1) caused significant tumor inhibition in this xenograft model ( Figure 20), indicating the interesting possibility of potential naked antibody-based therapy for specific multiple myeloma subtypes.

[0674] Kaplan-Meier analysis was performed to determine the survival rate after treatment. The CD46 ADC-treated group showed a significant survival advantage over the control group ( Figure 21 All mice that received four doses of 4 mg / kg CD46 ADC survived until the end of the experiment (day 212).

[0675] In addition to the aforementioned prostate cancer and multiple myeloma, CD46 has been found to be highly expressed in a wide range of cancer cell lines, including (but not limited to) colorectal cancer, pancreatic cancer, mesothelioma, lung cancer, breast cancer, ovarian cancer, bladder cancer, liver cancer, glioma, and neuroblastoma. In addition, anti-CD46 ADCs effectively kill these cells in vitro. For example, anti-CD46 ADCs are highly effective in killing colorectal cancer cells ( Figure 19 For YS5, Figure 22 For YS12 vs. HT29, and Figure 23 Targeting SB1HGNY against HT29), pancreatic cancer cells ( Figure 24 ), mesothelioma cells ( Figure 25 ) and ovarian cancer cells ( Figure 26 ).

[0676] To assess possible toxicity, the YS5 ADC was tested on a panel of control cells that either did not express CD46 (BPH-1) or expressed CD46 at intermediate levels (e.g., HS27). Figure 27 Regarding YS5 ADC against BPH-1, Figure 28 Regarding YS5 ADC vs HS27, Figure 29 Targeting YS5 ADC to standard T cells and Figure 30 Much reduced cytotoxicity was observed for the YS5 ADC against CD14-depleted peripheral blood mononuclear cells (PBMCs), suggesting that they do not readily take up the ADC. Differential internalization via macropinocytosis is a possible mechanism for the enhanced selectivity of the anti-CD46 ADC.

[0677] ADC toxicity was studied in vivo using transgenic mice expressing human CD46. There are no true murine orthologs of human CD46 and murine CD46 that perform physiological roles that are completely different from human CD46. Antibodies do not bind to murine CD46. Therefore, there are no good animal models other than transgenic models to evaluate possible anti-CD46 ADC toxicity. Anti-CD46 ADC was injected at 6 mg / kg into transgenic mice expressing human CD46, and the animals were monitored daily for overt signs of toxicity. The animals were sacrificed on day 14 and vital organs were harvested for histological examination of tissue damage. Figure 31As shown in Figure 2, there were no significant differences between mice treated with the anti-CD46 ADC and mice treated with the control ADC. At the dose of the ADC tested, no overt signs of toxicity were observed for the duration of this experiment. It should be understood that regulatory toxicology studies need to be conducted in non-human primates (e.g., cynomolgus macaques) in which CD46 is recognized by the anti-CD46 antibodies shown above.

[0678] Given that the human CD46 gene is located on the short arm of chromosome 1 (1q32.2), and given that 1q gains have been commonly observed in a variety of cancers with particularly poor prognoses, anti-CD46 antibody therapy, including but not limited to ADCs, may be applicable to a wide range of advanced malignancies with extreme therapeutic needs. For example, in prostate cancer, the region spanning 1q32.2 has been shown to gain in disseminated metastatic forms of the disease that are unresponsive to current therapies (Hanamura et al. (2006) Blood, 108:1724-1732), making metastatic castration-resistant prostate cancer an excellent candidate for treatment with our anti-CD46 ADC. In multiple myeloma, 1q gains are also commonly observed in relapsed patients (Id.), thus identifying an important patient population that our anti-CD46 ADCs may potentially help. Correlations between 1q gain (and specifically 1q32.2) and poor prognosis have also been found in other cancers, making 1q detection a potential biomarker for patient stratification and malignancy outcomes with our anti-CD46 ADC. FISH probes that detect 1q32.2 can be used to assess 1q status. Circulating DNA can also be used to detect 1q gain as a minimally invasive biomarker for CD46-expressing cancers with a poor prognosis (Fan et al. (2008) Proc. Natl. Acad. Sci. USA, 105:16266-16271).

[0679] The anti-CD46 antibodies described herein can be used as stand-alone imaging agents or companion diagnostics for anti-CD46 ADCs in imaging probes to monitor tumor status in vivo. We have previously labeled our initial anti-CD46 antibody, UA20, and demonstrated excellent in vivo imaging properties for prostate cancer targeting (He et al. (2010) J. Nucl. Med. 51:427-432). In addition to imaging, anti-CD46 antibodies can be used as immunohistochemistry-based biomarkers to assess CD46 expression in biopsies and archived patient samples, capture ELISA assays to assess serum CD46 levels, and FACS or chip-based assays to assess CD46 cell surface expression in disseminated and / or circulating tumor cells.

[0680] Example 2

[0681] CD46 ADC is highly active in an intrafemoral mCRPC xenograft model.

[0682] Because more than 95% of prostate cancer metastasizes to the bone, we further studied the efficacy of our anti-CD46 ADC in a bone xenograft model. We injected the metastatic castration-resistant prostate cancer (mCRPC) cell line LNCaP C4-2B carrying a firefly luciferase reporter into the femur of NSG mice to form an intraskeletal mCRPC xenograft model. After 7 days of transplantation, CD46 ADC (YS5-mcvcpab-MMAF) was injected once every 4 days for a total of 4 times. Tumor status was monitored by bioluminescence imaging during and after treatment. Figure 32 As shown in , CD46 ADC-treated mice displayed profound tumor suppression following the treatment period and persisting until the end of the experiment (day 65), indicating that our CD46 ADC is highly effective in this intramyocardial mCRPC xenograft model.

[0683] CD46 is highly expressed in CRPC and mCRPC tissues.

[0684] In addition to primary tumors, we performed immunohistochemical studies on tissue specimens from castration-resistant prostate cancer (CRPC) and metastatic castration-resistant prostate cancer (mCRPC). Figure 33 As shown in , CD46 is highly expressed in CRPC specimens. We further studied mCRPC specimens and found that CD46 was expressed in bone metastases ( Figure 34 ), lymph node metastasis ( Figure 35 ) and bladder metastasis ( Figure 36 ) with widespread distribution (100% of study cases, or 12 / 12) and strong expression in .

[0685] CD46 is overexpressed by the neuroendocrine subtype of prostate cancer.

[0686] Approximately 30% of patients are resistant to treatment with abiraterone and enzalutamide. The emergence of small cell / neuroendocrine prostate cancer can be a frequent event (approximately 30-40% of cases). Unlike adenocarcinomas, neuroendocrine prostate cancers do not usually express common markers such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). Therefore, we sought to investigate FACS CD46 expression in the neuroendocrine prostate cancer cell line H660. Figure 37 As shown in the left panel, CD46 is highly expressed by H660 cells. Western blot analysis confirmed that H660 cells expressed CD46 and the neuroendocrine marker neuron-specific enolase (NSE) ( Figure 37 , right). Our anti-CD46 antibody (YS5) was internalized by H660 cells and colocalized with the lysosomal marker LAMP1 ( Figure 38When incubated with H660 cells, our anti-CD46 ADC showed potent in vitro cytotoxic activity with EC50 < 1 nM ( Figure 39 ).

[0687] CD46 is further upregulated by prostate cancer cells after treatment with abiraterone or enzalutamide, rendering them susceptible to CD46 ADC impact.

[0688] We found that treatment of the mCRPC line LNCaP-C4-2B with 10 μM abiraterone for 7 days resulted in a significant upregulation of surface CD46 expression ( Figure 40 Interestingly, this upregulation was associated with enhanced tumor cell killing ( Figure 41 ), the EC50 value decreased from 169pM to 21pM. Similarly, when the neuroendocrine prostate cancer cell line H660 was cultured with 10μM enzalutamide for 7 days, a significant upregulation of cell surface CD46 was observed ( Figure 42 As observed in LNCaP-C4-2B cells, H660 cells became more sensitive to CD46 ADC after enzalutamide treatment, with a 4- to 5-fold decrease in EC50.

[0689] CD46 expression on additional tumors.

[0690] We found that CD46 is overexpressed in a variety of human cancers, in addition to prostate cancer and multiple myeloma. By immunohistochemical analysis, we found positive CD46 staining in 82% of colorectal cancers (81 / 99 cases), with 70 / 99 showing strong staining (71%) ( Figure 43 Interestingly, almost 100% of metastatic colorectal cancers express CD46 ( Figure 44 Liver metastasis, Figure 45 Lymph node metastasis, and Figure 46 bladder metastasis).

[0691] Positive CD46 staining was also observed in 41 / 50 mesothelioma cases (82%), with 31 / 50 showing strong staining (62%) ( Figure 47 In pancreatic cancer, positive CD46 staining was observed in 28 / 50 cases (56%) ( Figure 48 In glioblastoma multiforme (GBM), positive staining was observed in 30 / 40 (75%) cases ( Figure 49 ).

[0692] Positive staining was also observed in other tumors including, but not limited to, bladder, ovarian, gastric, lung, liver, breast, and lymphomas.

[0693] CD46 ADCs are effective against other tumors.

[0694] In addition to in vitro studies, we performed in vivo studies of CD46 ADCs in mesothelioma xenografts carried by NSG mice. Figure 50 As shown in Figure 3, YS5-mcvcpab-MMAF efficiently inhibited tumor xenograft progression.

[0695] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or variations thereof will occur to those skilled in the art and are included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Sequence Listing <110> Regents of the University of California <120> Macropinocytic human anti-CD46 antibodies and targeted cancer therapies <130> UCSFP037US <140> 2015800611385 <141> 2015-09-10 <150> US 62 / 049,973 <151> 2014-09-12 <150> PCT / US2015 / 049492 <151> 2015-09-10 <160> 78 <170> PatentIn version 3.5 <210> 1 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 1 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Leu Thr Val Asn Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 2 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 2 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Val Asn Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 3 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 3 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Val Asn Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 4 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VH Domain <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Ser Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Gly Asn Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 5 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Phe Ile Ser Tyr Asp Gly Asp Glu Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Trp Cys 85 90 95 Ala Lys Ala Ser Gly Tyr Gly Met Gly Ile Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 121 <212> PRT <213> artificial sequence <220> <223> Synthesized antibody VH structure <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Ile Ala Ala Ala Gly Arg Arg Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Ile Ala Ala Ala Gly Arg His Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser[[ID=۴۷]] 115 120 <210> 8 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Ile Ala Ala Ala Gly Arg His Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Val Gly Ser Thr Ala Ile Asn Tyr Val Arg Ala Tyr Thr 100 105 110 Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Ser Ser Thr Phe Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Gly Leu Tyr Ser Ser Gly Trp Ala Asn Trp Phe Asp Pro Arg 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VH Domain <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Met Gly Leu Ala Ala Ala Gly Leu Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 12 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Thr Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ser Ser Gly Ser Pro Gly Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 13 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 13 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn s 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Thr Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Gly Thr Ser Gly Tyr Asp Trp Ala Trp Phe Asp Leu Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VH Domain <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Met Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Tyr Tyr Gly Ser Gly Lys Asp Ala Phe Asp Ile 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 15 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Val Arg Gln Asp Gly Gly Gln Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Val Ser Gln Arg Asn Ser Gly Glu His Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VH domain <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Arg Ile Ala Ala Ala Gly Arg His Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 121 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Antibody VH Domain <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 n Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 九十 九十五 Ala Arg Asp Tyr Gly Arg Ile Ala Ala Ala Gly Arg Asn Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 121 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Antibody VH Domain <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr<​​​​​​​​​​​​​​​​​​​​100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 121 <212> PRT <213> artificial sequence <220> <223> Synthesized antibody VH structure <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Thr Asp Val Val Gly Val Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Ala Lys Val Met Gly Leu Ala Ala Ala Gly Leu Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> twenty one <211> 123 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VH domain <400> twenty one Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Ser Arg Pro Gly Gly Gly Tyr Ala Ser Gly Ser Thr Val Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> twenty two <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> twenty two Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95 Thr Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 23 <211> 109 <212> PRT <21B> Synthetic sequence <220> <223> Synthetic antibody VL domain <400> 23 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Note: There seems to be a tag error in the original text at line 28 where it says <210> 23. It should probably be something like <210> or <210> some_value. Also, in the translation, I've assumed <21B> in the translation of line 34 is what you meant as there's no <213> in the original. If these are incorrect assumptions, please correct the original text for a more accurate translation.20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95 Thr Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 24 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VL Domain <400> 24 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95 Thr Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 25 <211> 107 <212> PRT <213> artificial sequence <220> <223> Synthesized antibody VL structure <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Ser Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Ala Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100 105 <210> 26 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody VL domain <400> 26 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Val 20 25 30 Ser Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Phe Val Met Tyr 35 40 45 Gly Gln Asn Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Ile Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys His Ser Arg Asp Ser Ser Gly Thr His 85 90 95 Leu Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 27 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 27 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Ala Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Arg Thr Ser Asn Ile Gly Ser Asn 20 25 30 His Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 90 95 Ser Gly Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 28 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 28 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Thr His 85 90 95 Leu Glu Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 <210> 29 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 29 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Thr Leu Ser Thr Tyr Tyr Ala 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys His Ser Arg Asp Ile Ser Gly Asn Tyr 85 90 95 Leu Phe Ala Ser Gly Thr Lys Leu Thr Val Leu 100 105 <210> 30 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VL Domain <400> 30 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Ser Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Val Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 31 <211> 107 <212> PRT <213> artificial sequence <220> <223> Synthesized antibody VL structure <400> 31 Lys Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Thr Val Thr Ile Ala Cys Arg Ala Ser Arg Asp Ile Arg Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ile Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His Arg Leu Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100 105 <210> 32 <211> 111 <212> PRT <213> artificial sequence <220> <223> Synthesized antibody VL structure <400> 32 Asn Phe Met Leu Thr Gln Pro Ala Ser Leu Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Tyr Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Pro Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 33 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 33 Ser Tyr Val Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn Gln 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 34 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 34 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Thr Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Ile Leu Val Leu Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 35 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Synthetic antibody VL domain <400> 35 Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Gly Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln Asn Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Ser 85 90 95 Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Antibody VL Domain <400> 36 Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr 1 5 10 15 Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly 35 40 45 Glu Asn Ser Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser 50 55 60 Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Gl...

Claims

1. An immunoconjugate comprising: a) An isolated human IgG1 antibody that binds CD46, comprising an antibody with YS5 CDRs, wherein the CDRs comprise VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1 is GLTVNNYA as set forth at residues 26-33 of SEQ ID NO:1, the VH CDR2 is ISYDGNNK as set forth at residues 51-58 of SEQ ID NO:1, the VH CDR3 is AKGGGYFDL as set forth at residues 97-105 of SEQ ID NO:1, the VL CDR1 is SSNIGAGYD as set forth at residues 26-34 of SEQ ID NO:22, the VL CDR2 is GNN as set forth at residues 52-54 of SEQ ID NO:22, and the VL CDR3 is SSYTSGTWL as set forth at residues 91-99 of SEQ ID NO:22, b) MC-vc-PAB linker, and c) an effector comprising monomethyl auristatin F MMAF or monomethyl auristatin E MMAE.

2. The immunoconjugate of claim 1, wherein the effector is MMAE.

3. The immunoconjugate of claim 2, wherein the isolated human IgG1 antibody comprises: The variable light VL chain of the YS5 antibody shown in SEQ ID NO:22 and the variable heavy VH chain of the YS5 antibody shown in SEQ ID NO:

1.

4. The immunoconjugate of claim 3, wherein the ratio of the effector to the antibody is 2 or 4.

5. A pharmaceutical formulation comprising a unit dose of the immunoconjugate according to claim 4 formulated for intravenous administration.

6. Use of the immunoconjugate according to claim 4 in the manufacture of a medicament for inhibiting the growth and / or proliferation of cancer cells expressing or overexpressing CD46, wherein the cancer cells are selected from the group consisting of prostate cancer cells, multiple myeloma cells, colorectal cancer cells, pancreatic cancer cells, mesothelioma cells and ovarian cancer cells.

7. The use according to claim 6, wherein the cancer cells are castration-resistant prostate cancer cells.

8. The use according to claim 7, wherein the medicament is for administration in combination with abiraterone and / or enzalutamide.

9. The use according to claim 6, wherein the cancer cells are multiple myeloma cells.

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