Therapeutic antibodies and uses thereof

Abstract

To provide therapeutic agents for multiple myeloma.SOLUTION: The present invention relates to antibodies, e.g., full length antibodies or antigen binding fragments thereof, that specifically bind to BCMA (B-Cell Maturation Antigen) and CD3 (Cluster of Differentiation 3). The invention also relates to antibody conjugates (e.g., antibody-drug-conjugates) comprising the BCMA antibodies, compositions comprising the BCMA antibodies, and methods of using the BCMA antibodies and their conjugates for treating conditions associated with cells expressing BCMA (e.g., cancer or autoimmune disease). The invention further relates to heteromultimeric antibodies that specifically bind to CD3 and a tumor cell antigen, (e.g., bispecific antibodies that specifically bind to CD3 and BCMA). Compositions comprising such heteromultimeric antibodies, methods for producing and purifying such heterodimeric antibodies, and their use in diagnostics and therapeutics are also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies, e.g., full length antibodies or antigen-binding fragments thereof, that specifically bind to BCMA (B cell maturation antigen) and / or CD3 (cluster of differentiation 3). The present invention also relates to antibody conjugates (e.g., antibody-drug conjugates) comprising BCMA antibodies for treating conditions associated with cells expressing BCMA (e.g., cancer or autoimmune disease), compositions comprising BCMA antibodies, and methods of using BCMA antibodies and these conjugates. The present invention further relates to heteromultimeric antibodies (e.g., bispecific antibodies that specifically bind CD3 and BCMA) that specifically bind to CD3 and tumor cell antigens. Compositions comprising such heteromultimeric antibodies, methods for producing and purifying such heteromultimeric antibodies, and their use in diagnostics and therapeutics are also provided. [Background technology]

[0002] B-cell maturation antigen (BCMA, CD269, or TNFRSF17) is a member of the tumor necrosis factor receptor (TNFR) superfamily. BCMA was identified in malignant human T-cell lymphomas containing the t(4;16) translocation. The gene is preferentially expressed in the B-cell lineage, with highest expression in plasmablasts and plasma cells, antibody-secreting cells. BCMA binds two ligands, B-cell activating factor (BAFF) (also called B-lymphocyte stimulator (BLyS) and APOL-related leukocyte expressed ligand (TALL-1)) and proliferation-inducing ligand (APRIL), with affinities of 1 μM and 16 nM, respectively. Binding of APRIL or BAFF to BCMA promotes a signaling cascade involving NF-kappaB, Elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, which results in signals for cell survival and proliferation.

[0003] BCMA is also expressed in malignant B cells and in several cancers involving B lymphocytes, including multiple myeloma, plasmacytoma, Hodgkin's lymphoma, and chronic lymphocytic leukemia. In autoimmune diseases involving plasmablasts, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, BCMA-expressing antibody-producing cells secrete autoantibodies that attack themselves. Summary of the Invention [Problem to be solved by the invention]

[0004] In the case of multiple myeloma, approximately 24,000 new cases are newly diagnosed in the United States each year, accounting for approximately 15% of newly diagnosed blood cancers in the United States. On average, 11,000 deaths occur from multiple myeloma each year, with an average 5-year survival rate of approximately 44% and a median survival of 50-55 months. Current treatments for multiple myeloma focus on reducing plasma cell apoptosis and / or osteoclast activity (e.g., chemotherapy, thalidomide, lenalidomide, bisphosphonates, and / or proteasome inhibitors such as bortezomib (VELCADE®) or carfilzomib). However, multiple myeloma remains an incurable disease, with almost all patients developing resistance to these agents and eventually relapsing. Therefore, alternative treatments for multiple myeloma, such as the use of anti-BCMA antagonists, including antibodies and other immunotherapeutics (e.g., bispecific antibodies or antibody-drug conjugates), should provide superior therapeutic agents. [Means for solving the problem]

[0005] The invention disclosed herein is directed to therapeutic antibodies that bind BCMA and / or CD3. Antibody conjugates (e.g., antibody-drug conjugates) that include BCMA are also provided. Additionally, heteromultimeric antibodies (e.g., bispecific antibodies) that specifically bind CD3 and tumor cell antigens (e.g., bispecific antibodies that specifically bind CD3 and BCMA) are also provided.

[0006] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to B cell maturation antigen (BCMA), comprising: (a) (i) an antibody having the sequence SYX 1 MX 2 (In the formula, X 1 is A or P; X 2 is T, N, or S) (SEQ ID NO: 301), GFTFX 1 SY (wherein X 1 is G or S) (SEQ ID NO: 302), or GFTFX 1 SYX 2 MX 3 (In the formula, X 1 is G or S, and X 2 is A or P, and X 3 (ii) a heavy chain variable (VH) complementarity determining region 1 (CDR1) comprising the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (in the formula, X 1 is I, V, T, H, L, A, or C, and X 2 is S, D, G, T, I, L, F, M, or V, and X 3 is G, Y, L, H, D, A, S, or M, and X 4 is S, Q, T, A, F, or W, and X 5 is G or T, and X 6 is N, S, P, Y, W, or F, and X 7 is S, T, I, L, T, A, R, V, K, G, or C, and X 8 is F, Y, P, W, H, or G, and X 9 is V, R, or L, and X 10 is G or T) (SEQ ID NO: 305), or X 1 X 2 X 3 X 4 X 5 X6 (In the formula, X 1 is S, V, I, D, G, T, L, F, or M, and X 2 is G, Y, L, H, D, A, S, or M, and X 3 is S, G, F, or W, and X 4 is G or S, and X 5 is G or T, and X 6 is N, S, P, Y, or W) (SEQ ID NO: 306), and (iii) a VH CDR2 comprising the sequence VSPIX 1 X 2 X 3 (In the formula, X 1 is either A or Y, and X 2 is A or S, and X 3 is G, Q, L, P, or E) (SEQ ID NO: 307), or YWPMX 1 X 2 (In the formula, X 1 is D, S, T, or A, and X 2 (i) a VH region comprising a VH CDR3 comprising sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 (In the formula, X 1 is R, G, W, A, or C, and X 2 is A, P, G, L, C, or S, and X 3 is S, G, or R, and X 4 is Q, C, E, V, or I, and X 5 is S, P, G, A, R, or D, and X 6 is V, G, I, or L, and X 7 is S, E, D, P, or G, and X 8 is S, P, F, A, M, E, V, N, D, or Y, and X 9is I, T, V, E, S, A, M, Q, Y, H, R, or F, and X 10 is Y or F, and X 11 is L, W, or P, and X 12 (ii) a light chain variable (VL) CDR1 comprising the sequence X 1 ASX 2 RAX 3 (In the formula, X 1 is G or D, and X 2 is S or I, and X 3 is T or P) (SEQ ID NO: 310), and (iii) a VL CDR2 comprising the sequence QQYX 1 X 2 X 3 PX 4 T (where X 1 is G, Q, E, L, F, A, S, M, K, R, or Y, and X 2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C, and X 3 is W, F, or S, and X 4 is L or I) (SEQ ID NO: 311), or QQYX 1 X 2 X 3 PX 4 (In the formula, X 1 is G, Q, E, L, F, A, S, M, R, K, or Y, and X 2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y, and X 3 is W, S, or F, and X 4 The present invention provides an isolated antibody or antigen-binding fragment thereof comprising a VL region comprising a VL CDR3 comprising:

[0007] In another aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds BCMA, comprising the VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 125, 127, 313, 314, 363, or 365. 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 The present invention provides an isolated antibody or antigen-binding fragment thereof comprising a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in any one of claims 1 to 7, 79, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, 316, or 364.In some embodiments, the VH region comprises (i) a VH CDR1 comprising SEQ ID NO: 150, 151, 152, 156, or 157; (ii) a VH CDR2 comprising SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, or 172; and (iii) a VH CDR2 comprising SEQ ID NO: 155, 161, 197, 205, or 164. and / or the VL region comprises (i) a VL CDR1 comprising SEQ ID NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235, 297, or 299, (ii) a VL CDR2 comprising SEQ ID NO: 221, and (iii) a VL CDR3 comprising SEQ ID NO: 225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298, or 300. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 112, or a variant with one or several conservative amino acid substitutions in residues that are not within the CDRs, and / or the VL region comprises the amino acid sequence set forth in SEQ ID NO: 38, or a variant thereof with one or several amino acid substitutions in amino acids that are not within the CDRs. In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO: 357, and a heavy chain comprising the sequence set forth in SEQ ID NO: 358. In some embodiments, the antibody comprises a VH region produced by an expression vector having ATCC Accession No. PTA-122094. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC Accession No. PTA-122093.

[0008] In another aspect, the invention provides an isolated antibody comprising an acyl donor glutamine-containing tag engineered at a specific site of the BCMA antibody of the invention. In some embodiments, the tag is selected from the group consisting of Q, LQG, LLQGG (SEQ ID NO:318), LLQG (SEQ ID NO:454), LSLSQG (SEQ ID NO:455), GGGLLQGG (SEQ ID NO:456), GLLQG (SEQ ID NO:457), LLQ, GSPLAQSHGG (SEQ ID NO:458), GLLQGGG (SEQ ID NO:459), GLLQGG (SEQ ID NO:460), GLLQ (SEQ ID NO:461), LLQLLQGA (SEQ ID NO:462), LLQGA (SEQ ID NO:463), LLQYQGA (SEQ ID NO:464), LLQGSG (SEQ ID NO:465), LLQYQG (SEQ ID NO:466), LLQLLQG (SEQ ID NO:467), SLLQG (SEQ ID NO:468). , LLQLQ (SEQ ID NO:469), LLQLLQ (SEQ ID NO:470), LLQGR (SEQ ID NO:471), LLQGPP (SEQ ID NO:472), LLQGPA (SEQ ID NO:473), GGLLQGPP (SEQ ID NO:474), GGLLQGA (SEQ ID NO:475), LLQGPGK (SEQ ID NO:476), LLQGPG (SEQ ID NO:477), LLQGP (SEQ ID NO:478), LLQP (SEQ ID NO:479), LLQPGK (SEQ ID NO:480), LLQAPGK (SEQ ID NO:481), LLQGAPG (SEQ ID NO:482), LLQGAP (SEQ ID NO:483), and LLQLQG (SEQ ID NO:484).

[0009] In one variation, the invention provides an isolated antibody comprising an acyl donor glutamine-containing tag and an amino acid modification at position 222, 340, or 370 of the BCMA antibody of the invention. In some embodiments, the amino acid modification is a lysine to arginine substitution.

[0010] In some embodiments, the BCMA antibody of the invention further comprises a linker. In some embodiments, the linker is Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val- Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

[0011] In another aspect, the invention provides a conjugate of a BCMA antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment is conjugated to an agent selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide. In some embodiments, the agent is a cytotoxic agent, including but not limited to anthracyclines, auristatins, camptothecins, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterlins, spliceostatins, pladienolides, and stereoisomers, isosteres, analogs, or derivatives thereof.For example, cytotoxic agents include MMAD (monomethyl auristatin D), 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-1-yl), 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N -methyl-L-valinamide), 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), or 0121 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).

[0012] In some embodiments, the invention provides a conjugate comprising the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic agent). In some embodiments, the acyl donor glutamine-containing tag comprises the amino acid sequence LLQG (SEQ ID NO: 319) and / or GGLLQGPP (SEQ ID NO: 339), and the linker comprises acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl or amino-PEG6-C2. In some embodiments, the conjugate is selected from the group consisting of: 1) antibody-GGLLQGPP (SEQ ID NO: 339)-(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC))-0101, 2) antibody-LLQG (SEQ ID NO: 319)-amino-PEG6-C2-0131, and 3) antibody-LLQG (SEQ ID NO: 319)-amino-PEG6-C2-3377. In some embodiments, the conjugate further comprises an amino acid substitution from lysine to arginine at antibody position 222. In some embodiments, the conjugate further comprises an amino acid substitution at antibody position N297Q or N297A.

[0013] In another aspect, there is provided a method of producing a BCMA antibody as described herein, the method comprising culturing a host cell under conditions that result in the production of the BCMA antibody, and isolating the BCMA antibody from the host cell or culture.

[0014] In another aspect, the invention provides the use of a BCMA antibody or BCMA antibody conjugate as described herein in the manufacture of a medicament for treating a condition associated with BCMA expression (e.g. cancer or an autoimmune disorder). In some embodiments, there is provided the use of a BCMA antibody or BCMA antibody conjugate as described herein in the manufacture of a medicament for inhibiting tumor growth or progression. In some embodiments, there is provided the use of a BCMA antibody or BCMA antibody conjugate as described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing BCMA. In some embodiments, there is provided the use of a BCMA antibody or BCMA antibody conjugate as described herein in the manufacture of a medicament for inducing tumor regression.

[0015] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, or 400. The present invention provides an isolated antibody or antigen-binding fragment thereof, comprising a VH region comprising CDR3 and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable VL sequence set forth in SEQ ID NO:319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399. In some embodiments, the antibody comprises a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388, and / or a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387.In some embodiments, the VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NO: 331, 332, 333, 401, 402, 403, 407, 408, 415, 416, 418, 419, 420, 424, 425, 426, 446, 447, or 448; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449, or 450; and (iii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 335, 406, 423, 429, or 451. and / or the VL region comprises (i) a light chain variable (VL) CDR1 comprising the sequence set forth in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341, 433, 452, or 436, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453. In some embodiments, the VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NO: 331, 332, 333, 401, 407, or 408, (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 336, 404, 405, or 417, and (iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 335 or 406, and / or the VL region comprises (i) a light chain variable (VL) CDR1 comprising the sequence set forth in SEQ ID NO: 343 or 441, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341 or 436, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342 or 439. In some embodiments, the antibody comprises a VH region produced by an expression vector having ATCC Accession No. PTA-122513. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC Accession No. PTA-122512.

[0016] In another aspect, there is provided an isolated antibody that specifically binds to CD3 and competes with the anti-CD3 antibodies of the invention described herein.

[0017] In another aspect, the invention provides a bispecific antibody, the full length human antibody comprising a bispecific antibody first antibody variable domain capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cell, and a bispecific antibody second antibody variable domain capable of specifically binding to a target antigen, the first antibody variable domain comprising a VH CDR1, a VH CDR2, and a VH CDR3 of the heavy chain variable (VH) sequence set forth in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, or 400. The bispecific antibodies include a VH region comprising a VL CDR3 and / or a VL region comprising a VL CDR1, a VL CDR2, and a VL CDR3 of the light chain variable VL sequence set forth in SEQ ID NOs: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399.In some embodiments, the first antibody variable domain comprises (i) a VH CDR1 of a heavy chain variable (VH) sequence set forth in SEQ ID NO: 331, 332, 333, 401, 402, 403, 407, 408, 415, 416, 418, 419, 420, 424, 425, 426, 446, 447, or 448, (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449, or 450, and (iii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 335, 406, 423, 429, or 451. and / or a VL region comprising (i) a light chain variable (VL) CDR1 comprising the sequence set forth in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341, 433, 452, or 436, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453. In some embodiments, the first antibody variable domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388, and / or a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387, and the second antibody variable domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 112, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 38.

[0018] In some embodiments, the second antibody variable domain comprises: (a)(i) the sequence SYX 1 MX 2 (In the formula, X 1 is A or P, and X 2 is T, N, or S) (SEQ ID NO: 301), GFTFX 1 SY (wherein X 1is G or S) (SEQ ID NO: 302), or GFTFX 1 SYX 2 MX 3 (In the formula, X 1 is G or S, and X 2 is A or P, and X 3 (ii) a heavy chain variable (VH) complementarity determining region 1 (CDR1) comprising the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (in the formula, X 1 is I, V, T, H, L, A, or C, and X 2 is S, D, G, T, I, L, F, M, or V, and X 3 is G, Y, L, H, D, A, S, or M, and X 4 is S, Q, T, A, F, or W, and X 5 is G or T, and X 6 is N, S, P, Y, W, or F, and X 7 is S, T, I, L, T, A, R, V, K, G, or C, and X 8 is F, Y, P, W, H, or G, and X 9 is V, R, or L, and X 10 is G or T) (SEQ ID NO: 305), or X 1 X 2 X 3 X 4 X 5 X 6 (In the formula, X 1 is S, V, I, D, G, T, L, F, or M, and X 2 is G, Y, L, H, D, A, S, or M, and X 3 is S, G, F, or W, and X 4 is G or S, and X 5 is G or T, and X 6is N, S, P, Y, or W) (SEQ ID NO: 306), and (iii) a VH CDR2 comprising the sequence VSPIX 1 X 2 X 3 (In the formula, X 1 is either A or Y, and X 2 is A or S, and X 3 is G, Q, L, P, or E) (SEQ ID NO: 307), or YWPMX 1 X 2 (In the formula, X 1 is D, S, T, or A, and X 2 (i) a VH region comprising a VH CDR3 comprising sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 (In the formula, X 1 is R, G, W, A, or C, and X 2 is A, P, G, L, C, or S, and X 3 is S, G, or R, and X 4 is Q, C, E, V, or I, and X 5 is S, L, P, G, A, R, or D, and X 6 is V, G, or I, and X 7 is S, E, D, or P, and X 8 is S, P, F, A, M, E, V, N, D, or Y, and X 9 is I, T, V, E, S, A, M, Q, Y, H, or R, and X 10 is Y or F, and X 11 is L, W, or P, and X 12 (ii) a light chain variable (VL) CDR1 comprising the sequence X 1 ASX 2 RAX 3 (In the formula, X1 is G or D, and X 2 is S or I, and X 3 is T or P) (SEQ ID NO: 310), and (iii) a VL CDR2 comprising the sequence QQYX 1 X 2 X 3 PX 4 T (where X 1 is G, Q, E, L, F, A, S, M, K, R, or Y, and X 2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C, and X 3 is W, F, or S, and X 4 is L or I) (SEQ ID NO: 311), or QQYX 1 X 2 X 3 PX 4 (In the formula, X 1 is G, Q, E, L, F, A, S, M, R, K, or Y, and X 2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y, and X 3 is W, S, or F, and X 4wherein the VL CDR3 comprises a VL CDR4 which is L or I (SEQ ID NO: 312). In some embodiments, the second antibody variable domain comprises (i) a heavy chain variable (VH) CDR1 comprising the sequence set forth in SEQ ID NO: 150, 151, 152, 156, 157, 348, 349, 353, 354, or 355; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 350, 351, 356, or 357; and (iii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 155, 161, 197, 205,164, or 352, or 358. and / or a light chain variable (VL) region comprising (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235, 297, 299, or 361, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 221, 359, or 362, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 211, 225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298, 300, or 360.

[0019] In some embodiments, (a) the first antibody variable domain comprises a VH region comprising (i) a heavy chain variable (VH) complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NO: 331, 332, 333, 401, 407, or 408, (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 336, 417, 404, or 405, and (iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 335 or 406, and / or (i) a light chain variable (VL) CDR1 comprising the sequence set forth in SEQ ID NO: 343 or 441, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341 or 436, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342 or 439. and (b) the second antibody variable domain comprises a heavy chain VH region comprising (i) a heavy chain variable (VH) CDR1 comprising the sequence set forth in SEQ ID NO: 151, 156, or 157, (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 158 or 159, and (iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 155, and / or a light chain variable (VL) region comprising (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO: 209, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 225.

[0020] In some embodiments, the first and second antibody variable domains of the bispecific antibody both comprise amino acid modifications at positions 223, 225, and 228 in the hinge region and at positions 409 or 368 in the CH3 region (EU numbering scheme) of human IgG2 (SEQ ID NO: 493). In some embodiments, the bispecific antibodies described herein further comprise an amino acid modification at position 265 of human IgG2.

[0021] In another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies (e.g., BCMA, CD3, or bispecific) or conjugates thereof (e.g., BCMA antibody-drug conjugates) described herein.

[0022] In another aspect, the present invention also provides cell lines recombinantly producing any of the antibodies (e.g., BCMA, CD3, or bispecific) or conjugates thereof (e.g., BCMA antibody-drug conjugates) described herein.

[0023] In another aspect, the invention also provides nucleic acids encoding any of the antibodies (e.g., BCMA, CD3, or bispecific) or conjugates thereof (e.g., BCMA antibody-drug conjugates) described herein. The invention also provides nucleic acids encoding the heavy and / or light chain variable regions of any of these antibodies described herein.

[0024] The invention also provides kits comprising an effective amount of any of the antibodies (e.g., BCMA, CD3, or bispecific) or conjugates thereof (e.g., BCMA antibody-drug conjugates) described herein.

[0025] The invention also provides a method of treating a condition in a subject in need thereof (e.g. inhibiting tumor growth / progression, inhibiting metastasis of malignant cells expressing BCMA, inducing tumor regression in a subject having malignant cells expressing BCMA) comprising providing an isolated antibody (e.g. BCMA) or binding fragment, bispecific antibody (BCMA-CD3 bispecific), or conjugate thereof (e.g. BCMA antibody-drug conjugate) as described herein, and administering the antibody or conjugate to the subject.

[0026] Also provided is a method of treating a condition associated with malignant cells expressing a tumor antigen in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of the present invention. In some embodiments, the condition is cancer. In some embodiments, the cancer is multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CM), chronic myelogenous leukemia (CL), chronic myelogenous leukemia (CR), chronic myelogenous leukemia (CLL), chronic myelogenous leukemia (CML ... L), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenström's macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, nodular marginal zone B and B-cell-related cancers selected from the group consisting of diffuse large B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castremann's disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-related lymphomas. In some embodiments, the condition is an autoimmune disorder, such as systemic lupus erythematosus or rheumatoid arthritis.

[0027] In some embodiments, the antibodies described herein comprise a constant region. In some embodiments, the antibodies described herein are of human IgG1, IgG2 or IgG2Δa, IgG3, or IgG4 subclass. In some embodiments, the antibodies described herein comprise a glycosylated constant region. In some embodiments, the antibodies described herein comprise a constant region that has increased binding affinity to one or more human Fc gamma receptors. [Brief description of the drawings]

[0028] [Figure 1A] Graph showing dual-referenced sensorgrams with fit curves of the interaction of selected anti-BCMA antibodies of the invention with human BCMA. [Figure 1B] Graph showing dual-referenced sensorgrams with fit curves of the interaction of selected anti-BCMA antibodies of the invention with human BCMA. [Figure 1C] Graph showing dual-referenced sensorgrams with fit curves of the interaction of selected anti-BCMA antibodies of the invention with human BCMA. [Figure 1D] Graph showing dual-referenced sensorgrams with fit curves of the interaction of selected anti-BCMA antibodies of the invention with human BCMA. [Diagram 2] Graph showing in vivo efficacy study of various anti-BCMA ADCs in MM1S orthotopic multiple myeloma model including P6E01_VHVL-AcLys-Val-Cit-PABC-Aur0101, P5A2_VHVL-AcLys-Val-Cit-PABC-Aur0101, P5C1_VHVL-AcLys-Val-Cit-PABC-Aur0101, P4G4-AcLys-Val-Cit-PABC-Aur0101, and P1A11-AcLys-Val-Cit-PABC-Aur0101. NNC is a negative control non-BCMA antibody. "LCQ05" and "LCQ04" correspond to glutamine-containing transglutaminase tag SEQ ID NOs: 474 and 475, respectively. [Diagram 3]

[0023] Figure 1 depicts the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model including L3.PY / P6E01 antibody conjugated with 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMA antibody. "LCQ05" and H7c correspond to the glutamine-containing transglutaminase tags SEQ ID NO:474 and SEQ ID NO:454, respectively. [Figure 4] 13A-C are graphs depicting in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model comprising L3.PY / P6E01 antibody conjugated with 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMA antibody (Antibody-N297Q / K222R-AcLys-VC-PABC-0101). "LCQ05" and H7c correspond to the glutamine-containing transglutaminase tags SEQ ID NO: 474 and SEQ ID NO: 454, respectively. [Diagram 5]1 is a graph again depicting the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model. Anti-BCMA antibody COMBO_Rd4_0.6nM-C29 ("Combo C29 DI") is conjugated to H7c / N297A / K222R-amino-PEG6-C2-131 at doses ranging from 0.1 mg / kg, 0.38 mg / kg, 0.75 mg / kg, 1.5 mg / kg compared to NNC at 3 mg / kg, a control non-BCMA antibody (Antibody-N297Q / K222R-AcLys-VC-PABC-0101). H7c corresponds to the glutamine-containing transglutaminase tag SEQ ID NO:454. [Figure 6A] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 6B] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 6C] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 6D] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 6E] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 6F]1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. B cell depletion after a single dose of bispecific antibody is shown as a percentage of pre-study counts. [Figure 7A] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 7B] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 7C] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 7D] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 7E] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 7F] FIG. 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. CD8+ T cell kinetics were followed after a single dose of the bispecific antibody. [Figure 8A] 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of monovalent CD3 antibodies on T cell dynamics and proliferation was analyzed. [Figure 8B] 1 is a graph showing the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of monovalent CD3 antibodies on T cell dynamics and proliferation was analyzed. [Figure 9A]1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of anti-CD3 arm affinity on B cell depletion was analyzed. [Figure 9B] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of anti-CD3 arm affinity on B cell depletion was analyzed. [Figure 9C] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of anti-CD3 arm affinity on B cell depletion was analyzed. [Figure 9D] 1 is a graph depicting the in vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys. The effect of anti-CD3 arm affinity on B cell depletion was analyzed. [Figure 10A] 1 is a graph showing that selected anti-CD3 antibodies had thymidine incorporation readouts on human and cynomolgus monkey PBMCs. [Figure 10B] 1 is a graph showing that selected anti-CD3 antibodies had thymidine incorporation readouts on human and cynomolgus monkey PBMCs. [Figure 11A] FIG. 13 is a graph showing that all human anti-EpCam_h2B4 bispecific antibodies have cell killing activity in an in vitro setting. [Figure 11B] FIG. 13 is a graph showing that all human anti-EpCam_h2B4 bispecific antibodies have cell killing activity in an in vitro setting. [Figure 11C] FIG. 13 is a graph showing that all human anti-EpCam_h2B4 bispecific antibodies have cell killing activity in an in vitro setting. [Figure 11D] FIG. 13 is a graph showing that all human anti-EpCam_h2B4 bispecific antibodies have cell killing activity in an in vitro setting. [Figure 12] 1 is a graph showing that a single dose of human anti-BCMA / CD3 bispecific antibody resulted in tumor regression in an orthotopic MM1.S myeloma model in a dose-dependent manner. [Figure 13] 1 is a graph showing that two doses of human anti-BCMA / CD3 bispecific antibody increased tumor regression in an orthotopic Molp8 myeloma model. [Figure 14] 1 is a graph showing that anti-BCMA / CD3 bispecific antibodies alone or in combination with the standard of care for multiple myeloma (lenalidomide or bortezomib) are more effective than lenalidomide and bortezomib in combination in an orthotopic Molp8 tumor model. [Figure 15A] Graphs showing that carfilzomib, lenalidomide, and doxorubicin have no negative effect on the function of anti-BCMA / CD3 bispecific antibody on OPM2 cells compared to anti-BCMA / CD3 bispecific antibody alone. [Figure 15B] Graphs showing that carfilzomib, lenalidomide, and doxorubicin have no negative effect on the function of anti-BCMA / CD3 bispecific antibody on OPM2 cells compared to anti-BCMA / CD3 bispecific antibody alone. [Figure 15C] Graphs showing that carfilzomib, lenalidomide, and doxorubicin have no negative effect on the function of anti-BCMA / CD3 bispecific antibody on OPM2 cells compared to anti-BCMA / CD3 bispecific antibody alone. [Figure 16] Graph showing the synergistic effect on the function of an anti-BCMA / CD3 bispecific antibody when combined with carfilzomib and lenalidomide compared to each molecule alone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The invention disclosed herein provides antibodies and antibody conjugates (e.g., antibody-drug conjugates) that specifically bind to BCMA (e.g., human BCMA). The invention also provides polynucleotides encoding these antibodies and conjugates, compositions comprising these antibodies and conjugates, and methods of making these antibodies and conjugates. Additionally, the invention disclosed herein provides antibodies that specifically bind to CD3 (e.g., human CD3), and heterodimeric antibodies (e.g., bispecific antibodies) that specifically bind to CD3 and a tumor antigen (e.g., BCMA). The invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods of making and using these antibodies. The invention further provides methods for treating a condition associated with malignant BCMA expression in a subject, such as cancer or an autoimmune disease, using the antibodies (e.g., BCMA, CD3, or bispecific antibodies) or conjugates thereof (BCMA antibody-drug conjugates) described herein.

[0030] Common techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature, e.g., in Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989), Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by M. J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J. E. Elis, 1998), Academic Press; Animal Cell Culture (edited by R. I. Freshney, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J. B. Griffiths, and D. G. Newell, 1993-1998), J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Current Protocols in Molecular Biology (eds. JM Miller and MPCalos, 1987); Current Protocols in Molecular Biology (eds. FMAusubel et al., 1987); PCR: The Polymerase Chain Reaction (eds. Mullis et al., 1994); Current Protocols in Immunology (eds. JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CAJaneway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0031] definition An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located within the variable region of the immunoglobulin molecule. As used herein, the term refers to intact polyclonal or monoclonal antibodies as well as fragments thereof (Fab, Fab', F(ab')). 2, Fv), single chain (ScFv), and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, an immunoglobulin can be assigned to different classes. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0032] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., BCMA or CD3). The antigen-binding function of an antibody may be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab') and / or Fab' fragments. 2 , Fd fragments consisting of the VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single arm of an antibody, single-domain antibody (dAb) fragments (Ward et al., Nature, 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0033] An antibody, antibody conjugate, or polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., a BCMA protein or a CD3 protein) are terms well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, rapidly, for a greater duration and / or with greater affinity than it reacts or associates with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" if it binds to a target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a BCMA epitope or a CD3 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other BCMA epitopes, non-BCMA epitopes, other CD3 epitopes, or non-CD3 epitopes. By reading this definition, it is also understood that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0034] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and contribute, together with the CDRs from the other chain, to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J.Molec.Biol., 273:927-948). As used herein, CDRs can refer to CDRs defined by either approach, or by a combination of both approaches.

[0035] The "CDRs" of a variable domain are amino acid residues within a variable region as defined by Kabat, Chothia, both Kabat and Chothia's accumulation, AbM, contact, and / or conformation definitions, or any method of CDR determination known in the art. Antibody CDRs may be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The locations of CDRs may also be identified as structural loop structures as originally described by Chothia et al. See, e.g., Chothia et al., Nature, 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts, as presented in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. Another approach, referred to herein as the "conformational definition" of CDRs, allows the position of CDRs to be identified as residues that make an enthalpic contribution to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above methods, but they will nevertheless overlap with at least a portion of Kabat CDR, although they may be shortened or lengthened in light of predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly impact antigen binding.As used herein, CDR can refer to CDRs defined by any method known in the art, including a combination of methods.The methods used herein can utilize CDRs defined by any of these methods.For any given embodiment that contains more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0036] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495, 1975, or can be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.

[0037] As used herein, a "humanized" antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab', F(ab')) that contains minimal sequence derived from non-human immunoglobulin. 2Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are not specifically humanized (e.g., humanized antibody fragments, such as IgG, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG2, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG1, IgG2, IgG4, IgG5, IgG1, IgG2, IgG4, IgG1, IgG1, IgG2, IgG4, IgG5, IgG1, IgG2, IgG3, IgG4, IgG4, IgG5, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG5, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG2, IgG4 ...2, IgG4, IgG1, IgG4, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG4, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG4, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG4, IgG1, IgG4, IgG1, IgG4, IgG1, IgG2, IgG4, A humanized antibody will optimally also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies with modified Fc regions as described in WO99 / 58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0038] As used herein, a "human antibody" refers to an antibody that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies known to those of skill in the art or disclosed herein. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. Human antibodies can be generated using a variety of techniques known in the art. In one embodiment, human antibodies are selected from phage libraries, where the phage libraries express human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA), 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be produced by immunization of animals into which human immunoglobulin loci have been transgenic in place of endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This technique is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies may be prepared by immortalizing human B-lymphocytes that produce antibodies directed against a target antigen (such B-lymphocytes can be harvested from an individual or from single-cell cloning of cDNA, or immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991, and U.S. Patent No. 5,750,373.

[0039] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as, for example, an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0040] The terms "polypeptide", "oligopeptide", "peptide", and "protein" are used interchangeably herein to refer to a chain of amino acids of any length, preferably relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, which may include modified amino acids and / or may be interrupted by non-amino acids. The term also encompasses amino acid chains that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It is understood that polypeptides may exist as single chains or associated chains.

[0041] A "monovalent antibody" contains one antigen binding site per molecule (e.g., IgG or Fab). In some cases, a monovalent antibody can have more than one antigen binding site, but the binding sites are from different antigens.

[0042] A "monospecific antibody" contains two identical antigenic sites per molecule (e.g., IgG), such that the two binding sites bind the same epitope on the antigen. Thus, they compete with each other for binding to one antigen molecule. Most antibodies found in nature are monospecific. In some cases, a monospecific antibody can also be a monovalent antibody (e.g., Fab).

[0043] A "bivalent antibody" contains two antigen-binding sites per molecule (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0044] A "bispecific" or "dual specific" is a hybrid antibody that has two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which may be present on the same or different protein targets.

[0045] A "bifunctional" antibody has identical antigen-binding sites (ie, identical amino acid sequences) in its two arms, but each binding site can recognize two different antigens.

[0046] A "heteromultimer," "heteromultimeric complex," or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, where the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. A heteromultimer can include a "heterodimer" formed by the first and second polypeptides, or can form a more highly ordered tertiary structure in which polypeptides are present in addition to the first and second polypeptides.

[0047] A "heterodimer," "heterodimeric protein," "heterodimeric complex," or "heteromultimeric polypeptide" is a molecule comprising a first polypeptide and a second polypeptide, where the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.

[0048] "Hinge region," "hinge sequence," and variations thereof, as used herein, include their meanings known in the art, as exemplified in, e.g., Janeway et al., ImmunoBiology: the immune system in health and disease (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods, (1997), 209:193-202.

[0049] "Immunoglobulin-like hinge region," "immunoglobulin-like hinge sequence," and variations thereof, as used herein refer to the hinge region and hinge sequence of an immunoglobulin-like or antibody-like molecule (e.g., an immunoadhesin). In some embodiments, the immunoglobulin-like hinge region may be or may be derived from any IgG1, IgG2, IgG3, or IgG4 subtype, including chimeric forms thereof, or may be from IgA, IgE, IgD, or IgM, for example, a chimeric IgG1 / 2 hinge region.

[0050] The term "immune effector cell" or "effector cell" as used herein refers to a cell within the natural repertoire of cells in the human immune system that can be activated to affect the viability of a target cell. Target cell viability can include cell survival, proliferation, and / or the ability to interact with other cells.

[0051] The antibodies of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques, or other techniques readily apparent in the art (see, e.g., Jayasena, SD, Clin. Chem., 45:1628-50, 1999, and Fellouse, FA et al., J. MoI. Biol., 373(4):924-40, 2007).

[0052] As known in the art, "polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembling the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a label component. Other types of modifications include, for example, "caps", substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any of the hydroxyl groups normally present in the sugar can be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogues of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogues, alpha- or beta-anomeric sugars, epimeric sugars, such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogues, and abasic nucleoside analogues, such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, phosphate, P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR. 2 ("amidate"), P(O)R, P(O)OR', CO, or CH 2 ("formacetal"), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl, which may contain ether (-O-) linkages. Not all linkages in a polynucleotide need be identical. The above statement applies to all polynucleotides referred to herein, including RNA and DNA.

[0053] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0054] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0055] "Host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide of the invention.

[0056] As known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The numbering of residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.

[0057] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A suitable FcR is a native sequence human FcR. Additionally, a suitable FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0058] The term "compete" as used herein with respect to antibodies means that a first antibody or antigen-binding fragment (or portion) thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody, compared to the binding of the first antibody in the absence of the second antibody. The remaining options, in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, may, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0059] A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays known in the art for evaluating such antibody effector functions.

[0060] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[0061] The term "effector function" refers to the biological activity attributed to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement-dependent cytotoxicity (CDC), phagocytosis, C1q binding, and down-regulation of cell surface receptors (e.g., B cell receptor; BCR). See, for example, U.S. Patent No. 6,737,056. Such effector functions generally require that the Fc region is combined with a binding domain (e.g., an antibody variable domain), and can be assessed using various assays known in the art for evaluating such antibody effector functions. An exemplary measurement of effector function is by Fcγ3 and / or C1q binding.

[0062] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[0063] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., antibody) complexed with a cognate antigen. To assess complement activation, for example, the CDC assay described in Gazzano-Santoro et al., J.Immunol.Methods, 202:163 (1996) can be carried out.

[0064] As used herein, "treatment" is an approach for obtaining a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in the proliferation of neoplastic or cancerous cells (or destruction of these cells), inhibition of metastasis of neoplastic cells, remission of the BCMA-associated disease (e.g., cancer or autoimmune disease), reduction of symptoms resulting from the BCMA-associated disease (e.g., cancer or autoimmune disease), increasing the quality of life of a person suffering from the BCMA-associated disease (e.g., cancer or autoimmune disease), reduction in the dosage of other medications required to treat the BCMA-associated disease (e.g., cancer or autoimmune disease), slowing the progression of the BCMA-associated disease (e.g., cancer or autoimmune disease), curing the BCMA-associated disease (e.g., cancer or autoimmune disease), and / or increasing the survival of patients with the BCMA-associated disease (e.g., cancer or autoimmune disease).

[0065] "Ameliorating" refers to the lessening or improvement of one or more symptoms compared to not administering the BCMA antibody or BCMA antibody conjugate. "Ameliorating" also includes a shortening or reduction in the duration of the symptoms.

[0066] As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, disease, including the disease, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes that present during disease development. For therapeutic use, beneficial or desired results include clinical results, such as reducing the incidence or ameliorating one or more symptoms of various BCMA-related diseases or conditions (such as multiple myeloma), reducing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of BCMA-related disease in patients. An effective dosage can be administered in one or more administrations. For purposes of the present invention, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0067] An "individual" or "subject" is a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0068] As used herein, "vector" refers to a construct that can deliver one or more genes or sequences of interest into a host cell and preferably express them in the host cell.Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0069] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0070] As used herein, "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any material that, when combined with active ingredient, allows the ingredient to retain biological activity and is non-reactive with the immune system of a subject. Examples include, but are not limited to, standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and any of various types of wetting agents. Suitable diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions that include such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A.Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st edition, Mack Publishing, 2005).

[0071] The term "acyl donor glutamine-containing tag" or "glutamine tag" as used herein refers to a polypeptide or protein that contains one or more Gln residues that act as transglutaminase amine acceptors. See, e.g., WO2012059882 and WO2015015448.

[0072] The term "k" on " or "k a " refers herein to the rate constant for the association of an antibody to an antigen. Specifically, the rate constant (k on / k a and k off / k d ) and the equilibrium dissociation constant are measured using whole antibody (i.e., bivalent) and monomeric BCMA protein.

[0073] The term "k" off " or "k d " as used herein refers to the rate constant for dissociation of an antibody from the antibody / antigen complex.

[0074] The term “K D " as used herein refers to the equilibrium dissociation constant of an antibody-antigen interaction.

[0075] Reference herein to a value or parameter with "about" includes (and describes) embodiments directed to that value or parameter itself. For example, a statement referring to "about X" includes the statement of "X." Numeric ranges are inclusive of the numbers defining the range.

[0076] Whenever an embodiment is described herein using the phrase "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0077] Where aspects or embodiments of the invention are described in terms of a Markush group or other classification of options, the invention encompasses not only the entire recited group as a whole, as well as each member of the group individually and all possible subgroups of the main group, but also the main group lacking one or more of the group members. The invention also envisions the explicit exclusion of any one or more of the group members in the claimed invention.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0079] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0080] BCMA ANTIBODIES AND METHODS FOR MAKING SAME The present invention provides antibodies that bind to BCMA (e.g., human BCMA (e.g., SEQ ID NO: 353 or Accession No: Q02223-2)) and are characterized by any one or more of the following properties: (a) treat, prevent, or ameliorate one or more symptoms of a condition associated with malignant cells expressing BCMA (e.g., a B-cell related cancer such as multiple myeloma) in a subject; (b) inhibit tumor growth or progression in a subject (having a malignant tumor that expresses BCMA); (c) inhibit metastasis of cancer (malignant) cells expressing BCMA in a subject (having one or more malignant cells that express BCMA); (f) induce regression (e.g., long-term regression) of tumors expressing BCMA; (d) exert cytotoxic activity on malignant cells expressing BCMA; and (e) block BCMA interaction with other yet to be identified factors.

[0081] In one aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to B cell maturation antigen (BCMA) is provided, the antibody or antigen-binding fragment thereof comprising: (a) (i) the sequence SYX 1 MX 2 (In the formula, X 1 is A or P, and X 2 is T, N, or S) (SEQ ID NO: 301), 4GFTFX 1 SY (wherein X 1 is G or S) (SEQ ID NO: 302), or GFTFX 1 SYX 2 MX 3 (In the formula, X 1 is G or S, and X 2 is A or P, and X 3 (ii) a heavy chain variable (VH) complementarity determining region 1 (CDR1) comprising the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (in the formula, X 1is I, V, T, H, L, A, or C, and X 2 is S, D, G, T, I, L, F, M, or V, and X 3 is G, Y, L, H, D, A, S, or M, and X 4 is S, Q, T, A, F, or W, and X 5 is G or T, and X 6 is N, S, P, Y, W, or F, and X 7 is S, T, I, L, T, A, R, V, K, G, or C, and X 8 is F, Y, P, W, H, or G, and X 9 is V, R, or L, and X 10 is G or T) (SEQ ID NO: 305), or X 1 X 2 X 3 X 4 X 5 X 6 (In the formula, X 1 is S, V, I, D, G, T, L, F, or M, and X 2 is G, Y, L, H, D, A, S, or M, and X 3 is S, G, F, or W, and X 4 is G or S, and X 5 is G or T, and X 6 is N, S, P, Y, or W) (SEQ ID NO: 306), and (iii) a VH CDR2 comprising the sequence VSPIX 1 X 2 X 3 (In the formula, X 1 is either A or Y, and X 2 is A or S, and X 3 is G, Q, L, P, or E) (SEQ ID NO: 307), or YWPMX 1 X 2 (In the ceremony 、 X 1 is D, S, T, or A, and X 2 is I, S, L, P, or D) (SEQ ID NO: 308), and / or (i) a VH region comprising a VH CDR3 comprising sequence X 1 X 2 X3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 (In the formula, X 1 is R, G, W, A, or C, and X 2 is A, P, G, L, C, or S, and X 3 is S, G, or R, and X 4 is Q, C, E, V, or I, and X 5 is S, P, G, A, R, or D, and X 6 is V, G, I, or L, and X 7 is S, E, D, P, or G, and X 8 is S, P, F, A, M, E, V, N, D, or Y, and X 9 is I, T, V, E, S, A, M, Q, Y, H, R, or F, and X 10 is Y or F, and X 11 is L, W, or P, and X 12 (ii) a light chain variable (VL) CDR1 comprising the sequence X 1 ASX 2 RAX 3 (In the formula, X 1 is G or D, and X 2 is S or I, and X 3 is T or P) (SEQ ID NO: 310), and (iii) a VL CDR2 comprising the sequence QQYX 1 X 2 X 3 PX 4 T (where X 1 is G, Q, E, L, F, A, S, M, K, R, or Y, and X 2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C, and X 3 is W, F, or S, and X 4 is L or I) (SEQ ID NO: 311), or QQYX 1 X 2 X3 PX 4 (In the formula, X 1 is G, Q, E, L, F, A, S, M, R, K, or Y, and X 2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y, and X 3 is W, S, or F, and X 4 is L or I) (SEQ ID NO: 312).

[0082] In another aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds BCMA, wherein the antibody comprises a VH CDR1, a VH CDR2 and a VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 112, 125, 127, 313, 314, 363, or 365. and / or SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 80, 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, 316, or 364.

[0083] In some embodiments, an antibody having any one of the partial light chain sequences listed in Table 1 and / or any one of the partial heavy chain sequences listed in Table 1 is provided.

[0084] [Table 1-1]

[0085] [Table 1-2]

[0086] [Table 1-3]

[0087] [Table 1-4]

[0088] [Table 1-5]

[0089] [Table 1-6]

[0090] [Table 1-7]

[0091] [Table 1-8]

[0092] [Table 1-9]

[0093]

Table 1-10

[0094]

Table 1-11

[0095]

Table 1-12

[0096]

Table 1-13

[0097]

Table 1-14

[0098]

Table 1-15

[0099]

Table 1-16

[0100]

Table 1-17

[0101]

Table 1-18

[0102]

Table 1-19

[0103]

Table 1-20

[0104]

Table 1-21

[0105]

Table 1-22

[0106]

Table 1-23

[0107]

Table 1-24

[0108]

Table 1-25

[0109]

Table 1-26

[0110]

Table 1-27

[0111]

Table 1-28

[0112]

Table 1-29

[0113]

Table 1-30

[0114] [Table 1-31]

[0115] [Table 1-32]

[0116] [Table 1-33]

[0117] In Table 1, the underlined sequences are the CDR sequences according to Kabat and the bold are the CDR sequences according to Chothia, except for the heavy chain CDR2 sequences below where the Chothia CDR sequences are underlined and the Kabat CDR sequences are in bold. P5A2_VHVL、A02_Rd4_0.6nM_C06、A02_Rd4_0.6nM_C09、A02_Rd4_6nM_C16、A02_Rd4_6nM_C03、A02_Rd4_6nM_C01、A02_Rd4_6nM_C26、A02_Rd4_6nM_C 25、A02_Rd4_6nM_C22、A02_Rd4_6nM_C19、A02_Rd4_0.6nM_C03、A02_Rd4_6 nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6nM_C18、A02_Rd4_6nM_C10、A02_R d4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C2 6、A02_Rd4_0.6nM_C13、A02_Rd4_0.6nM_C01、A02_Rd4_6nM_C08、P5C1_VHV L、C01_Rd4_6nM_C24、C01_Rd4_6nM_C26、C01_Rd4_6nM_C10、C01_Rd4_0.6n M_C27、C01_Rd4_6nM_C20、C01_Rd4_6nM_C12、C01_Rd4_0.6nM_C16、C01_Rd 4_0.6nM_C09、C01_Rd4_6nM_C09、C01_Rd4_0.6nM_C03、C01_Rd4_0.6nM_C 06、C01_Rd4_6nM_C04、COMBO_Rd4_0.6nM_C22、COMBO_Rd4_6nM_C21、COMBO _Rd4_6nM_C10、COMBO_Rd4_0.6nM_C04、COMBO_Rd4_6nM_C25、COMBO_Rd4_0.6nM_C21、COMBO_Rd4_6nM_C11、COMBO_Rd4_0.6nM_C20、COMBO_Rd4_6nM_C 09、COMBO_Rd4_6nM_C08、COMBO_Rd4_0.6nM_C19、COMBO_Rd4_0.6nM_C02、 COMBO_Rd4_0.6nM_C23、COMBO_Rd4_0.6nM_C29、COMBO_Rd4_0.6nM_C09、CO MBO_Rd4_6nM_C12、COMBO_Rd4_0.6nM_C30、COMBO_Rd4_0.6nM_C14、COMBO_ Rd4_6nM_C07、COMBO_Rd4_6nM_C02、COMBO_Rd4_0.6nM_C05、COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, andCOMBO_Rd4_0.6nM_C11.

[0118] The present invention also provides CDR portions of antibodies against BCMA, including Chothia, Kabat CDRs, and CDR contact regions. Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments having more than one CDR, the CDRs may be either Kabat, Chothia, combined CDRs, or combinations thereof. Table 2 provides examples of CDR sequences provided herein.

[0119] [Table 2-1]

[0120] [Table 2-2]

[0121] [Table 2-3]

[0122] [Table 2-4]

[0123] [Table 2-5]

[0124] [Table 2-6]

[0125]

Table 2-7

[0126]

Table 2-8

[0127]

Table 2-9

[0128]

Table 2-10

[0129]

Table 2-11

[0130]

Table 2-12

[0131]

Table 2-13

[0132]

Table 2-14

[0133]

Table 2-15

[0134]

Table 2-16

[0135]

Table 2-17

[0136]

Table 2-18

[0137]

Table 2-19

[0138]

Table 2-20

[0139]

Table 2-21

[0140]

Table 2-22

[0141]

Table 2-23

[0142]

Table 2-24

[0143]

Table 2-25

[0144]

Table 2-26

[0145]

Table 2-27

[0146]

Table 2-28

[0147]

Table 2-29

[0148]

Table 2-30

[0149]

Table 2-31

[0150]

Table 2-32

[0151]

Table 2-33

[0152]

Table 2-34

[0153]

Table 2-35

[0154]

Table 2-36

[0155]

Table 2-37

[0156]

Table 2-38

[0157] In some embodiments, the present invention binds to BCMA and P6E01 / P6E01, P6E01 / H3.AQ, L1.LGF / L3.KW / P6E01; L1.LGF / L3.NY / P6E01, L1.GDF / L3.NY / P6E01, L1.LGF / L3.KW / H3.AL, L1.LGF / L3.KW / H3.AP, L1.LGF / L3.KW / H3.AQ, L1.LGF / L3.PY / H3.AP, L1.LGF / L3.PY / H3.AQ, L1.LGF / L3.NY / H3.AL, L1.LGF / L3.NY / H3.AP, L1.LGF / L3.NY / H3.AQ, L1.GDF / L3.KW / H3.AL, L1.GDF / L3.KW / H3.AP, L1.GDF / L3.KW / H3.AQ, L1.GDF / L3.PY / H3.AQ, L1.GDF / L3.NY / H3.AL, L1.GDF / L3.NY / H3.AP, L1.GDF / L3.NY / H3.AQ, L3.KW / P6E01, L3.PY / P6E01, L3.NY / P6E01, L3.PY / L1.PS / P6E01、L3.PY / L1.AH / P6E01、L3.PY / L1.FF / P6E01、L3.PY / L1.PH / P6E01、L3.PY / L3.KY / P6E01、L3.PY / L3.KF / P6E01、L3.PY / H2.QR、L3.PY / H2.DY、L3.PY / H2.YQ、L3.PY / H2.LT、L3.PY / H2.HA、L3.PY / H2.QL、L3.PY / H3.YA、L3.PY / H3.AE、L3.PY / H3.AQ、L3.PY / H3.TAQ、L3.PY / P6E01、L3.PY / L1.PS / H2.QR、L3.PY / L1.PS / H2.DY、L3.PY / L1.PS / H2.YQ、L3.PY / L1.PS / H2.LT、L3.PY / L1.PS / H2.HA、L3.PY / L1.PS / H2.QL、L3.PY / L1.PS / H3.YA、L3.PY / L1.PS / H3.AE、L3.PY / L1.PS / H3.AQ、L3.PY / L1.PS / H3.TAQ、L3.PY / L1.AH / H2.QR、L3.PY / L1.AH / H2.DY、L3.PY / L1.AH / H2.YQ、L3.PY / L1.AH / H2.LT、L3.PY / L1.AH / H2.HA、L3.PY / L1.AH / H2.QL、L3.PY / L1.AH / H3.YA、L3.PY / L1.AH / H3.AE、L3.PY / L1.AH / H3.AQ、L3.PY / L1.AH / H3.TAQ、L3.PY / L1.FF / H2.QR、L3.PY / L1.FF / H2.DY、L3.PY / L1.FF / H2.YQ、L3.PY / L1.FF / H2.LT、L3.PY / L1.FF / H2.HA、L3.PY / L1.FF / H2.QL、L3.PY / L1.FF / H3.YA、L3.PY / L1.FF / H3.AE、L3.PY / L1.FF / H3.AQ、L3.PY / L1.FF / H3.TAQ、L3.PY / L1.PH / H2.QR、L3.PY / L1.PH / H2.HA、L3.PY / L1.PH / H3.AE、L3.PY / L1.PH / H3.AQ、L3.PY / L1.PH / H3.TAQ、L3.PY / L3.KY / H2.QR、L3.PY / L3.KY / H2.DY、L3.PY / L3.KY / H2.YQ L3.PY / L3.KY / H2.LT、L3.PY / L3.KY / H2.HA、L3.PY / L3.KY / H2.QL、L3.PY / L3.KY / H3.YA L3.PY / L3.KY / H3.TAQ、L3.PY / L3.KF / H2.DY、L3.PY / L3.KF / H2.YQ、L3.PY / L3.KF / H2.LT L3.PY / L3.KF / H2.QL、L3.PY / L3.KF / H3.YA、L3.PY / L3.KF / H3.AE、L3.PY / L3.KF / H3.AQ L3.PY / L3.KF / H3.TAQ、P5A2_VHVL、A02_Rd4_0.6nM_C06、A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16、A02_Rd4_6nM_C03、A02_Rd4_6nM_C01、A02_Rd4_6nM_C26 A02_Rd4_6nM_C25、A02_Rd4_6nM_C22、A02_Rd4_6nM_C19、A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6nM_C18、A02_Rd4_6nM_C10 A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C26 A02_Rd4_0.6nM_C13、A02_Rd4_0.6nM_C01、A02_Rd4_6nM_C08、P5C1_VHVL、C01_Rd4_6nM_C24、C01_Rd4_6nM_C26、C01_Rd4_6nM_C10、C01_Rd4_0.6nM_C27 C01_Rd4_6nM_C20、C01_Rd4_6nM_C12、C01_Rd4_0.6nM_C16、C01_Rd4_0.6nM_C09 C01_Rd4_6nM_C09、C01_Rd4_0.6nM_C03、C01_Rd4_0.6nM_C06、C01_Rd4_6nM_C04 COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_6nM_C1 1, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0. COMBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09, COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14, COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05, COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, COMBO_Rd4_0.6nM_C11, COMBO_Rd4_0.6nM_C29, P4G4, or P1A11 are provided.

[0158] In some embodiments, the invention provides an antibody or antigen-binding fragment that specifically binds to BCMA, wherein the antibody or antigen-binding fragment comprises a VH region comprising the sequence set forth in SEQ ID NO:112 and / or comprises a VL region comprising the sequence set forth in SEQ ID NO:38. In some embodiments, the antibody comprises a light chain comprising the sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 357), and a light chain comprising the sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 358).

[0159] In some embodiments, the invention provides an antibody or antigen-binding fragment that specifically binds to BCMA, wherein the antibody or antigen-binding fragment comprises a VH region comprising the sequence set forth in SEQ ID NO: 2, 32, 42, or 78, and / or a VL region comprising the sequence set forth in SEQ ID NO: 6, 16, 43, or 85.

[0160] In some embodiments, the present invention also provides CDR portions of antibodies to BCMA antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that imbue the antibody with specificity for an antigen. Generally, CDR contact regions include residue positions within the CDRs and Vernier zones that are constrained to maintain the proper loop structure of the antibody to bind a specific antigen. See, e.g., Makabe et al., J.Biol.Chem., 283:1156-1166, 2007. Determining CDR contact regions is well within the skill of the art.

[0161] The binding affinity (K of the BCMA antibodies described herein to BCMA (such as human BCMA (e.g., (SEQ ID NO: 353)) DIn some embodiments, the binding affinity may be about 6500 nM, 6000 nM, 5986 nM, 5567 nM, 5500 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2134 nM, 2000 nM, 1500 nM, 1000 nM, 750 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 193 nM, 100 nM, 90nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 10nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM, or 0.002nM. In some embodiments, the binding affinity is less than any of 6500nM, 6000nM, 5500nM, 5000nM, 4000nM, 3000nM, 2000nM, 1000nM, 900nM, 800nM, 250nM, 200nM, 100nM, 50nM, 30nM, 20nM, 10nM, 7.5nM, 7nM, 6.5nM, 6nM, 5nM, 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, 1nM, or 0.5nM.

[0162] In some embodiments, the invention encompasses compositions, including pharmaceutical compositions, comprising antibodies described herein or made using the methods and having the properties described herein. As used herein, compositions comprise one or more antibodies that bind to BCMA and / or one or more polynucleotides that comprise sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients, such as pharma- ceutically acceptable excipients, including buffers, as are well known in the art.

[0163] The present invention also provides a method for making any of these antibodies. The antibodies of the present invention can be made by procedures known in the art. The polypeptides can be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Antibody polypeptides, particularly shorter polypeptides up to about 50 amino acids, are conveniently produced by chemical synthesis. Methods of chemical synthesis are known in the art and commercially available. For example, antibodies can be produced by automated polypeptide synthesizers using solid phase methods. See also U.S. Patent Nos. 5,807,715; 4,816,567; and 6,331,415.

[0164] The present invention also encompasses fusion proteins comprising one or more fragments or regions from the antibodies of the present invention. In one embodiment, SEQ ID NOs: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 78, 79, 80, 81, 9, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 80, 315, 36, or 364 Fusion polypeptides are provided that comprise at least 10 contiguous amino acids of the indicated variable light chain region and / or at least 10 amino acids of the variable heavy chain region shown in SEQ ID NO:2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 112, 125, 127, 313, 314, 363, or 365. In other embodiments, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable light chain region and / or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide is selected from SEQ ID NOs: 1 and 2, 1 and 3, 4 and 2, 5 and 2, 6 and 2, 4 and 7, 4 and 8, 4 and 3, 9 and 8, 9 and 3, 10 and 7, 10 and 8, 10 and 3, 11 and 7, 11 and 8, 11 and 3, 12 and 3, 13 and 7, 13 and 8, 14 and 3, 15 and 2, 16 and 2, 17 and 2, 18 and 2, 19 and 2, 20 and 2, 21 and 2, 22 and 2, 23 and 2, 16 and 24, 16 and 25, 16 and 26, 16 and 27,16 and 28, 16 and 29, 16 and 30, 16 and 31, 16 and 3, 16 and 32, 16 and 2, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 18 and 29, 18 and 30, 18 and 31, 18 and 3, 18 and 32, 19 and 24, 19 and 25, 19 and 26, 19 and 27, 19 and 28, 19 and 29, 19 and 30, 19 and 31, 19 and 3, 19 and 32, 20 and 24, 20 and 25, 20 and 26, 20 and and 27, 20 and 28, 20 and 29, 20 and 30, 20 and 31, 20 and 3, 20 and 32, 21 and 24, 21 and 28, 21 and 31, 21 and 3, 21 and 32, 22 and 24, 22 and 25, 22 and 26, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 32, 23 and 25, 23 and 26, 23 and 27, 23 and 29, 23 and 30, 23 and 31, 23 and 3, 23 and 32, 34 and 33, 36 and 35, 38 and 37, 40 and 39, 41 and 33, 43 and 42, 45 and 44, 47 and 46, 49 and 48, 51 and 50, 53 and 52, 55 and 54, 57 and 56, 59 and 58, 61 and 60, 63 and 62, 65 and 64, 67 and 66, 69 and 68, 71 and 70, 73 and 72, 75 and 74, 77 and 76, 79 and 78, 317 and 78, 79 and 78, 81 and 78, 82 and 78, 84 and 83, 85 and 78, 86 and 78, 88 and 87, 89 and 78 , 90 and 78, 91 and 78, 93 and 92, 94 and 78, 96 and 95, 98 and 97, 38 and 78, 102 and 101, 103 and 78, 105 and 104, 107 and 106, 108 and 78, 109 and 78, 111 and 110, 38 and 112, 113 and 112, 115 and 114, 116 and 76, 117 and 112, 119 and 118, 121 and 120, 123 and 122, 124 and 112, 126 and 125, 128 and 127, 80 and 363,or 364 and 365. In another embodiment, the fusion polypeptide comprises one or more CDRs. In yet another embodiment, the fusion polypeptide comprises CDR H3 (VH CDR3) and / or CDR L3 (VL CDR3). For purposes of the present invention, a fusion protein contains one or more antibodies and another amino acid sequence that is not attached to the native molecule, e.g., a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags", e.g., FLAG tag or 6His tag. Tags are well known in the art.

[0165] The invention also provides isolated polypeptides encoding the antibodies of the invention, as well as vectors and host cells comprising the polynucleotides.

[0166] In one embodiment, the polynucleotide is the antibody P6E01 / P6E01, P6E01 / H3.AQ, L1.LGF / L3.KW / P6E01;L1.LGF / L3.NY / P6E01, L1.GDF / L3.NY / P6E01, L1.L GF / L3.KW / H3.AL, L1.LGF / L3.KW / H3.AP, L1.LGF / L3.KW / H3.AQ, L1.LGF / L3.PY / H3.AP, L1.LGF / L3.PY / H3.AQ, L1.LGF / L3.NY / H3 .AL, L1.LGF / L3.NY / H3.AP, L1.LGF / L3.NY / H3.AQ, L1.GDF / L3.KW / H3.AL, L1.GDF / L3.KW / H3.AP, L1.GDF / L3.KW / H3.AQ, L1.GDF / L3.PY / H3.AQ, L1.GDF / L3.NY / H3.AL, L1.GDF / L3.NY / H3.AP, L1.GDF / L3.NY / H3.AQ, L3.KW / P6E01, L3.PY / P6E01, L3.NY / P6E01, L3.PY / L1.PS / P6E01、L3.PY / L1.AH / P6E01、L3.PY / L1.FF / P6E01、L3.PY / L1.PH / P6E01、L3.PY / L3.KY / P6E01、L3.PY / L3.KF / P6E01、L3.PY / H2.QR、L3.PY / H2.DY、L3.PY / H2.YQ、L3.PY / H2.LT、L3.PY / H2.HA、L3.PY / H2.QL、L3.PY / H3.YA、L3.PY / H3.AE、L3.PY / H3.AQ、L3.PY / H3.TAQ、L3.PY / P6E01、L3.PY / L1.PS / H2.QR、L3.PY / L1.PS / H2.DY、L3.PY / L1.PS / H2.YQ、L3.PY / L1.PS / H2.LT、L3.PY / L1.PS / H2.HA、L3.PY / L1.PS / H2.QL、L3.PY / L1.PS / H3.YA、L3.PY / L1.PS / H3.AE、L3.PY / L1.PS / H3.AQ、L3.PY / L1.PS / H3.TAQ、L3.PY / L1.AH / H2.QR、L3.PY / L1.AH / H2.DY、L3.PY / L1.AH / H2.YQ、L3.PY / L1.AH / H2.LT、L3.PY / L1.AH / H2.HA、L3.PY / L1.AH / H2.QL、L3.PY / L1.AH / H3.YA、L3.PY / L1.AH / H3.AE、L3.PY / L1.AH / H3.AQ、L3.PY / L1.AH / H3.TAQ、L3.PY / L1.FF / H2.QR、L3.PY / L1.FF / H2.DY、L3.PY / L1.FF / H2.YQ、L3.PY / L1.FF / H2.LT、L3.PY / L1.FF / H2.HA、L3.PY / L1.FF / H2.QL、L3.PY / L1.FF / H3.YA、L3.PY / L1.FF / H3.AE、L3.PY / L1.FF / H3.AQ、L3.PY / L1.FF / H3.TAQ、L3.PY / L1.PH / H2.QR、L3.PY / L1.PH / H2.HA、L3.PY / L1.PH / H3.AE、L3.PY / L1.PH / H3.AQ、L3.PY / L1.PH / H3.TAQ、L3.PY / L3.KY / H2.QR、L3.PY / L3.KY / H2.DY、L3.PY / L3.KY / H2.YQ L3.PY / L3.KY / H2.LT、L3.PY / L3.KY / H2.HA、L3.PY / L3.KY / H2.QL、L3.PY / L3.KY / H3.YA L3.PY / L3.KY / H3.TAQ、L3.PY / L3.KF / H2.DY、L3.PY / L3.KF / H2.YQ、L3.PY / L3.KF / H2.LT L3.PY / L3.KF / H2.QL、L3.PY / L3.KF / H3.YA、L3.PY / L3.KF / H3.AE、L3.PY / L3.KF / H3.AQ L3.PY / L3.KF / H3.TAQ、P5A2_VHVL、A02_Rd4_0.6nM_C06、A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16、A02_Rd4_6nM_C03、A02_Rd4_6nM_C01、A02_Rd4_6nM_C26 A02_Rd4_6nM_C25、A02_Rd4_6nM_C22、A02_Rd4_6nM_C19、A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6nM_C18、A02_Rd4_6nM_C10 A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C26 A02_Rd4_0.6nM_C13、A02_Rd4_0.6nM_C01、A02_Rd4_6nM_C08、P5C1_VHVL、C01_Rd4_6nM_C24、C01_Rd4_6nM_C26、C01_Rd4_6nM_C10、C01_Rd4_0.6nM_C27 C01_Rd4_6nM_C20、C01_Rd4_6nM_C12、C01_Rd4_0.6nM_C16、C01_Rd4_0.6nM_C09 C01_Rd4_6nM_C09、C01_Rd4_0.6nM_C03、C01_Rd4_0.6nM_C06、C01_Rd4_6nM_C04 COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_6nM_C1 1, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0. The antibody of interest may be maintained in a vector within the host cell, which may then be propagated and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0167] The present invention also encompasses the scFv of the antibody of the present invention. Single chain variable region fragments are generated by linking the light and / or heavy chain variable regions using a short linking peptide (Bird et al., Science, 242:423-426, 1988). An example of a linking peptide is (GGGGS) 3(SEQ ID NO: 498), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have also been designed and used (Bird et al., 1988, supra). The linker should be a short flexible polypeptide, preferably consisting of less than about 20 amino acid residues. The linker can then be modified for additional functions, such as attachment of drugs or attachment to solid supports. Single-chain variants can be produced recombinantly or synthetically. For synthetic production of scFvs, automated synthesizers can be used. For recombinant production of scFvs, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, eukaryotic, such as yeast, plant, insect, or mammalian cells, or prokaryotic, such as E. coli. A polynucleotide encoding the scFv of interest can be generated by routine manipulation, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0168] Other forms of single chain antibodies are also included, such as diabodies or minibodies. Diabodies are bivalent, bispecific antibodies expressed with a linker in which the heavy chain variable (VH) and light chain variable (VL) domains are on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen binding sites (see, for example, Holliger, P. et al., Proc. Natl. Acad Sci. USA, 90:6444-6448, 1993; Poljak, RJ et al., Structure, 2:1121-1123, 1994). Minibodies contain the VL and VH domains of a natural antibody fused to the hinge region and CH3 domain of an immunoglobulin molecule. See, for example, US 5,837,821.

[0169] In another aspect, the invention provides compositions (such as pharmaceutical compositions) comprising any of the polynucleotides of the invention. In some embodiments, the compositions comprise an expression vector comprising a polynucleotide encoding any of the antibodies described herein. In yet other embodiments, the compositions comprise either or both of the polynucleotides set forth in SEQ ID NO:486 and SEQ ID NO:485 below: COMBO_Rd4_0.6nM_C29 heavy chain variable region GAAGTCCAACTCCTCGAATCCGGTGGCGGCCTTGTCCAGCCTGGAGGTTCCTTGCGCCTGTCATGTGCCGCCAGCGGATTCACCTTCTCGTCCTACCCGATGTCGTGGGTCCGCCAGGCTCCGGGAAAGGGCCTGGAATGGGTGTCAGCCATCGGAGGATCGGGGGCTCCCTGCCC TACGCCGATATCGTGAAGGGAAGGTTCACCATTAGCCGGGACAACTCCAAGAACACTCTGTACCTCCAAATGAACAGCCTGAGAGCGGAGGACACCGCAGTGTACTATTGCGCCGGTACTGGCCAATGGACATCTGGGGCCAGGGGACTCTGGTCACCGTCTCCTCA (SEQ ID NO: 486) COMBO_Rd4_0.6nM_C29 light chain variable region GAGATCGTGCTGACTCAGTCCCCTGGAACCCTGTCCCTGTCACCTGGCGAAAGAGCTACCTTGTCCTGTCGCGCATCACAATCCGTGTCGTCGAGCTATCTCGCGTGGTACCAGCAGAAGCCCGGACAGGCCCCAAGGCTGCTTATGTACGACGCCTCCATC CGGGCCACTGGTATCCCCGACCGCTTCGGGCTCCGGAAGCGGCACCGACTTCACCCTGACTATTTCCCGGCTCGAACCGGAGGATTTCGCCGTGTACTACTGCCAACAGTACCAGAGCTGGCCGCTGACGTTTGGGCAGGGGACCAAGGTCGAAATCAAA (SEQ ID NO:485)

[0170] In other embodiments, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO:488 and SEQ ID NO:487: L3.PY / H3TAQ heavy chain variable region GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCGGCAGCTACGCTATGACCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAATACCTTCT ACGCCGAGAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACACGGGTGTCCCCTATCGCCGCGCAGATGGATTATTGGGCCAGGGCACTCTGGTCACCGTCTCCTCA (SEQ ID NO:488) L3.PY / H3TAQ heavy chain variable region GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGAGCGTGTCCAGCAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTATGGCGCCTCTTCTA GAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCACTACCCTTATCCCCCAGCTTCACATTTGGCCAGGGCACCAAGGTGGAGATCAAA (SEQ ID NO:487)

[0171] In yet other embodiments, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO:490 and SEQ ID NO:489: A02_Rd4_0.6nM_C01 Heavy chain variable region GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTACTGCGTCTGGTGGTTCTA CTTACTATGCCGATGTGGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGTCGTTGTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA (SEQ ID NO:490) A02_Rd4_0.6nM_C01 Light chain variable region GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCGGCGTATTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATGAGCGTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG (SEQ ID NO:489)

[0172] In other embodiments, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO:492 and SEQ ID NO:491 below: A02_Rd4_0.6nM_C16 heavy chain variable region GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTTTGGTGGTTCTA CTTACTATGCCGATATCGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA (SEQ ID NO:492) A02_Rd4_0.6nM_C16 light chain variable region GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGATCTGTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATCAGACTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG (SEQ ID NO:491)

[0173] Expression vectors and administration of polynucleotide compositions are further described herein.

[0174] In another aspect, the invention provides a method of making any of the polynucleotides described herein.

[0175] Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond in a one-to-one manner to DNA molecules, and mRNA, which does not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present invention, and polynucleotides may, but need not, be linked to other molecules and / or supporting materials.

[0176] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. A polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not diminished compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. A variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity with a polynucleotide sequence encoding a native antibody or portion thereof.

[0177] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0178] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS, 4:11~17; Robinson, ED, 1971, Comb. Theor., 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol., 4:406~425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of In Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA, 80:726-730.

[0179] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, and the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20 percent or less, typically 5-15 percent, or 10-12 percent additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0180] Variants may also, or alternatively, be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0181] Suitable "moderately stringent conditions" include a prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C to 65°C in 5x SSC; followed by two washes for 20 minutes each at 65°C with 2x, 0.5x, and 0.2x SSC containing 0.1% SDS.

[0182] As used herein, "highly stringent conditions" or "high stringency conditions" refers to (1) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50° C.; (2) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at pH 6.5, with denaturing agents such as formamide, e.g., 750 mM sodium chloride, 75 mM sodium citrate, at 42° C., with denaturing agents such as formamide ... or (3) using 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a wash in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0183] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein. Some of these polynucleotides bear minimal homology with the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes that comprise the polynucleotide sequences provided herein are within the scope of the present invention. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and / or database sequence comparison.

[0184] The polynucleotide of the present invention can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail here.Those skilled in the art can use the sequence provided herein and commercially available DNA synthesizers to generate desired DNA sequence.

[0185] To prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence can be inserted into a suitable vector, as further discussed herein, and the vector can then be introduced into a suitable host cell for replication and amplification. A polynucleotide can be inserted into a host cell by any means known in the art. A cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0186] Instead, PCR allows the duplication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0187] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as set forth in Sambrook et al., 1989, supra.

[0188] A suitable cloning vector can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended to be used, but a useful cloning vector will generally have the ability to replicate autonomously, can have a single target for a specific restriction endonuclease, and / or can have a marker gene that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers, such as BioRad, Strategene, and Invitrogen.

[0189] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present invention. It is implied that an expression vector must be replicable in a host cell as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT Publication No. WO87 / 04462. Vector components generally include, but are not limited to, one or more of the following: signal sequence; origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0190] The vector containing the polynucleotide of interest can be introduced into the host cell by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus).The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0191] The present invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cell expresses the cDNA at a level about 5-fold greater, more preferably 10-fold greater, and even more preferably 20-fold greater than that of the corresponding endogenous antibody or protein of interest, if present, in the host cell. Screening the host cells for specific binding to BCMA or a BCMA domain (e.g., domains 1-4) can be done by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

[0192] Representative materials of the present invention were deposited at the American Type Culture Collection (ATCC) on April 15, 2015. The vector having ATCC Accession No. PTA-122094 is a polynucleotide encoding a humanized BCMA antibody heavy chain variable region, and the vector having ATCC Accession No. PTA-122093 is a polynucleotide encoding a humanized BCMA antibody light chain variable region. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty), which ensures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and ATCC which assures perpetual and unrestricted availability of the progeny of the cultures of the deposit to the public after issuance of the pertinent U.S. patent, or after the public disclosure of any U.S. or foreign patent application, whichever occurs earlier, and to persons determined by the Director of the U.S. Patent and Trademark Office to be entitled pursuant to 35 U.S.C. § 122 and applicable Director regulations thereunder (including 37 CFR § 1.14, with specific reference to 886 OG 638).

[0193] The agent for this application has agreed that if a culture of the deposited material, when cultured under appropriate conditions, dies or is lost or destroyed, the material will be promptly replaced upon notice with another identical one. Availability of the deposited material is not to be construed as a license to practice the invention in violation of the rights granted under the authority of any government pursuant to its patent laws.

[0194] BCMA antibody conjugates The invention also provides conjugates (or immunoconjugates) of the BCMA antibodies or antigen-binding fragments thereof described herein, where the antibodies or antigen-binding fragments are conjugated to a targeted immunotherapy agent (e.g., a cytotoxic agent) either directly or indirectly via a linker (e.g., an antibody-drug conjugate). For example, a cytotoxic agent can be linked or conjugated to the BCMA antibodies or antigen-binding fragments thereof described herein for targeted localized delivery of the cytotoxic agent moiety to tumors (e.g., BCMA-expressing tumors).

[0195] Methods for conjugating cytotoxic or other therapeutic agents to antibodies have been described in various publications. For example, chemical modifications can be made in antibodies through lysine side chain amines or through cysteine ​​sulfhydryl groups activated by reducing interchain disulfide bonds to carry out conjugation reactions. See, for example, Tanaka et al., FEBS Letters, 579:2092-2096, 2005, and Gentle et al., Bioconjugate Chem., 15:658-663, 2004. Engineered reactive cysteine ​​residues at specific sites of antibodies for specific drug conjugation with defined stoichiometric ratios have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. Conjugation using acyl donor glutamine-containing tags or endogenous glutamine (i.e., the ability to form covalent bonds as acyl donors) that have been made reactive by engineering the polypeptide in the presence of transglutaminase and amines (e.g., cytotoxic agents that contain or have attached thereto reactive amines) is also described in International Applications WO2012 / 059882 and WO2015015448.

[0196] In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag engineered into a specific site of the antibody (e.g., at the carboxyl terminus, amino terminus, or another site in the BCMA antibody). In some embodiments, the tag comprises the amino acid glutamine (Q) or the amino acid sequence LQG, LLQGG (SEQ ID NO:318), LLQG (SEQ ID NO:454), LSLSQG (SEQ ID NO:455), GGGLLQGG (SEQ ID NO:456), GLLQG (SEQ ID NO:457), LLQ, GSPLAQSHGG (SEQ ID NO:458), GLLQGGG (SEQ ID NO:459), GLLQGG (SEQ ID NO:460), GLLQ (SEQ ID NO:461), LLQLLQGA (SEQ ID NO:462), LLQGA (SEQ ID NO:463), LLQYQGA (SEQ ID NO:464), LLQGSG (SEQ ID NO:465), LLQYQG (SEQ ID NO:466), LLQLLQG (SEQ ID NO:467), LLQLLQG (SEQ ID NO:468), LLQLLQG (SEQ ID NO:469), LLQLLQG (SEQ ID NO:470), LLQLLQG (SEQ ID NO:471), LLQLLQGA (SEQ ID NO:472), LLQGA (SEQ ID NO:473), LLQYQGA (SEQ ID NO:474), LLQGSG (SEQ ID NO:475), LLQYQG (SEQ ID NO:476), LLQLLQG (SEQ ID NO:477), LLQLLQG (SEQ ID NO:478), LLQLLQG (SEQ ID NO:479), LLQLLQ 467), SLLQG (SEQ ID NO: 468), LLQLQ (SEQ ID NO: 469), LLQLLQ (SEQ ID NO: 470), LLQGR (SEQ ID NO: 471), LLQGPP (SEQ ID NO: 472), LLQGPA (SEQ ID NO: 473), GGLLQGPP (SEQ ID NO: 474), GGLLQGA (SEQ ID NO: 475), LLQGPGK (SEQ ID NO: 476), LLQGPG (SEQ ID NO: 477), LLQGP (SEQ ID NO: 478), LLQP (SEQ ID NO: 479), LLQPGK (SEQ ID NO: 480), LLQAPGK (SEQ ID NO: 481), LLQGAPG (SEQ ID NO: 482), LLQGAP (SEQ ID NO: 483), and LLQLQG (SEQ ID NO: 484).

[0197] In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag engineered at a specific site in the antibody, the tag comprising the amino acid sequence GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475) engineered at the carboxyl terminus of the light chain of the BCMA antibody. In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag engineered at a specific site in the antibody, the tag comprising the amino acid sequence LLQG (SEQ ID NO: 454) engineered after residue T135 in the heavy chain of the BCMA antibody. In other embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag engineered at a specific site in the antibody, the tag comprising the amino acid sequence LLQGA (SEQ ID NO: 463) or LLQGPP (SEQ ID NO: 472) engineered at the carboxyl terminus of the heavy chain of the BCMA antibody, the lysine residue at the carboxyl terminus of the heavy chain being deleted. In some embodiments, the BCMA antibody or conjugate described herein comprises an amino acid substitution at position 297 (EU numbering scheme) of the BCMA antibody. For example, the amino acid asparagine (N) may be substituted with glutamine (Q) or alanine (A) at position 297 of the BCMA antibody.

[0198] Also provided is an isolated antibody comprising an acyl donor glutamine-containing tag and an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody, wherein the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. Thus, in some embodiments, an acyl donor glutamine-containing tag (e.g., Q, LQG, LLQGG (SEQ ID NO:318), LLQG (SEQ ID NO:454), LSLSQG (SEQ ID NO:455), GGGLLQGG (SEQ ID NO:456), GLLQG (SEQ ID NO:457), LLQ, GSPLAQSHGG (SEQ ID NO:458), GLLQGGG (SEQ ID NO:459), GLLQGG (SEQ ID NO:460), GLLQ (SEQ ID NO:461), LLQLLQGA (SEQ ID NO:462), LLQGA (SEQ ID NO:463), LLQYQGA (SEQ ID NO:464), LLQGSG (SEQ ID NO:465), LLQYQG (SEQ ID NO:466), LLQLLQG (SEQ ID NO:467) , SLLQG (SEQ ID NO: 468), LLQLQ (SEQ ID NO: 469), LLQLLQ (SEQ ID NO: 470), LLQGR (SEQ ID NO: 471), LLQGPP (SEQ ID NO: 472), LLQGPA (SEQ ID NO: 473), GGLLQGPP (SEQ ID NO: 474), GGLLQGA (SEQ ID NO: 475), LLQGPGK (SEQ ID NO: 476), LLQGPG (SEQ ID NO: 477), LLQGP (SEQ ID NO: 478), LLQP (SEQ ID NO: 479), LLQPGK (SEQ ID NO: 480), LLQAPGK (SEQ ID NO: 481), LLQGAPG (SEQ ID NO: 482), LLQGAP (SEQ ID NO: 483), and LLQLQG (SEQ ID NO: 484)), and an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody is provided. In some embodiments, the amino acid modification is a lysine to arginine substitution (eg, K222R, K340R, or K370R).

[0199] In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence GGLLQGPP (SEQ ID NO: 474) engineered at the C-terminus of the BCMA antibody light chain, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence GGLLQGA (SEQ ID NO: 475) engineered at the C-terminus of the BCMA antibody light chain, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence LLQGA (SEQ ID NO: 463) engineered at the C-terminus of the BCMA antibody heavy chain, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody, wherein the lysine residue at the carboxy terminus of the heavy chain has been deleted. In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence LLQG (SEQ ID NO: 454) engineered after residue T135 in the heavy chain of the BCMA antibody, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody.

[0200] In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising an engineered glutamine at position 297 in the BCMA antibody, or an amino acid substitution of asparagine (N) to another amino acid at position 297, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody. For example, in some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence GGLLQGPP (SEQ ID NO: 474) engineered at the C-terminus of the BCMA antibody light chain, an amino acid substitution of asparagine (N) to glutamine (Q) at position 297 of the BCMA antibody, and an amino acid substitution of lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, the BCMA antibody or conjugate described herein comprises an acyl donor glutamine-containing tag comprising the sequence LLQG (SEQ ID NO: 454) engineered after residue T135 of the heavy chain of the BCMA antibody, an asparagine (N) to alanine (A) amino acid substitution at position 297 of the BCMA antibody, and a lysine to arginine amino acid substitution at position 222 (EU numbering scheme) of the antibody.

[0201] Agents that may be conjugated to the BCMA antibodies or antigen-binding fragments of the invention may include, but are not limited to, a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, or an oligonucleotide.

[0202] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatins, combretastatins, duocarmycins, pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, camptothecins, tubulysins, hemiasterlins, spliceostatins, pladienolides, and stereoisomers, isosteres, analogs, or derivatives thereof.

[0203] Anthracyclines are derived from the bacteria Streptomyces and are used to treat a wide range of cancers, including leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0204] Dolastatins and their peptide analogs and derivatives, the auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity (see, e.g., U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother., 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethylauristatin D or monomethyldolastatin 10), MMAF (monomethylauristatin F or N-methylvaline-valine-dolaisoloiin-dolaproine-phenylalanine), MMAE (monomethylauristatin E or N-methylvaline-valine-dolaisoloiin-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (such as those described in U.S. Publication No. 2013 / 0129753). In some embodiments, the auristatin is 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0205] [ka]

[0206] In some embodiments, the auristatin is 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), which has the following structure:

[0207] [ka]

[0208] In some embodiments, the auristatin is OMe (N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0209] [ka]

[0210] In other embodiments, the auristatin is 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0211] [ka]

[0212] In other embodiments, the auristatin is 0121 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0213] [ka]

[0214] Camptothecin is a cytotoxic quinoline alkaloid that inhibits the enzyme topoisomerase I. Examples of camptothecin and its derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites such as SN-38.

[0215] Combretastatins are natural phenols that have vascular disrupting properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

[0216] Duocarmycins and CC-1065 are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary duocarmycins and CC-1065s include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, (+)-CC-1065, and compounds having the structure:

[0217] [ka] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide, having the structure:

[0218] [ka] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide, structure:

[0219] [ka] Having N~2~-acetyl-L-lysyl-L-valyl-N~5~-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(4-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}pentacyclo[4.2.0.0~2,5~. and 4,7-dihydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide.

[0220] Enediynes are a class of antitumor bacterial products characterized by the presence of 9- and 10-membered rings, or ring systems with conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and derivatives thereof.

[0221] Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and is used as an antitumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0222] Hemiasterlin and its analogs (eg, HTI-286) bind to tubulin, disrupt normal microtubule dynamics, and depolymerize microtubules in stoichiometric amounts.

[0223] Maytansine or their derivatives, maytansinoids, inhibit cell proliferation by inhibiting microtubule formation during mitosis through inhibition of tubulin polymerization. See Remillard et al., Science, 189:1002-1005, 1975. Exemplary maytansine and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives, and the ansamitocins.

[0224] Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers (IGNs) are antitumor agents that contain one or more imine functional groups or their equivalents that bind to double-stranded DNA. PBDs and IGN molecules are based on the natural product anthramycin and interact with DNA in a sequence-selective manner, preferring purine-guanine-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

[0225] Spliceostatins and pladienolides are antitumor compounds that inhibit splicing and interact with the spliceosome, SF3b. ​​Examples of spliceostatins include, but are not limited to, spliceostatin A, FR901464, and

[0226] [ka] and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate, having the structure: Examples of pladienolides include, but are not limited to, pladienolide B, pladienolide D, or E7107.

[0227] Taxanes are diterpenes that act as antitubulin or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

[0228] Tubulysins are natural products isolated from strains of myxobacteria that have been shown to depolymerize microtubules and induce mitotic arrest. Exemplary tubulysins include, but are not limited to, tubulysin A, tubulysin B, and tubulysin D.

[0229] Vinca alkaloids are also antitubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0230] Thus, in some embodiments, the cytotoxic agent is MMAD (monomethyl auristatin D), 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2 S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 0131-OMe (N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 0121 (2-methyl-L-propyl-N-[(3R,4 S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate.

[0231] In some embodiments, the agent is an immunomodulatory agent. Examples of immunomodulatory agents include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and analogs thereof, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factors referred to as "S1 factors," erythropoietin, and thrombopoietin, or combinations thereof.

[0232] In some embodiments, the agent moiety is an imaging agent (eg, a fluorophore or chelator), such as fluorescein, rhodamine, lanthanide phosphors, and derivatives thereof, or a radioisotope bound to a chelator. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelators include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

[0233] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101).

[0234] In some embodiments, therapeutic or diagnostic radioisotopes, or other labels (e.g., PET or SPECT labels) can be incorporated into the agent for conjugation to the BCMA antibodies or antigen-binding fragments described herein. Examples of radioisotopes or other labels include, but are not limited to, 3 H, 11 C. 13 N, 14 C. 15 N, 15 O. 35 S, 18 F, 32 P, 33 P, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, 76 Br, 77 Br, 86 Y, 89 Zr, 90 Y, 94 Tc, 95 Ru, 97 Ru, 99 Tc, 103 Ru, 105 Rh, 105 Ru, 107 Hg, 109 Pd, 111 Ag, 111 In, 113 In, 121 Te, 122 Te, 123 I, 124 I, 125 I, 125 Te, 126 I, 131 I, 131 In, 133 I, 142 Pr, 143 Pr, 153 Pb, 153 Sm, 161 Tb, 165 Tm, 166Dy, 166 H, 167 Tm, 168 Tm, 169 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 197 Pt, 198 Au, 199 Au, 201 Tl, 203 Hg, 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 224 Ac, or 225 There is Ac.

[0235] In some embodiments, the agent is a therapeutic protein, including but not limited to, toxins, hormones, enzymes, and growth factors.

[0236] Examples of toxin proteins (or polypeptides) include, but are not limited to, diphtheria (e.g., diphtheria A chain), Pseudomonas exotoxins and endotoxins, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii proteins, dianthin proteins, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis proteins, and the like. officinalis inhibitors, mitogenin, restrictocin, phenomycin, enomycin, the trichothecenes, inhibitor cystine knot (ICK) peptides (eg, ceratotoxin), and conotoxins (eg, KIIIA or SmIIIa).

[0237] In some embodiments, the agent is a biocompatible polymer. The BCMA antibodies or antigen-binding fragments described herein can be conjugated to a biocompatible polymer to increase serum half-life and biological activity and / or extend in vivo half-life. Examples of biocompatible polymers include water-soluble polymers, such as polyethylene glycol (PEG) or derivatives thereof, and zwitterion-containing biocompatible polymers (e.g., phosphorylcholine-containing polymers).

[0238] In some embodiments, the agent is an oligonucleotide, such as an antisense oligonucleotide.

[0239] In another aspect, the invention provides a conjugate of an antibody or antigen-binding fragment described herein, comprising the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic agent), wherein the acyl donor glutamine-containing tag has been engineered into a specific site on the antibody or antigen-binding fragment (e.g., at the carboxyl terminus of the heavy or light chain, after residue T135 in the antibody heavy chain, or at another site), and the tag is linked to a linker (e.g., one or more reactive amines (e.g., primary amine NH 2 )) which linker is conjugated to a cytotoxic agent (e.g., MMAD or other auristatins, such as, for example, 0101, 0131, or 3377).

[0240] Examples of linkers containing one or more reactive amines include, but are not limited to, Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or aminoPEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzenesulfon ... carboxycarbonyl), amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, or Ac-Lys-putrescine.

[0241] In some embodiments, the conjugate is: 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101; 2) antibody-AcLys-VC-PABC-0101 and comprises N297Q; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprises N297Q; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131 and comprises N297A; 5) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377 and comprises N297A; 6) antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101. In some embodiments, an acyl donor glutamine-containing tag, for example, including GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475), is engineered at the C-terminus of the antibody light chain. In other embodiments, an acyl donor glutamine-containing tag (e.g., LLQGA (SEQ ID NO: 463) or LLQGPP (SEQ ID NO: 472)) is engineered at the C-terminus of the antibody heavy chain, and the C-terminal lysine residue is deleted. In some embodiments, an acyl donor glutamine-containing tag, for example, including LLQG (SEQ ID NO: 454), is engineered after residue T135 in the antibody heavy chain, or replaces amino acid residues E294-N297 in the antibody heavy chain.P6E01 / P6E01, P6E01 / H3.AQ, L1.LGF / L 3.KW / P6E01;L1.LGF / L3.NY / P6E01、L1.GDF / L3.NY / P6E01、L1.L GF / L3.KW / H3.AL、L1.LGF / L3.KW / H3.AP、L1.LGF / L3.KW / H3.AQ、L1.LGF / L3.PY / H3.AP、L1.LGF / L3.PY / H3.AQ、L1.LGF / L3.NY / L3.H3. .AL、L1.LGF / L3.NY / H3.AP、L1.LGF / L3.NY / H3.AQ、L1.GDF / L3.KW / H3.AL、L1.GDF / L3.KW / H3.AP、L1.GDF / L3.KW / H3.AQ. L3.PY / H3.AQ、L1.GDF / L3.NY / H3.AL、L1.GDF / L3.NY / H3.AP、L1.GDF / L3.NY / H3.AQ、L3.KW / P6E01、L3.PY / P6E01、L1.3.01 / NY L3.PY / L1.PS / P6E01、L3.PY / L1.AH / P6E01、L3.PY / L1.FF / P6E01、L3.PY / L1.PH / P6E01、L3.PY / L3.KY / P6E01、L3.PY / L3.KF / P6E01、L3.PY / H2.QR、L3.PY / H2.DY、L3.PY / H2.YQ、L3.PY / H2.LT、L3.PY / H2.HA、L3.PY / H2.QL、L3.PY / H3.YA、L3.PY / H3.AE、L3.PY / H3.AQ、L3.PY / H3.TAQ、L3.PY / P6E01、L3.PY / L1.PS / H2.QR、L3.PY / L1.PS / H2.DY、L3.PY / L1.PS / H2.YQ、L3.PY / L1.PS / H2.LT、L3.PY / L1.PS / H2.HA、L3.PY / L1.PS / H2.QL、L3.PY / L1.PS / H3.YA、L3.PY / L1.PS / H3.AE、L3.PY / L1.PS / H3.AQ、L3.PY / L1.PS / H3.TAQ、L3.PY / L1.AH / H2.QR、L3.PY / L1.AH / H2.DY、L3.PY / L1.AH / H2.YQ、L3.PY / L1.AH / H2.LT、L3.PY / L1.AH / H2.HA、L3.PY / L1.AH / H2.QL、L3.PY / L1.AH / H3.YA、L3.PY / L1.AH / H3.AE、L3.PY / L1.AH / H3.AQ、L3.PY / L1.AH / H3.TAQ、L3.PY / L1.FF / H2.QR、L3.PY / L1.FF / H2.DY、L3.PY / L1.FF / H2.YQ、L3.PY / L1.FF / H2.LT、L3.PY / L1.FF / H2.HA、L3.PY / L1.FF / H2.QL、L3.PY / L1.FF / H3.YA、L3.PY / L1.FF / H3.AE、L3.PY / L1.FF / H3.AQ、L3.PY / L1.FF / H3.TAQ、L3.PY / L1.PH / H2.QR、L3.PY / L1.PH / H2.HA、L3.PY / L1.PH / H3.AE、L3.PY / L1.PH / H3.AQ、L3.PY / L1.PH / H3.TAQ、L3.PY / L3.KY / H2.QR、L3.PY / L3.KY / H2.DY、L3.PY / L3.KY / H2.YQ L3.PY / L3.KY / H2.LT、L3.PY / L3.KY / H2.HA、L3.PY / L3.KY / H2.QL、L3.PY / L3.KY / H3.YA L3.PY / L3.KY / H3.TAQ、L3.PY / L3.KF / H2.DY、L3.PY / L3.KF / H2.YQ、L3.PY / L3.KF / H2.LT L3.PY / L3.KF / H2.QL、L3.PY / L3.KF / H3.YA、L3.PY / L3.KF / H3.AE、L3.PY / L3.KF / H3.AQ L3.PY / L3.KF / H3.TAQ、P5A2_VHVL、A02_Rd4_0.6nM_C06、A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16、A02_Rd4_6nM_C03、A02_Rd4_6nM_C01、A02_Rd4_6nM_C26 A02_Rd4_6nM_C25、A02_Rd4_6nM_C22、A02_Rd4_6nM_C19、A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6nM_C18、A02_Rd4_6nM_C10 A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C26 A02_Rd4_0.6nM_C13、A02_Rd4_0.6nM_C01、A02_Rd4_6nM_C08、P5C1_VHVL、C01_Rd4_6nM_C24、C01_Rd4_6nM_C26、C01_Rd4_6nM_C10、C01_Rd4_0.6nM_C27 C01_Rd4_6nM_C20、C01_Rd4_6nM_C12、C01_Rd4_0.6nM_C16、C01_Rd4_0.6nM_C09 C01_Rd4_6nM_C09、C01_Rd4_0.6nM_C03、C01_Rd4_0.6nM_C06、C01_Rd4_6nM_C04 COMBO_Rd4_0.6nM_C22, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_0.6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_0.6nM_C25, COMBO_Rd4_0.6nM_C2 6nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C0 8. COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23. COMBO_Rd4_0.6nM_C29 COMBO_Rd4_0.6nM_C09 COMBO_Rd4_6nM_C12 COMB O_Rd4_0.6nM_C30、COMBO_Rd4_0.6nM_C14、COMBO_Rd4_6nM_C07、COMBO_Rd 4_6nM_C02、COMBO_Rd4_0.6nM_C05、COMBO_Rd4_0.6nM_C17、COMBO_Rd4_6n M_C22, COMBO_Rd4_0.6nM_C11, COMBO_Rd4_0.6nM_C29, P4G4, and P1A11.

[0242] In one variation, the conjugate further comprises an amino acid substitution at position 222 from lysine to arginine. Thus, for example, the conjugates are: 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101, comprising K222R; 2) antibody-AcLys-VC-PABC-0101, comprising N297Q and K222R; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101, comprising N297Q and K222R; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131, comprising N297A and K222R; 5) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377, comprising N297A and K222R; 6) antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101, comprising K222R. In some embodiments, an acyl donor glutamine-containing tag, for example, including GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475), is engineered at the C-terminus of the antibody light chain. In other embodiments, an acyl donor glutamine-containing tag (e.g., LLQGA (SEQ ID NO: 473) or LLQGPP (SEQ ID NO: 472)) is engineered at the C-terminus of the antibody heavy chain, and the C-terminal lysine residue is deleted. In some embodiments, an acyl donor glutamine-containing tag, for example, including LLQG (SEQ ID NO: 454), is engineered after residue T135 in the antibody heavy chain, or replaces amino acid residues E294-N297 in the antibody heavy chain.P6E01 / P6E01, P6E01 / H3.AQ, L1.LGF / L 3.KW / P6E01;L1.LGF / L3.NY / P6E01、L1.GDF / L3.NY / P6E01、L1.L GF / L3.KW / H3.AL、L1.LGF / L3.KW / H3.AP、L1.LGF / L3.KW / H3.AQ、L1.LGF / L3.PY / H3.AP、L1.LGF / L3.PY / H3.AQ、L1.LGF / L3.NY / L3.H3. .AL、L1.LGF / L3.NY / H3.AP、L1.LGF / L3.NY / H3.AQ、L1.GDF / L3.KW / H3.AL、L1.GDF / L3.KW / H3.AP、L1.GDF / L3.KW / H3.AQ. L3.PY / H3.AQ、L1.GDF / L3.NY / H3.AL、L1.GDF / L3.NY / H3.AP、L1.GDF / L3.NY / H3.AQ、L3.KW / P6E01、L3.PY / P6E01、L1.3.01 / NY L3.PY / L1.PS / P6E01、L3.PY / L1.AH / P6E01、L3.PY / L1.FF / P6E01、L3.PY / L1.PH / P6E01、L3.PY / L3.KY / P6E01、L3.PY / L3.KF / P6E01、L3.PY / H2.QR、L3.PY / H2.DY、L3.PY / H2.YQ、L3.PY / H2.LT、L3.PY / H2.HA、L3.PY / H2.QL、L3.PY / H3.YA、L3.PY / H3.AE、L3.PY / H3.AQ、L3.PY / H3.TAQ、L3.PY / P6E01、L3.PY / L1.PS / H2.QR、L3.PY / L1.PS / H2.DY、L3.PY / L1.PS / H2.YQ、L3.PY / L1.PS / H2.LT、L3.PY / L1.PS / H2.HA、L3.PY / L1.PS / H2.QL、L3.PY / L1.PS / H3.YA、L3.PY / L1.PS / H3.AE、L3.PY / L1.PS / H3.AQ、L3.PY / L1.PS / H3.TAQ、L3.PY / L1.AH / H2.QR、L3.PY / L1.AH / H2.DY、L3.PY / L1.AH / H2.YQ、L3.PY / L1.AH / H2.LT、L3.PY / L1.AH / H2.HA、L3.PY / L1.AH / H2.QL、L3.PY / L1.AH / H3.YA、L3.PY / L1.AH / H3.AE、L3.PY / L1.AH / H3.AQ、L3.PY / L1.AH / H3.TAQ、L3.PY / L1.FF / H2.QR、L3.PY / L1.FF / H2.DY、L3.PY / L1.FF / H2.YQ、L3.PY / L1.FF / H2.LT、L3.PY / L1.FF / H2.HA、L3.PY / L1.FF / H2.QL、L3.PY / L1.FF / H3.YA、L3.PY / L1.FF / H3.AE、L3.PY / L1.FF / H3.AQ、L3.PY / L1.FF / H3.TAQ、L3.PY / L1.PH / H2.QR、L3.PY / L1.PH / H2.HA、L3.PY / L1.PH / H3.AE、L3.PY / L1.PH / H3.AQ、L3.PY / L1.PH / H3.TAQ、L3.PY / L3.KY / H2.QR、L3.PY / L3.KY / H2.DY、L3.PY / L3.KY / H2.YQ L3.PY / L3.KY / H2.LT、L3.PY / L3.KY / H2.HA、L3.PY / L3.KY / H2.QL、L3.PY / L3.KY / H3.YA L3.PY / L3.KY / H3.TAQ、L3.PY / L3.KF / H2.DY、L3.PY / L3.KF / H2.YQ、L3.PY / L3.KF / H2.LT L3.PY / L3.KF / H2.QL、L3.PY / L3.KF / H3.YA、L3.PY / L3.KF / H3.AE、L3.PY / L3.KF / H3.AQ L3.PY / L3.KF / H3.TAQ、P5A2_VHVL、A02_Rd4_0.6nM_C06、A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16、A02_Rd4_6nM_C03、A02_Rd4_6nM_C01、A02_Rd4_6nM_C26 A02_Rd4_6nM_C25、A02_Rd4_6nM_C22、A02_Rd4_6nM_C19、A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6nM_C18、A02_Rd4_6nM_C10 A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C26 A02_Rd4_0.6nM_C13、A02_Rd4_0.6nM_C01、A02_Rd4_6nM_C08、P5C1_VHVL、C01_Rd4_6nM_C24、C01_Rd4_6nM_C26、C01_Rd4_6nM_C10、C01_Rd4_0.6nM_C27 C01_Rd4_6nM_C20、C01_Rd4_6nM_C12、C01_Rd4_0.6nM_C16、C01_Rd4_0.6nM_C09 C01_Rd4_6nM_C09、C01_Rd4_0.6nM_C03、C01_Rd4_0.6nM_C06、C01_Rd4_6nM_C04 COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_6 nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23, CO MBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09, COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14, COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05, COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, COMBO_Rd4_0.6nM_C11, COMBO_Rd4_0.6nM_C29, or P4G4, or P1A11.

[0243] CD3 Antibodies and Methods for Producing Them The present invention further provides antibodies that bind to CD3 (e.g., human CD3 (SEQ ID NO: 502; or Accession No.: NM_000733.3)).

[0244] In one embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a VH CDR1, a VH CDR2, and a VH CDR3 of the VH sequence set forth in SEQ ID NOs: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, or 400. Provided is an isolated antibody or antigen-binding fragment thereof comprising a VH region comprising CDR3 and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the variable light chain (VL) sequence set forth in SEQ ID NO:319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399.

[0245] In another embodiment, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, wherein the VH region comprises: (i) a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NO: 331, 332, 333, 401, 402, 403, 407, 408, 415, 416, 418, 419, 420, 424, 425, 426, 446, 447, or 448; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449, or 450; and (iii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 335, 406, 423, 429, or 451. and / or a VL region comprising (i) a light chain variable (VL) CDR1 comprising the sequence set forth in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341, 433, 452, or 436, and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453.

[0246] In some embodiments, an antibody having any one of the partial light chain sequences listed in Table 3 and / or any one of the partial heavy chain sequences listed in Table 3 is provided.

[0247] [Table 3-1]

[0248] [Table 3-2]

[0249] [Table 3-3]

[0250] [Table 3-4]

[0251] [Table 3-5]

[0252] In Table 3, the underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0253] The present invention also provides CDR portions of antibodies against CD3, including Chothia, Kabat CDRs, and CDR contact regions. Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments having more than one CDR, the CDRs may be either Kabat, Chothia, combined CDRs, or combinations thereof. Table 4 provides examples of CDR sequences provided herein.

[0254] [Table 4-1]

[0255] [Table 4-2]

[0256] [Table 4-3]

[0257] [Table 4-4]

[0258] [Table 4-5]

[0259] [Table 4-6]

[0260] [Table 4-7]

[0261] [Table 4-8]

[0262] The invention also provides isolated polynucleotides encoding the antibodies of the invention, as well as vectors and host cells containing the polynucleotides.

[0263] In one embodiment, the polynucleotide is selected from the group consisting of antibodies h2B4, h2B4-VH-wt VL_TK, h2B4-VH-hnps VL_TK, h2B4-VH-yaes VL_TK, h2B4-VH-yads VL_TK, h2B4-VH-yaps VL_TK, h2B4-VH-hnps VL_TK-S55Y, h2B4-VH-hnps VL_TK-S105Q, h2B4-vH-hnps The antibody of interest may be encoded in a vector in a host cell, and the host cell may then be propagated and frozen for future use. Vectors (including expression vectors) and host cells are described further herein.

[0264] The present invention also encompasses fusion proteins comprising one or more fragments or regions from the antibodies of the present invention. In one embodiment, a fusion polypeptide is provided that comprises at least 10 contiguous amino acids of the variable light chain region set forth in SEQ ID NO: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 445, 352, 355, 443, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399, and / or at least 10 amino acids of the variable heavy chain region set forth in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 354, 356, 444, 442, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, or 400. In other embodiments, fusion polypeptides are provided that comprise at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable light chain region and / or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide comprises a light chain variable region and / or a heavy chain variable region as set forth in any of the sequence pairs selected from among SEQ ID NOs: 319 and 320, 321 and 322, 323 and 324, 325 and 326, 327 and 328, 329 and 330, 344 and 345, 346 and 347, 348 and 349, 350 and 351, 445 and 444, 352 and 354, 355 and 356, 443 and 442, 377 and 378, 379 and 380, 381 and 382, ​​383 and 384, 385 and 386, 387 and 388, 389 and 390, 391 and 392, 393 and 394, 395 and 396, 397 and 398, or 399 and 400. In another embodiment, the fusion polypeptide comprises one or more CDRs. In yet another embodiment, the fusion polypeptide comprises CDR H3 (VH CDR3) and / or CDR L3 (VL CDR3).For the purposes of the present invention, a fusion protein contains one or more antibodies and another amino acid sequence that is not attached to a natural molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags", such as FLAG tags or 6His tags. Tags are well known in the art.

[0265] Fusion polypeptides can be created by methods known in the art, e.g., synthetically or recombinantly. Typically, the fusion proteins of the invention are made by preparing and expressing polynucleotides encoding them using recombinant methods described herein, although they can also be prepared by other means known in the art, including, e.g., chemical synthesis.

[0266] Representative materials of the CD3 antibody of the present invention were deposited at the American Type Culture Collection (ATCC) on September 11, 2015. A vector having ATCC accession number PTA-122513 is a polynucleotide encoding a humanized CD3 antibody heavy chain variable region, and a vector having ATCC accession number PTA-122512 is a polynucleotide encoding a humanized CD3 antibody light chain variable region. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations Thereunder (Budapest Treaty), which ensures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and ATCC which assures perpetual and unrestricted availability of the progeny of the cultures of the deposit to the public after issuance of the pertinent U.S. patent, or after the public disclosure of any U.S. or foreign patent application, whichever occurs earlier, and to persons determined by the Director of the U.S. Patent and Trademark Office to be entitled pursuant to 35 U.S.C. § 122 and applicable Director regulations thereunder (including 37 CFR § 1.14, with specific reference to 886 OG 638).

[0267] The agent for this application has agreed that if a culture of the deposited material, when cultured under appropriate conditions, dies or is lost or destroyed, the material will be promptly replaced upon notice with another identical one. Availability of the deposited material is not to be construed as a license to practice the invention in violation of the rights granted under the authority of any government pursuant to its patent laws.

[0268] Bispecific antibodies and methods of making them Bispecific antibodies, monoclonal antibodies, having binding specificities for at least two different antigens can be prepared using the antibodies disclosed herein. Methods for producing bispecific antibodies are known in the art (e.g., Suresh et al., Methods in Enzymology, 121:210, 1986). Traditionally, recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy-light chain pairs with two heavy chains having different specificities (Millstein and Cuello, Nature, 305, 537-539, 1983).

[0269] According to one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant region sequences. The fusion is preferably with an immunoglobulin heavy chain constant region, including at least a portion of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows for great flexibility in adjusting the mutual ratio of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptides used in the construct provide optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in equal ratios provides high yields or when the ratio is not particularly important.

[0270] In one approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates separation of the desired bispecific compound from undesired immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94 / 04690.

[0271] In another approach, bispecific antibodies are constructed with amino acid modifications in a first hinge region in one arm, where the substituted / replaced amino acid in the first hinge region has an opposite charge to the corresponding amino acid in the second hinge region in the other arm. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0272] In another approach, the formation of the desired heteromultimeric or heterodimeric protein (e.g., bispecific antibody) is enhanced by modifying or manipulating the interface between the first and second immunoglobulin-like Fc regions (e.g., hinge and / or CH3 regions). In this approach, the bispecific antibody can be composed of a CH3 region, which includes a first CH3 polypeptide and a second CH3 polypeptide, which interact together to form a CH3 interface, and one or more amino acids in the CH3 interface destabilize homodimer formation and are not electrostatically unfavorable to homodimer formation. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0273] In another approach, bispecific antibodies can be generated using a glutamine-containing peptide tag engineered into an antibody directed to an epitope (e.g., BCMA) in one arm, and another peptide tag (e.g., a Lys-containing peptide tag or reactive endogenous Lys) engineered into a second antibody directed to a second epitope in another arm in the presence of transglutaminase. This approach is described in International Patent Application No. PCT / IB2011 / 054899 (WO2012 / 059882).

[0274] In another aspect of the invention, the heterodimeric protein (e.g., bispecific antibody) described herein comprises a full-length human antibody, wherein a first antibody variable domain of the heterodimeric protein is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells, and a second antibody variable domain of the heterodimeric protein is capable of specifically binding to a target antigen. In some embodiments, the human antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the heterodimeric protein comprises an immunologically inert Fc region.

[0275] The human immune effector cell can be any of a variety of immune effector cells known in the art. For example, the immune effector cell can be a member of the human lymphoid cell lineage, including, but not limited to, T cells (e.g., cytotoxic T cells), B cells, and natural killer (NK) cells. The immune effector cell can also be a member of the human myeloid lineage, including, but not limited to, monocytes, neutrophil granulocytes, and dendritic cells. Such immune effector cells can have a cytotoxic or apoptotic effect on target cells, or other desired effect, when activated by binding of an effector antigen.

[0276] Effector antigen is an antigen (e.g., protein or polypeptide) expressed by human immune effector cells. Examples of effector antigens that can be bound by heterodimeric protein (e.g., heterodimeric antibody or bispecific antibody) include, but are not limited to, human CD3 (or CD3 (cluster of differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

[0277] The target cell may be a cell that is native or foreign to humans. In a natural target cell, the cell may be transformed into a malignant cell or may be pathologically modified (e.g., a natural target cell infected with a virus, a malaria parasite, or a bacterium). In a foreign target cell, the cell is an invading pathogen, such as a bacterium, a malaria parasite, or a virus.

[0278] A target antigen is expressed on a target cell in a disease state (e.g., an inflammatory disease, a proliferative disease (e.g., cancer), an immunological disorder, a neurological disease, a neurodegenerative disease, an autoimmune disease, an infectious disease (e.g., a viral or parasitic infection), an allergic reaction, a graft versus host disease, or a host versus graft disease). A target antigen is not an effector antigen. Examples of target antigens include, but are not limited to, BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor)-2 / neu, HER-3, HER-4, EGFR (epidermal growth factor receptor), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl GM1, polySA, GD2, carboanhydrase IX (MN / CA IX), CD44v6, Shh (sonic hedgehog), Wue-1, plasma cell antigen, (membrane-bound) IgE, MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, and MIS (Mullerian inhibitory substance) receptor type II, sTn (sialylated Tn antigen; tag-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, and CD63.

[0279] In some embodiments, the heterodimeric proteins (e.g., bispecific antibodies) described herein comprise a full-length human antibody, wherein a first antibody variable domain of the heterodimeric protein is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen (e.g., CD3 antigen) located on the human immune effector cells, and a second antibody variable domain of the heterodimeric protein is capable of specifically binding to a target antigen (e.g., CD20 antigen or EpCAM), and wherein the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225R), and (P228E or P228R)) in the hinge region of human IgG2 (SEQ ID NO: 493), and at positions 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in the CH3 region.

[0280] In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 221 and 228 (e.g., (D221R or D221E) and (P228R or P228E)) in the hinge region and at positions 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in the CH3 region of human IgG1 (SEQ ID NO: 494).

[0281] In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at position 228 (e.g., (P228E or P228R)) in the hinge region and at position 409 or 368 (e.g., R409 or L368E (EU numbering scheme)) in the CH3 region of human IgG4 (SEQ ID NO: 495).

[0282] In another embodiment, the first antibody variable domain of the heterodimeric protein comprises the VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, or 400. and / or a VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence set forth in SEQ ID NOs: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399. and the heterodimeric protein second antibody variable domain comprises a VL region comprising a VL CDR3, and the heterodimeric protein second antibody variable domain comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 125, 127, 313, 314, 363, or 365. and / or SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 , 77, 79, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, or 364.

[0283] In another embodiment, the first antibody variable domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388, and / or a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387, and the second antibody variable domain comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 112, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 38.

[0284] Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins containing antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

[0285] In some embodiments, the BCMA or CD3 antibodies described herein are monoclonal antibodies, e.g., the BCMA or CD3 antibodies are humanized or chimeric monoclonal antibodies.

[0286] In some embodiments, the antibody comprises a modified constant region, such as, without limitation, a constant region with increased potential to elicit an immune response. For example, the constant region may be modified to have increased affinity for an Fc gamma receptor, such as FcγRI, FcγRIIA, or FcγIII.

[0287] In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert, i.e., has reduced potential to elicit an immune response. In some embodiments, the constant region is modified as described in Eur.J.Immunol.29:2613-2624, 1999; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 98099518. The Fc can be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc can be human IgG2 (IgG2Δa) containing mutations A330P331 to S330S331, where the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur.J.Immunol., 29:2613-2624, 1999. In some embodiments, the antibody comprises a constant region of IgG4 comprising the following mutations (Armour et al., Molecular Immunology, 40, 585-593, 2003): E233F234L235 to P233V234A235 (IgG4Δc), numbering is with reference to wild type IgG4. In yet another embodiment, the Fc is human IgG4 E233F234L235 to P233V234A235 (IgG4Δb) with the deletion G236. In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb, or IgG4Δc) containing the hinge stabilizing mutation S228 to P228 (Aalberse et al., Immunology, 105, 9-19, 2002). In another embodiment, the Fc can be an aglycosylated Fc.

[0288] In some embodiments, the constant region is aglycosylated by mutating the oligosaccharide attachment residue (such as Asn297) and / or flanking residues that are part of a glycosylation recognition sequence in the constant region. In some embodiments, the constant region is enzymatically aglycosylated for N-linked glycosylation. The constant region may be aglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

[0289] In some embodiments, the constant region has a modified constant region that eliminates or reduces Fc gamma receptor binding. For example, the Fc can be human IgG2 containing the mutation D265, where the amino acid residues are numbered with reference to the wild-type IgG2 sequence (SEQ ID NO: 493). Thus, in some embodiments, the constant region has the sequence shown in SEQ ID NO: 496: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The polypeptide has a modified constant region having the formula:

[0290] In some embodiments, the constant region comprises the sequence set forth in SEQ ID NO:497: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The polypeptide has a modified constant region having the formula:

[0291] One way to determine the binding affinity of an antibody to BCMA or CD3 is by measuring the binding affinity of the monofunctional Fab fragment of the antibody. To obtain the monofunctional Fab fragment, the antibody (e.g., IgG) can be cleaved with papain or recombinantly expressed. The affinity of the BCMA Fab fragment of the antibody can be determined by surface plasmon resonance (Biacore™ 3000™ Surface Plasmon Resonance (SPR) System, Biacore™, INC, Piscataway NJ) with a pre-immobilized streptavidin sensor chip (SA), or anti-mouse Fc or anti-human Fc using HBS-EP running buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v / v surfactant P20). Biotinylated or Fc-fused human BCMA can be diluted in HBS-EP buffer to concentrations below 0.5 μg / mL and injected into individual chip channels using various contact times to achieve two ranges of antigen density, 50-200 response units (RU) for detailed kinetic studies or 800-1,000 RU for screening assays. Regeneration studies showed that 25 mM NaOH in 25% v / v ethanol effectively removed bound Fab while preserving the activity of BCMA on the chip over 200 injections. Typically, serial dilutions of purified Fab samples (spanning concentrations of 0.1-10×) were used to determine the K D The kinetic association rate (k) is estimated to be 100 μL / min for 1 min, allowing a maximum dissociation time of 2 h. The concentration of the Fab protein is determined by ELISA and / or SDS-PAGE electrophoresis using Fabs of known concentration (determined by amino acid analysis) as standards. on ) and dissociation rate (k off) are simultaneously obtained by globally fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994), Methods Enzymology, 6, 99-110) using the BIAevaluation program. D ) value is k off / k on The binding affinity of an antibody is calculated as: This protocol is suitable for use in determining the binding affinity of an antibody to any BCMA, including human BCMA, BCMA from another mammal (such as mouse BCMA, rat BCMA, or primate BCMA), and different forms of BCMA (e.g., glycosylated BCMA). Antibody binding affinity is typically measured at 25°C, but may also be measured at 37°C.

[0292] The antibodies described herein can be produced by any method known in the art. For the production of hybridoma cell lines, the route and schedule of immunization of the host animal is generally in accordance with the established and routine techniques of antibody stimulation and production, as further described herein. General techniques for the production of human and mouse antibodies are known in the art and / or described herein.

[0293] It is contemplated that any mammalian subject, including a human, or antibody-producing cells derived therefrom can be manipulated to serve as a basis for the production of mammalian, including human, and hybridoma cell lines.Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of an immunogen, including those described herein.

[0294] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature, 256:495-497, 1975, or as modified by Buck, DW et al., In Vitro, 18:377-381, 1982. Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. In general, the technique fuses myeloma cells and lymphoid cells using a fusing agent such as polyethylene glycol or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to eliminate unhybridized parent cells. Any of the media described herein, supplemented with serum or free of serum, can be used to culture hybridomas secreting monoclonal antibodies. As an alternative to cell fusion techniques, EBV-immortalized B cells may be used to produce the monoclonal antibodies of the subject invention. Hybridomas are expanded and subcloned as necessary, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0295] Hybridomas which may be used as a source of antibodies include all derivatives, progeny cells of the parent hybridoma that produce monoclonal antibodies specific for BCMA, CD3, or portions thereof.

[0296] Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids, if necessary, by conventional immunoglobulin purification procedures, such as ammonium sulfate fractionation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesired activity, if present, can be removed, for example, by running the preparation over an adsorbent made of the immunogen attached to a solid phase, and eluting or releasing the desired antibody from the immunogen. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N=C=NR, where R and R 1 are different alkyl groups) to immunize a host animal with a protein that is immunogenic in the species being immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or human BCMA or CD3 conjugated to soybean trypsin inhibitor, or a fragment containing the target amino acid sequence, to obtain a population of antibodies (e.g., monoclonal antibodies).

[0297] If necessary, the antibody of interest (monoclonal or polyclonal) can be sequenced, and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and then the host cell can be propagated and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be carried out by cloning antibody genes from B cells by means known in the art. For example, see Tiller et al., J.Immunol.Methods, 329, 112, 2008; U.S. Patent No. 7,314,622.

[0298] Alternatively, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve affinity or other properties of the antibody.For example, when the antibody is used in clinical trials and treatments in humans, the constant region can be engineered to more closely resemble human constant regions to avoid immune responses.It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to BCMA or CD3, and greater potency in inhibiting BCMA.

[0299] There are four general steps in humanizing a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., determining which antibody framework regions will be used during the humanization process; (3) the actual humanization methodology / techniques; and (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

[0300] Several "humanized" antibody molecules have been described that contain antigen-binding sites derived from non-human immunoglobulins, including chimeric antibodies with rodent or modified rodent V regions and their associated CDRs fused to human constant regions.See, for example, Winter et al., Nature, 349:293-299, 1991; Lobuglio et al., Proc.Nat.Acad.Sci.USA, 86:4220-4224, 1989; Shaw et al., J Immunol., 138:4534-4538, 1987, and Brown et al., Cancer Res., 47:3577-3583, 1987. Other references describe rodent CDRs grafted into human supporting framework regions (FRs) before being fused to the appropriate human antibody constant regions. See, e.g., Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; and Jones et al., Nature, 321:522-525, 1986. Other references describe rodent CDRs supported by engineered rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize unwanted immune responses directed toward rodent anti-human antibody molecules that limit the duration and efficacy of therapeutic use of these moieties in human recipients. For example, the antibody constant region can be engineered so that it is immunologically inert (e.g., does not induce complement lysis). See, e.g., PCT Publication No. PCT / GB99 / 01441; UK Patent Application No. 9809951.8. Other methods for humanizing antibodies, which may also be utilized, are disclosed by Daugherty et al., Nucl. Acids Res., 19:2471-2476, 1991, and in U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861, and in PCT Publication No. WO 01 / 27160.

[0301] The general principles for humanizing antibodies discussed above are also applicable to customizing antibodies for use in, for example, dogs, cats, primates, horses, and cattle. Furthermore, one or more of the embodiments of humanizing an antibody described herein may be combined with, for example, CDR grafting, framework mutations, and CDR mutations.

[0302] In one variation, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to generate more desirable (e.g., fully human antibodies) or more robust immune responses can also be used to generate humanized or human antibodies. Examples of such technology are Xenomouse™ from Abgenix, Inc. (Fremont, CA), and HuMAb-Mouse® and TCMouse™ from Medarex, Inc. (Princeton, NJ).

[0303] Alternatively, the antibodies may be recombinantly produced and expressed using any method known in the art. In another alternative, the antibodies may be recombinantly produced by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol., 12:433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature, 348:552-553, 1990) can be used to generate humanized antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into major or minor coat protein genes of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats. For reviews, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology, 3:564-571, 1993. Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628, 1991, isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Mark et al., J. Mol. Biol., 222:581-597, 1991, or Griffith et al., EMBO J., 12:725-734, 1993.In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells displaying high affinity surface immunoglobulins will preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be mimicked by using a technique known as "chain shuffling". (Marks et al., Bio / Technol., 10:779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with a repertoire of naturally occurring variants of V domain genes obtained from non-immunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for generating very large phage antibody repertoires (also known as "mother of all libraries") was described by Waterhouse et al., Nucl. Acids Res., 21:2265-2266, 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinities and specificities as the starting rodent antibody. According to this method, also called "epitope imprinting", the heavy or light chain V domain genes of a rodent antibody obtained by phage display techniques are replaced with a repertoire of human V domain genes to create rodent-human chimeras. The selection of the antigen leads to the isolation of human variable regions capable of restoring a functional antigen binding site, i.e., the epitope governs (imprints) the choice of partner. When the process is repeated to replace the remaining rodent V domains, a human antibody is obtained (see PCT Publication No. WO 93 / 06213). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique results in completely human antibodies, which have no framework or CDR residues of rodent origin.

[0304] Antibodies can be made recombinantly by first isolating the antibody and antibody-producing cells from a host animal, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in a host cell (e.g., CHO cell). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or transgenic milk. Methods for expressing antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters et al., Vaccine, 19:2756, 2001; Lonberg, N. and D. Huszar, Int. Rev. Immunol, 13:65, 1995; and Pollock et al., J Immunol Methods, 231:147, 1999. Methods for making derivatives of antibodies, e.g., humanized, single chain, etc., are known in the art.

[0305] Immunoassays and flow cytometric sorting techniques, such as fluorescence activated cell sorting (FACS), can also be used to isolate antibodies that are specific for BCMA, CD3, or a tumor antigen of interest.

[0306] The antibodies described herein can be bound to many different carriers. The carriers can be active and / or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble for the purposes of the present invention. Those skilled in the art will know other suitable carriers for binding antibodies, or will be able to ascertain such using routine experimentation. In some embodiments, the carrier comprises a moiety that targets the myocardium.

[0307] The DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a suitable source of such DNA. Once isolated, the DNA can be placed into an expression vector (such as the expression vector disclosed in PCT Publication No. WO87 / 04462), which is then transfected into host cells that do not otherwise produce immunoglobulin protein, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the monoclonal antibody in the recombinant host cell. See, for example, PCT Publication No. WO87 / 04462. The DNA may also be modified, for example, by substituting the coding sequence for the human heavy and light chain constant regions for the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci., 81:6851, 1984, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In that manner, "chimeric" or "hybrid" antibodies are prepared, having the binding specificity of the monoclonal antibodies herein.

[0308] The BCMA or tumor antigen of interest antibodies described herein can be identified or characterized using methods known in the art to detect and / or measure a reduction in BCMA or other tumor antigen expression levels. In some embodiments, BCMA antibodies are identified by incubating a candidate agent with BCMA and monitoring binding and / or a concomitant reduction in BCMA expression levels. Binding assays can be performed using purified BCMA polypeptides or using cells that naturally express or have been transfected to express BCMA polypeptides. In one embodiment, the binding assay is a competitive binding assay, where the ability of a candidate antibody to compete for BCMA binding with a known BCMA antibody is assessed. The assay can be performed in a variety of formats, including ELISA formats.

[0309] After initial identification, the activity of candidate BCMA, CD3, or other tumor antigen antibodies can be further confirmed and refined by bioassays known to test targeted biological activity. Alternatively, bioassays can be used to directly screen candidates. Some of the methods for identifying and characterizing antibodies are described in detail in the Examples.

[0310] BCMA, CD3 or other tumor antigen antibodies can be characterized using methods known in the art. For example, one method is to identify the epitope that it binds to, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including elucidation of the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In an additional example, epitope mapping can be used to determine the sequence that an antibody binds to. Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch. Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with BCMA, CD3, or other tumor antigen antibodies. In another example, the epitope to which BCMA, CD3, or other tumor antigen antibodies bind can be determined in a systematic screen by using overlapping peptides derived from the BCMA, CD3, or other tumor antigen sequence and determining binding by the BCMA, CD3, or other tumor antigen antibody. According to gene fragment expression assays, the open reading frame encoding BCMA, CD3, or other tumor antigen is fragmented randomly or by specific gene construction and the reactivity of the expressed fragments of BCMA, CD3, or other tumor antigen with the antibody being tested is determined. Gene fragments can be generated, for example, in vitro, by PCR, then transcribed and translated into proteins in the presence of radioactive amino acids.Binding of the antibody to the radiolabeled BCMA, CD3, or other tumor antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutant BCMA, CD3, or other tumor antigens in which various fragments of BCMA, CD3, or other tumor antigen proteins have been replaced (swapped) with sequences from BCMA from another species (e.g., mouse), or closely related but antigenically distinct proteins (e.g., Trop-1). By assessing antibody binding to mutant BCMA, CD3, or other tumor antigens, the importance of particular BCMA, CD3, or other tumor antigen fragments to antibody binding can be assessed.

[0311] Yet another method that can be used to characterize BCMA, CD3, or other tumor antigen antibodies is to use competition assays with other antibodies known to bind to various fragments on the same antigen, i.e., BCMA, CD3, or other tumor antigen, to determine whether the BCMA, CD3, or other tumor antigen antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art.

[0312] Expression vectors can be used to directly express BCMA, CD3, or other tumor antigen antibodies.Those skilled in the art are familiar with the administration of expression vectors to obtain the expression of exogenous proteins in vivo.See, for example, U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471.Expression vector administration includes local or systemic administration, including injection, oral administration, particle gun, or catheter insertion administration, and local administration.In another embodiment, expression vectors are directly administered into sympathetic trunks or ganglia, or into coronary artery, atrium, ventricle, or pericardium.

[0313] Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, edited by JA Wolff, 1994; Wu et al., J.Biol.Chem., 263:621, 1988; Wu et al., J.Biol.Chem., 269:542, 1994; Zenke et al., Proc.Natl.Acad.Sci.USA, 87:3655, 1990; and Wu et al., J.Biol.Chem., 266:338, 1991. Therapeutic compositions containing polynucleotides are administered in the range of about 100ng to about 200mg of DNA for local administration in gene therapy protocols. Concentration ranges of DNA from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg can also be used during gene therapy protocols. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy, 1:51, 1994; Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 6:148, 1994). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequences can be constitutive or regulated.

[0314] Viral vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and European Patent No. 0345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-2111), and the like). VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), as well as adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984, and WO95 / 00655). Administration of DNA linked to a killed adenovirus, as described in Curiel, Hum. Gene Ther., 1992, 3:147, can also be used.

[0315] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycation-condensed DNA (see, for example, Curiel, Hum. Gene Ther., 3:147, 1992) with or without being linked to killed adenovirus alone; ligand-linked DNA (see, for example, Wu, J. Biol. Chem., 264:16985, 1989); eukaryotic cell delivery vehicle cells (see, for example, U.S. Patent No. 5,814,482; PCT Publication Nos. WO95 / 07994; WO96 / 17072; WO95 / 30763; and WO97 / 42338), as well as nuclear charge neutralization or fusion with cell membrane. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes which can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP 0524968. Additional techniques are described in Philip, Mol. Cell Biol., 14:2411, 1994 and Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

[0316] In some embodiments, the invention encompasses compositions, including pharmaceutical compositions, comprising antibodies described herein or made using the methods and having the properties described herein. As used herein, compositions comprise one or more antibodies that bind to CD3 and a tumor antigen (e.g., BCMA), and / or one or more polynucleotides that comprise sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients, such as pharma- ceutically acceptable excipients, including buffers, as are well known in the art.

[0317] The present invention also provides a method for making any of these antibodies. The antibodies of the present invention can be made by procedures known in the art. The polypeptides can be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Antibody polypeptides, particularly shorter polypeptides up to about 50 amino acids, are conveniently produced by chemical synthesis. Methods of chemical synthesis are known in the art and commercially available. For example, antibodies can be produced by automated polypeptide synthesizers using solid phase methods. See also U.S. Patent Nos. 5,807,715; 4,816,567; and 6,331,415.

[0318] Heteroconjugate antibodies, which comprise two covalently linked antibodies, are also within the scope of the present invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (PCT Publications WO91 / 00360 and WO92 / 200373; EP03089). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are described in U.S. Pat. No. 4,676,980.

[0319] Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0320] In recombinant humanized antibody, Fcγ part can be modified to avoid interaction with Fcγ receptor and complement and immune system.Techniques for preparing such antibodies are described in WO99 / 58572.For example, when antibody is used in clinical trials and treatment in humans, constant region can be engineered to more closely resemble human constant region to avoid immune response.See, for example, U.S. Patent Nos. 5,997,867 and 5,866,692.

[0321] The present invention encompasses modifications to the antibodies and polypeptides of the variants of the present invention described herein, including functionally equivalent antibodies that do not significantly affect the properties of the antibody, as well as variants with enhanced or reduced activity and / or affinity. For example, amino acid sequences can be mutated to obtain antibodies with the desired binding affinity to BCMA and / or CD3. Modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitution of amino acid residues, deletion or addition of one or more amino acids that do not significantly and adversely change the functional activity or mature (increase) the affinity of the polypeptide to its ligand, or the use of chemical analogs.

[0322] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0323] Substitution variants have at least one amino acid residue in an antibody molecule removed and a different residue inserted in its place. The most interesting sites for substitution mutagenesis are hypervariable regions, but FR changes are also contemplated. Conservative substitutions are shown in Table 5 under the heading of "conservative substitutions". If such substitutions change biological activity, more substantial changes can be introduced, as named "exemplary substitutions" in Table 5 or further described below in reference to amino acid classes, and the products can be screened.

[0324] [Table 5]

[0325] Substantial alterations in the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the area of ​​the substitution, for example as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintenance of side chain bulk. Naturally occurring amino acid residues are divided into groups based on common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) uncharged polar: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His.

[0326] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0327] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody also may be replaced, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0328] Amino acid modifications can range from changing or modifying one or more amino acids to a complete redesign of a region, such as a variable region. Changes in the variable region can alter binding affinity and / or specificity. In some embodiments, no more than 1-5 conservative amino acid substitutions are made in the CDR domain. In other embodiments, no more than 1-3 conservative amino acid substitutions are made in the CDR domain. In yet other embodiments, the CDR domain is CDR H3 and / or CDR L3.

[0329] Modifications include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions within their constant regions (Jefferis and Lund, Chem. Immunol., 65:111-128, 1997; Wright and Morrison, TibTECH, 15:26-32, 1997). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., Mol. Immunol., 32:1311-1318, 1996; Wittwe and Howard, Biochem., 29:4175-4180, 1990), as well as the intramolecular interactions between moieties of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech., 7:409-416, 1996), which may affect the conformation and presented three-dimensional surface of the glycoprotein. Oligosaccharides may also function to target a given glycoprotein to a particular molecule based on a specific recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, have been reported to have improved ADCC activity (Umana et al., Mature Biotech., 17:176-180, 1999).

[0330] Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugar N-acetylgalactosamine, galactose, or xylose to one of the hydroxyamino acids, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0331] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0332] The glycosylation pattern of an antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used to express recombinant glycoproteins, e.g., antibodies, as potential therapeutic agents, is rarely a native cell, variations in the glycosylation pattern of an antibody can be expected (see, e.g., Hse et al., J.Biol.Chem., 272:9062-9070, 1997).

[0333] In addition to the choice of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, medium formulation, culture density, oxygenation, pH, purification schemes, etc. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261, and 5,278,299). Glycosylation or certain types of glycosylation can be enzymatically removed from glycoproteins using, for example, Endo H, N-glycosidase F, Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3. Furthermore, recombinant host cells can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

[0334] Other methods of modification include the use of coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to attach labels for immunoassays. Modified polypeptides can be made using procedures established in the art and screened using standard assays known in the art, some of which are described below and in the Examples.

[0335] In some embodiments of the invention, the antibody comprises a modified constant region, such as a constant region with increased affinity to human Fc gamma receptors, and is immunologically inactive or partially inactive, e.g., does not induce complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), does not activate macrophages, or has reduced activity (compared to unmodified antibodies) in any one or more of the following: induce complement-mediated lysis, stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or activate microglia. Different modifications of the constant region can be used to achieve optimal levels and / or combinations of effector functions. See, e.g., Morgan et al., Immunology, 86:319-324, 1995; Lund et al., J. Immunology, 157:4963-9, 157:4963-4969, 1996; Idusogie et al., J. Immunology, 164:4178-4184, 2000; Tao et al., J. Immunology, 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews, 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild type IgG2 sequence). Eur. J. Immunol., 1999, 29:2613-2624. In yet other embodiments, the constant region is aglycosylated instead of N-linked glycosylation. In some embodiments, the constant region is aglycosylated instead of N-linked glycosylation by mutating a glycosylated amino acid residue or a flanking residue that is part of an N-glycosylation recognition sequence in the constant region. For example, N-glycosylation site N297 can be mutated to A, Q, K, or H.See Tao et al., J. Immunology, 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews, 163:59-76, 1998. In some embodiments, the constant region is aglycosylated instead of N-linked glycosylation. The constant region can be aglycosylated instead of N-linked glycosylation enzymatically (such as by removal of carbohydrate with the enzyme PNGase) or by expression in a glycosylation-deficient host cell.

[0336] Other antibody modifications include antibodies modified as described in PCT Publication No. WO99 / 58572. These antibodies contain, in addition to a binding domain directed to a target molecule, an effector domain having an amino acid sequence substantially homologous to all or a portion of the constant region of a human immunoglobulin heavy chain. These antibodies can bind a target molecule without inducing significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain can specifically bind FcRn and / or FcγRIIb. These typically bind two or more human immunoglobulin heavy chain Cs. H The antibody modified in this manner is particularly suitable for use in long-term antibody therapy to avoid inflammatory and other adverse reactions to conventional antibody therapy.

[0337] The present invention includes affinity matured embodiments. For example, affinity matured antibodies can be generated by procedures known in the art (Marks et al., Bio / Technology, 10:779-783, 1992; Barbas et al., Proc Nat. Acad. Sci, USA, 91:3809-3813, 1994; Schier et al., Gene, 169:147-155, 1995; Yelton et al., J. Immunol., 155:1994-2004, 1995; Jackson et al., J. Immunol., 154(7):3310-9, 1995; Hawkins et al., J. Mol. Biol., 226:889-896, 1992; and PCT Publication No. WO2004 / 058184).

[0338] The following methods can be used to tune the affinity of an antibody and to characterize the CDRs. One way of characterizing the CDRs of an antibody and / or changing (e.g., improving) the binding affinity of a polypeptide such as an antibody is called "library scanning mutagenesis". In general, library scanning mutagenesis works as follows: One or more amino acid positions in the CDR are replaced with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using art-recognized methods. This generates a small library of clones (in some embodiments, one for each amino acid position being analyzed), each with a complexity of two or more members (when two or more amino acids are substituted per position). In general, the library also includes clones that contain natural (unsubstituted) amino acids. A small number of clones from each library, for example, about 20-80 clones (depending on the complexity of the library), are screened for binding affinity to the target polypeptide (or other binding target) to identify candidates with enhanced, identical, reduced, or no binding. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or more. Biacore™ detects that the starting antibody already has a relatively high affinity, for example, a K of about 10 nM or less. D Screening using Biacore™ surface plasmon resonance is described in the Examples herein.

[0339] Binding affinity can be determined using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN Immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast display. Binding affinity can also be screened using a suitable bioassay.

[0340] In some embodiments, every amino acid position within the CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids using art-recognized mutagenesis methods, some of which are described herein. This generates a small library of clones (in some embodiments, one for each amino acid position being analyzed), each with a complexity of 20 members (if all 20 amino acids are substituted at every position).

[0341] In some embodiments, the library to be screened contains substitutions in two or more positions, which may be in the same CDR or in two or more CDRs. Thus, the library may contain substitutions at two or more positions in one CDR. The library may contain substitutions at two or more positions in two or more CDRs. The library may contain substitutions at three, four, five, or more positions, which are found in two, three, four, five, or six CDRs. The substitutions may be prepared using low redundancy codons. See, for example, Table 2 in Balint et al., Gene, 137(1):109-18, 1993.

[0342] The CDRs may be CDRH3 and / or CDRL3. The CDRs may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and / or CDRH3. The CDRs may be Kabat CDRs, Chothia CDRs, or extended CDRs.

[0343] Candidates with improved binding can be sequenced, thereby identifying CDR substitution mutants that improve affinity (also called "improved" substitutions). Candidates that bind can also be sequenced, thereby identifying CDR substitutions that retain binding.

[0344] Multiple rounds of screening can be performed. For example, candidates with improved binding (each containing an amino acid substitution at one or more positions in one or more CDRs) are also useful for designing a second library that contains at least the original and substituted amino acids at each improved CDR position (i.e., the amino acid position in the CDR where the substitution mutant showed improved binding). The preparation and screening or selection of this library is further discussed below.

[0345] Library scanning mutagenesis also provides a means to characterize CDRs, inasmuch as frequent clones with improved, the same, reduced, or no binding also provide information on the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, it is identified as an unlikely position required for antigen binding. Conversely, if a CDR position retains binding at only a small percentage of substitutions, it is identified as a position that is important for CDR function. Thus, library scanning mutagenesis generates information on positions in CDRs that can be changed to many different amino acids (including all 20 amino acids) and positions in CDRs that cannot be changed or can only be changed to a few amino acids.

[0346] Candidates with improved affinity can be combined in a second library, which contains the improved amino acid, the original amino acid at that position, and can further contain additional substitutions at that position, depending on the complexity of the library desired or tolerated using the desired screening or selection method. In addition, if necessary, adjacent amino acid positions can be randomized to at least two or more amino acids. Randomization of adjacent amino acids can allow additional conformational flexibility in the mutant CDR, which in turn can allow or facilitate the introduction of a larger number of improving mutations. The library can also contain substitutions at positions that did not show improved affinity in the first round of screening.

[0347] The second library is screened or selected for library members with improved and / or altered binding affinity using any method known in the art, including screening using Biacore™ surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.

[0348] The present invention also provides compositions comprising antibodies conjugated (e.g., linked) to an agent (such as biotin or avidin) that facilitates coupling to a solid support. For simplicity, reference will be made generally to antibodies with the understanding that these methods apply to any of the BCMA antibody embodiments described herein. Conjugation generally refers to the linking of these components as described herein. Linking, which is generally to fix these components in close association at least for administration, can be accomplished in any number of ways. For example, a direct reaction between an agent and an antibody is possible when each has a substituent that can react with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, of one can react with a carbonyl-containing group, such as an anhydride or acid halide, of the other, or an alkyl group that contains a good leaving group (e.g., a halide).

[0349] In another aspect, the invention provides a method of making any of the polynucleotides described herein.

[0350] Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond in a one-to-one manner to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present invention, and polynucleotides may, but need not, be linked to other molecules and / or supporting materials.

[0351] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. A polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not diminished compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. A variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity with a polynucleotide sequence encoding a native antibody or portion thereof.

[0352] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0353] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS, 4:11~17; Robinson, ED, 1971, Comb. Theor., 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol., 4:406~425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of In Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA, 80:726-730.

[0354] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, and the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20 percent or less, typically 5-15 percent, or 10-12 percent additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0355] Variants may also, or alternatively, be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0356] Suitable "moderately stringent conditions" include a prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C to 65°C in 5x SSC; followed by two washes for 20 minutes each at 65°C with 2x, 0.5x, and 0.2x SSC containing 0.1% SDS.

[0357] As used herein, "highly stringent conditions" or "high stringency conditions" refers to (1) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50° C.; (2) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at pH 6.5, with denaturing agents such as formamide, e.g., 750 mM sodium chloride, 75 mM sodium citrate, at 42° C., with denaturing agents such as formamide ... or (3) using 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a wash in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0358] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein. Some of these polynucleotides bear minimal homology with the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes that comprise the polynucleotide sequences provided herein are within the scope of the present invention. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and / or database sequence comparison.

[0359] The polynucleotide of the present invention can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail here.Those skilled in the art can use the sequence provided herein and commercially available DNA synthesizers to generate desired DNA sequence.

[0360] To prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence can be inserted into a suitable vector, as further discussed herein, and the vector can then be introduced into a suitable host cell for replication and amplification. A polynucleotide can be inserted into a host cell by any means known in the art. A cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0361] Instead, PCR allows the duplication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0362] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as set forth in Sambrook et al., 1989, supra.

[0363] A suitable cloning vector can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended to be used, but a useful cloning vector will generally have the ability to replicate autonomously, can have a single target for a specific restriction endonuclease, and / or can have a marker gene that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers, such as BioRad, Strategene, and Invitrogen.

[0364] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present invention. It is implied that an expression vector must be replicable in a host cell as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT Publication No. WO87 / 04462. Vector components generally include, but are not limited to, one or more of the following: signal sequence; origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0365] The vector containing the polynucleotide of interest can be introduced into the host cell by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus).The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0366] The present invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cell expresses the cDNA at a level about 5-fold greater, more preferably 10-fold greater, and even more preferably 20-fold greater than that of the corresponding endogenous antibody or protein of interest, if present, in the host cell. Screening the host cells for specific binding to BCMA or a BCMA domain (e.g., domains 1-4) can be done by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

[0367] Methods of using bispecific antibodies [therapeutic use] The antibodies (e.g., BCMA, CD3, or bispecific) and antibody conjugates (e.g., BCMA antibody-drug conjugates) of the invention are useful in a variety of applications, including, but not limited to, therapeutic and diagnostic treatment methods.

[0368] In one aspect, the present invention provides a method for treating a condition associated with BCMA expression in a subject. In some embodiments, the method for treating a condition associated with BCMA expression in a subject comprises administering to a subject in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising a BCMA antibody or BCMA antibody conjugate as described herein. Conditions associated with BCMA expression include, but are not limited to, aberrant BCMA expression, altered or ectopic BCMA expression, malignant cells expressing BCMA, and proliferative disorders (e.g., cancer), or autoimmune disorders.

[0369] In another aspect, the present invention provides methods for treating B cell-associated cancers or malignant cells expressing a tumor antigen. In some embodiments, methods are provided for treating B cell-associated cancer in a subject in need thereof, comprising a) providing a bispecific antibody as described herein, and b) administering the bispecific antibody to the patient. In some embodiments, methods are provided for treating a condition associated with malignant cells expressing a tumor antigen in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bispecific antibody as described herein.

[0370] Thus, in some embodiments, there is provided a method of treating cancer in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising an antibody (e.g., a BCMA or CD3-BCMA bispecific antibody) or BCMA antibody conjugate described herein. As used herein, cancer includes, but is not limited to, multiple myeloma, malignant plasma cell neoplasms, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic osteomyelitis (OA), osteoporosis ... Myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenström's macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma , nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, inflammation-associated diffuse large B-cell lymphoma, ALK-positive large B-cell lymphoma The cancer may be a B cell-related cancer, including diffuse large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma occurring in HHV8-associated multicentric Castremann's disease, unclassified B cell lymphoma with features intermediate between diffuse large B cell lymphoma and Burkitt's lymphoma, unclassified B cell lymphoma with features intermediate between diffuse large B cell lymphoma and classical Hodgkin lymphoma, and other B cell-related lymphomas.

[0371] In some embodiments, there is provided a method of inhibiting tumor growth or progression in a subject having malignant cells expressing BCMA, comprising administering to a subject in need thereof an effective amount of a composition comprising a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate as described herein. In other embodiments, there is provided a method of inhibiting metastatic cells expressing BCMA in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate as described herein. In other embodiments, there is provided a method of inducing tumor regression in malignant cells in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate as described herein.

[0372] In some embodiments, there is provided a method of treating an autoimmune disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate described herein.

[0373] As used herein, autoimmune disorders include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus (type I), multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, Hashimoto's encephalopathy, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren's syndrome, acute disseminated encephalomyelitis, agammaglobulinemia, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, and rheumatoid arthritis. syndrome), antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Behçet's disease , Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, sweat gland abscess, idiopathic thrombocytopenic purpura, narcolepsy, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatic fever, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis.

[0374] In another aspect, the invention provides an effective amount of a composition (e.g., pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for treating a condition associated with BCMA expression (e.g., cancer or autoimmune disorder) in a subject in need thereof. In some embodiments, an effective amount of a composition (e.g., pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inhibiting tumor growth or progression in a subject having malignant cells expressing BCMA is provided. In some embodiments, an effective amount of a composition (e.g., pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inhibiting metastasis of malignant cells expressing BCMA in a subject in need thereof. In some embodiments, an effective amount of a composition (e.g., pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inducing tumor regression in a subject having malignant cells expressing BCMA is provided.

[0375] In another aspect, the invention provides an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for use in treating a condition associated with BCMA expression (e.g., cancer or an autoimmune disorder) in a subject in need thereof. In some embodiments, an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inhibiting tumor growth or progression in a subject having malignant cells that express BCMA is provided. In some embodiments, an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inhibiting metastasis of malignant cells that express BCMA in a subject in need thereof is provided. In some embodiments, an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein for inducing tumor regression in a subject having malignant cells that express BCMA is provided.

[0376] In another aspect, the invention provides the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein in the manufacture of a medicament for treating a condition associated with BCMA expression (e.g., cancer or an autoimmune disorder). In some embodiments, there is provided the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein in the manufacture of a medicament for inhibiting tumor growth or progression. In some embodiments, there is provided the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing BCMA. In some embodiments, there is provided the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein in the manufacture of a medicament for inducing tumor regression.

[0377] In another aspect, methods are provided for detecting, diagnosing, and / or monitoring conditions associated with BCMA expression. For example, the antibodies described herein (e.g., BCMA or CD3-BCMA bispecific) can be labeled with a detectable moiety, such as imaging agents and enzyme-substrate labels. The antibodies described herein can also be used in in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or staining reagents.

[0378] In some embodiments, the methods described herein further comprise treating the subject with an additional form of therapy, which in some embodiments is an additional anti-cancer therapy, including but not limited to chemotherapy, radiation, surgery, hormonal therapy, and / or additional immunotherapy.

[0379] In some embodiments, the additional form of therapy involves administering one or more therapeutic agents in addition to an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein. The one or more therapeutic agents include, but are not limited to, a second antibody (e.g., an anti-VEGF (vascular endothelial growth factor) antibody (e.g., AVASTIN®), an anti-HER2 antibody (e.g., HERCEPTIN®), an anti-CD25 antibody, an anti-CD33 antibody, an anti-CD20 antibody (e.g., RITUXAN®), an anti-mucin-like glycoprotein antibody, an anti-TNF antibody, and / or an epidermal growth factor receptor (EGFR) antibody (e.g., ERBITUX®). ), angiogenesis inhibitors, cytotoxic agents (e.g., anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone), taxanes (e.g., paclitaxel and docetaxel), dolastatins, duocarmycins, enediynes, geldanamycin, maytansine, puromycin, vinca alkaloids (e.g., vincristine), topoisomerase inhibitors (e.g., enantiomeric compounds), toposide), tubulysin, pyrimidine analogues (e.g., fluorouracil), platinum-containing agents (e.g., cisplatin, carboplatin, and oxaliplatin), alkylating agents (e.g., melphalan, cyclophosphamide, or carmustine), and hemiasterlin), immunomodulators (e.g., prednisone and lenalidomide (REVLIMID®)), anti-inflammatory agents (e.g., dexamethasone), aromatase inhibitors (e.g., anaphylaxis, The agent may be a chemotherapeutic agent including an anti-cancer drug such as brozole, exemestane, letrozole, vorozole, formestane, or testolactone), a proteasome inhibitor (e.g., bortezomib, such as VELCADE® ([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid, or carfilzomib), as well as other agents such as tamoxifen.

[0380] For example, in some embodiments, methods of treating multiple myeloma are provided that include administering to a patient in need thereof an effective amount of a composition comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein and one or more other therapeutic agents, such as a chemotherapeutic agent (e.g., doxorubicin or carfilzomib), or thalidomide or a derivative thereof (e.g., lenalidomide (REVLIMID®)). In some embodiments, the one or more other therapeutic agents are selected from the group consisting of bortezomib (e.g., VELCADE®), melphalan, prednisone, doxorubicin, lenalidomide, thalidomide, prednisone, carmustine, etoposide, cisplatin, cyclophosphamide, carfilzomib, and vincristine. In some embodiments, the other therapeutic agent is bortezomib (e.g., VELCADE®), melphalan, lenalidomide (REVLIMID®), carfilzomib, doxorubicin, or prednisone. Thus, provided are methods of treating multiple myeloma, comprising administering to a patient in need thereof an effective amount of a composition comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein and one or more other therapeutic agents selected from the group consisting of bortezomib, lenalidomide, carfilzomib, and doxorubicin. In some embodiments, the patient is relapsed or refractory to a previous multiple myeloma therapy.

[0381] The antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be administered to an individual via any suitable route. It should be understood by those skilled in the art that the examples described herein are not intended to be limiting, but rather illustrative of the techniques available. Thus, in some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate is administered to an individual in accordance with known methods, such as, for example, intravenous administration, such as a bolus, or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, intracranial, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, by insufflation, intrathecal, oral, inhalation, or via localized routes. Administration can be systemic, e.g., intravenous, or localized. Commercially available nebulizers for liquid formulations, including jet and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, or lyophilized powders can be nebulized after reconstitution. Alternatively, the antibody (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or can be inhaled as a lyophilized and milled powder.

[0382] In one embodiment, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate, or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0383] Various formulations of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be used for administration. In some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be administered as is. In some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate and pharmaceutically acceptable excipients can be present in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate administration of a pharmacologically active substance. For example, excipients can give form or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for various osmolalities, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy, 21st Edition, Mack Publishing, 2005.

[0384] In some embodiments, the agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Thus, the agents can be combined with a pharma- ceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, etc. The particular administration regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and that individual's medical history.

[0385] The antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates described herein can be administered using any suitable method, including by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). The antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can also be administered via inhalation, as described herein. In general, for administration of antibodies (e.g., BCMA or CD3-BCMA bispecific) and BCMA antibody conjugates, the initial candidate dosage may be about 2 mg / kg. For purposes of the present invention, a typical daily dosage may range from about 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors discussed above. For example, dosages of about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg, and about 25 mg / kg may be used. For repeated administration over several days or more, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or sufficient therapeutic levels are achieved, for example, to inhibit or delay tumor growth / progression or metastasis of cancer cells. An exemplary dosing regimen includes administration of an initial dose of about 2 mg / kg of antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate, followed by a weekly maintenance dose of about 1 mg / kg, or a biweekly maintenance dose of about 1 mg / kg thereafter. Other exemplary dosing regimens include administration of escalating doses (e.g., an initial dose of 1 mg / kg, followed by one or more escalations to higher doses every week or longer). Other dosing regimens may also be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, in some embodiments, dosing from one to four times per week is contemplated. In other embodiments, dosing once a month or once every other month or every three months is contemplated. The progress of this therapy is easily monitored by conventional techniques and assays. Dosing regimens, including the antibodies (e.g., BCMA or CD3-BCMA bispecifics used) or BCMA antibody conjugates, may be varied over time.

[0386] For purposes of the present invention, the appropriate dosage of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate will depend on the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate (or composition thereof) used, the type and severity of the condition being treated, whether the agent is administered for therapeutic purposes, previous therapy, the patient's medical history and response to the agent, the patient's clearance rate for the administered agent, and the discretion of the attending physician. Typically, the clinician will administer the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate until a dosage is reached that achieves the desired result. Dosage and / or frequency may vary over the course of treatment. Empirical considerations such as half-life will generally contribute to determining the dosage. For example, antibodies that are compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of symptoms, such as tumor growth inhibition or delay. Alternatively, a sustained continuous release formulation of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0387] In one embodiment, the dosage of the antibody (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate may be empirically determined in individuals who have been given one or more administrations of the antibody (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. Individuals are given increasing doses of the antibody (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. Indicators of disease may be employed to assess efficacy.

[0388] Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate according to the methods of the invention may be continuous or intermittent depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate may be essentially continuous over a preselected period of time, or may be a series of spaced doses.

[0389] In some embodiments, there may be more than one antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. There may be at least one, at least two, at least three, at least four, at least five different, or more, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates. In general, these antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates may have complementary activities that do not adversely affect each other. For example, one or more of the following antibodies may be used: a first BCMA or CD3 antibody directed against one epitope on BCMA or CD3 and a second BCMA or CD3 antibody directed against a different epitope on BCMA or CD3.

[0390] Therapeutic formulations of the antibodies (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates used in accordance with the invention are prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the antibody having the desired degree of purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington, The Science and Practice of Pharmacy, 21st ed., Mack Publishing, 2005). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, such as phosphate, citric acid, and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as blood. such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0391] Liposomes containing antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030, 1980; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0392] Active ingredient can also be encapsulated in microcapsules, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, prepared by coacervation techniques or by interfacial polymerization, respectively.Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005.

[0393] Sustained release preparations can be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0394] Preparations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0395] Compositions according to the invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation.

[0396] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical carriers, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it means that the active ingredient is evenly distributed throughout the composition, so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0397] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions which include a surfactant will conveniently contain between 0.05 and 5% surfactant, and may be between 0.1 and 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added if required.

[0398] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition, or alternatively it may be dissolved in an emulsion formed upon mixing of oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil), and phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) with water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5 and 20% oil. Fat emulsions may contain fat droplets between 0.1 and 1.0 μm, particularly between 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.

[0399] The emulsion composition may be prepared by mixing an antibody (e.g. BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol, and water).

[0400] Compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as set forth above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions, preferably in sterile pharma- ceutically acceptable solvents, can be nebulized by using a gas. Nebulized solutions may be breathed directly from the nebulizer, or the nebulizer may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0401] composition The compositions used in the methods of the invention comprise an effective amount of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein. Examples of such compositions and how to formulate them are also described in the previous section and below. In some embodiments, the compositions comprise one or more antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates. For example, the BCMA antibody or CD3-BCMA bispecific antibody recognizes human BCMA or CD3-BCMA. In some embodiments, the BCMA or CD3-BCMA antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the BCMA antibody or CD3-BCMA antibody comprises a constant region that can elicit a desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the BCMA antibody or CD3-BCMA antibody comprises a constant region that does not elicit an unwanted or undesired immune response, such as antibody-mediated lysis or ADCC.

[0402] It will be understood that a composition may comprise more than one type of antibody (e.g. BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate (e.g. a mixture of BCMA antibodies or CD3-BCMA bispecific antibodies that recognize different epitopes of BCMA or CD3 and BCMA). Other exemplary compositions comprise more than one BCMA antibody, CD3-BCMA antibody, or BCMA antibody conjugate that recognizes the same epitope, or different species of BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate that bind to different epitopes of BCMA (e.g. human BCMA) or CD3 and BCMA (human CD3 and BCMA).

[0403] The compositions used in the present invention may further comprise a pharma- ceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington: The Science and practice of Pharmacy, 21st ed., 2005, Lippincott Williams and Wilkins, ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the appropriate doses and concentrations, and include buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, Such as gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0404] kit The present invention also provides kits for use in the methods. The kits of the invention include one or more containers containing a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate as described herein, and instructions for use according to any of the methods of the invention described herein. Typically, the instructions include a description of the administration of the BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate for the therapeutic treatment described above.

[0405] Instructions for use of the BCMA antibodies, CD3-BCMA bispecific antibodies, or BCMA antibody conjugates described herein will generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided in kits of the invention will typically be written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions contained on a magnetic or optical storage disk) are also acceptable.

[0406] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices such as mini-pumps, etc. are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate. The container may further comprise a second medicament active agent.

[0407] The kit may provide additional components, such as buffers, interpretive information, etc. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0408] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES

[0409] Example 1 Determining the kinetics and affinity of the hBCMA / human IgG interaction at 25°C and / or 37°C This example determines the kinetics and affinity of various anti-BCMA antibodies at 25°C and 37°C.

[0410] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). Arrays of anti-BCMA antibodies were prepared using the amine coupling method on a Bio-Rad GLC sensor chip similar to that described in Abdiche et al., Anal. Biochem., 411, 139-151 (2011). The analysis temperature for immobilization was 25 °C and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) direction by injecting a mixture of 1 mM ECD and 0.25 mM NHS at a flow rate of 30 μL / min for 3 min. IgG was immobilized on the activated spots by injecting them at 20 μg / mL in 10 mM acetate pH 4.5 buffer at 30 μL / min for 1.5 min in the ligand (perpendicular) direction. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5 at 30 μL / min for 3 min in the analyte direction.

[0411] The analysis temperature for hBCMA binding analysis was 37 °C or 25 °C in a running buffer of HBS-T+ supplemented with 1 mg / mL BSA. A kinetic titration method was used for the interaction analysis as described in Abdiche et al. The hBCMA (human BCMA) analyte was injected in the analyte direction using a series of injections from low to high concentrations. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM (a 5-member series with a 5-fold dilution factor and a top concentration of 50 nM). The association time for a given analyte dilution was 2 min. Immediately after the 50 nM hBCMA injection, dissociation was monitored for 2 h. Prior to hBCMA analyte injection, buffer was injected five times using the same association and dissociation times in the hBCMA analyte cycle to prepare a buffer blank sensorgram for double referencing purposes (double referencing as described in Myszka, J. Mol. Recognit., 12, 279-284 (1999)).

[0412] Sensorgrams were double referenced and fitted to a 1:1 Langmuir using a mass transport kinetic titration model in BIAevaluation software version 4.1.1 (GE Lifesciences, Piscataway, NJ). The sensorgrams and fits are shown in Figure 1, and the kinetic and affinity parameters of various anti-BCMA antibodies of the invention are shown in Tables 6A-6C.

[0413] [Table 6]

[0414] [Table 7-1]

[0415] [Table 7-2]

[0416] [Table 7-3]

[0417] [Table 7-4]

[0418] [Table 8-1]

[0419] [Table 8-2]

[0420] Example 2 Flow cytometry of human anti-BCMA antibodies on BCMA-positive tumor cells This example demonstrates binding of BCMA positive tumor cells by various BCMA antibodies of the invention.

[0421] Binding of human anti-hBCMA expressed in mouse IgG2a was assessed on BCMA expressing cells (KMS12BM, L363, MM1S, and KMS12PE) by flow cytometry. 250,000 cells were incubated with 0.5 μg of antibody in 100 μL of binding buffer (PBS (phosphate buffered solution) + 0.2% BSA (bovine serum albumin)) followed by incubation with Alex Fluor647 conjugated anti-mouse IgG (Biolegend). Table 7 shows the MFI (mean fluorescence intensity) on BCMA positive tumor cells by various BCMA antibodies (e.g., Combo_Rd4_0.6nM_C29, A02_Rd4_6nM_C01, A02_Rd4_6nM_C16, and P6E01 / H3TAQ).

[0422] [Table 9]

[0423] Example 3 Cytotoxicity of anti-BCMA ADCs in BCMA-positive cells This example illustrates the efficacy of anti-BCMA ADCs in BCMA positive cells.

[0424] Human anti-BCMA (L3.PY / P6E01, L3.PY / H3.TAQ, Combo_Rd4_0.6nM_C29, A02_Rd4_6nM_C01, and A02_Rd4_6nM_C16) antibodies were expressed as human IgG1 subtype engineered with a glutamine-containing transglutaminase ("Q") tag for drug-antibody ratios (DAR) of 2, 4, and 6 (e.g., LCQ05, H7c, N297A, N297Q, N297A / H7c, N297Q / LCQ05). TG17 corresponds to SEQ ID NO: 472 (LLQGPP), LCQ05 corresponds to SEQ ID NO: 474 (GGLLQGPP), and H7c corresponds to SEQ ID NO: 454 (LLQG), respectively, conjugated with AcLys-Val-Cit-PABC-Aur0101 (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Aur3377, or amino-PEG6-C2-Aur0131 as shown in Table 8. In one case, the transglutaminase tag can be engineered into the light chain, the heavy chain, or a combination of the light and heavy chains. In other cases, the transglutaminase tag (e.g., Q) is engineered into a site of the antibody, such as position 297 of human IgG (EU numbering scheme). For example, the wild type amino acid asparagine (N) is replaced with glutamine or alanine at position 297 of the BCMA antibody of the invention (N297Q or N297A). Anti-BCMA antibody conjugation to Aur0101, Aur3377, and Aur0131 was then achieved via a microbial transglutaminase-catalyzed transamidation reaction between an anti-BCMA antibody bearing a glutamine or glutamine tag targeted at a specific site (e.g., at the carboxyl or amino terminus of the heavy or light chain, at position 297, or at another site on the antibody) and an amine-containing derivative of the payload (e.g., MMAD, Aur0101, Aur3377, or Aur0131). In some cases, the wild type amino acid lysine at positions 222, 340, or 370 (according to the EU numbering scheme) was replaced with the amino acid arginine ("K222R", "K340R", or "K370R").For example, the K222R substitution was found to have the unexpected effect of resulting in more homogeneous antibody and payload conjugates, better intermolecular cross-linking between antibody and payload, and / or significantly reduced interchain cross-linking with the glutamine tag on the C-terminus of the antibody light chain.

[0425] In the transamidation reaction, glutamine on the antibody acted as the acyl donor and an amine-containing compound acted as the acyl acceptor (amine donor). Purified anti-BCMA antibodies at concentrations of 1–150 μM were incubated with a 5–100 molar excess of acyl acceptor ranging between 5 μM and 15 mM in the presence of 0.23–0.55% (w / v) Streptoverticillium mobaraense transglutaminase (ACTIVA™, Ajinomoto, Japan) in 10–1000 mM NaCl, and 25 mM MES, HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] or Tris HCl buffers with a pH range of 6.2–8.8. Reaction conditions were adjusted for each individual acyl acceptor derivative, with optimal efficiency and specificity typically observed at 33 μM antibody, 0.67 mM derivative, and 0.378% (w / v) transglutaminase in 75 mM NaCl, 25 mM Tris HCl, pH 8.5. After incubation for 1-24 hours at 20-37°C, antibodies were purified on Butyl Sepharose High Performance (Butyl HP) resin (GE Healthcare, Waukesha, WI) using standard chromatographic methods known to those skilled in the art, such as commercially available hydrophobic interaction chromatography from GE Healthcare.

[0426] Target-expressing (MM1.S, KMS12BM, and L363) cells were then seeded in clear bottom plates at 3000 cells / well. Cells were treated in triplicate with 4-fold serially diluted antibody-drug conjugates. Cell viability was determined 96 hours after treatment by CellTiter-Glo® Luminescent Cell Viability Assay 96 (Promega, Madison WI). Relative cell viability was determined as a percentage of untreated control. EC50 was calculated by Prism software. Table 8 shows that all human anti-BCMA antibodies of the invention conjugated to the cytotoxic agents 0101, 3377, and 0131 by transglutaminase tag and linker exert potent cell killing activity in BCMA expressing cells.

[0427] [Table 10]

[0428] Example 4 Anti-BCMA ADCs induce tumor regression in orthotopic multiple myeloma models This example illustrates the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model.

[0429] An in vivo efficacy study of the BCMA ADC was performed using the multiple myeloma cell line MM1.S, which expresses luciferase and GFP (green fluorescent protein) in an orthotopic model. Ten million MM1.S LucGFP cells were injected intravenously through the tail vein into 6-8 week old female CB17 / SCID animals. D-Luciferin (Regis Technologies, Morton Grove, IL) was injected intraperitoneally (200 μL per animal at 15 mg / mL) followed by anesthesia with isofluorane, followed by whole body bioluminescence imaging (BLI) to allow monitoring of whole body tumor burden. Bioluminescent signals emitted by the interaction of luciferase and luciferin expressed by tumor cells were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When total flux reached an average of 1-3E6 for all animals, animals were randomized into groups and administered a single dose of human anti-BCMA antibody conjugated at the C-terminus of the antibody light chain with LCQ05 / K222R-vc0101 and control conjugates by bolus tail vein injection. Animals were sacrificed when they exhibited hind limb paralysis, an endpoint of the MM1.S orthotopic model. FIG. 2 shows that a single dose of various human anti-BCMA ADCs at 3 mg / kg inhibits tumor progression compared to a negative control (NNC), including P6E01 / P6E01-AcLys-Val-Cit-PABC-Aur0101; P5A2_VHVL-AcLys-Val-Cit-PABC-Aur0101; P5C1_VHVL-AcLys-Val-Cit-PABC-Aur0101; P4G4-AcLys-Val-Cit-PABC-Aur0101; and P1A11-AcLys-Val-Cit-PABC-Aur0101.

[0430] This study demonstrates that treatment with BCMA-ADC inhibits the progression of multiple myeloma.

[0431] Example 5 Anti-BCMA ADCs induce tumor regression and inhibition in orthotopic multiple myeloma models This example also illustrates the in vivo efficacy of anti-BCMA ADCs in the MM1.S orthotopic multiple myeloma model.

[0432] An in vivo efficacy study of the BCMA ADC was performed using the multiple myeloma cell line MM1.S expressing luciferase and GFP in an orthotopic model. Ten million MM1.S LucGFP cells were injected intravenously through the tail vein into 6-8 week old female CB17 / SCID animals. D-luciferin (Regis Technologies, Morton Grove, IL) was injected intraperitoneally (200 μL per animal at 15 mg / mL) followed by anesthesia with isofluorane, followed by whole body bioluminescence imaging (BLI) to allow monitoring of whole body tumor burden. Bioluminescent signals emitted by the interaction of luciferase and luciferin expressed by tumor cells were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When total flux reached an average of 1-3E6 for all animals, animals were randomized into groups; 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugates were administered by bolus tail vein injection. Animals were sacrificed when they exhibited hind limb paralysis, an endpoint of the MM1.S orthotopic model. Figure 3 shows that a single dose of 1) H7c / N297A / K222R-amino-PEG6-C2-0131 and 2) human anti-BCMA L3.PY / P6E01 antibody conjugated to H7c / N297A / K222R-amino-PEG6-C2-3377 resulted in tumor regression.Single doses of 1) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 2) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, and 3) human anti-BCMA L3.PY / P6E01 antibody conjugated with N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101 resulted in tumor inhibition.

[0433] Thus, this study demonstrates that treatment with a BCMA-ADC induces regression and inhibits progression of multiple myeloma.

[0434] Example 6 Anti-BCMA ADCs induce tumor inhibition in orthotopic multiple myeloma models This example also illustrates the in vivo efficacy of anti-BCMA ADCs in the KMS12BM orthotopic multiple myeloma model.

[0435] An in vivo efficacy study of the BCMA ADC was performed using the multiple myeloma cell line KMS12BM expressing luciferase and GFP in an orthotopic model. Female NSG animals aged 6-8 weeks were irradiated with 100 cGy and 24 hours after irradiation, 10 million KMS12BM LucGFP cells were injected intravenously via the tail vein. D-Luciferin (Regis Technologies, Morton Grove, IL) was injected intraperitoneally (200 μL per animal at 15 mg / mL) followed by anesthesia with isofluorane and subsequent whole-body bioluminescence imaging (BLI) to allow monitoring of whole-body tumor burden. Bioluminescence signals emitted by the interaction of luciferase and luciferin expressed by tumor cells were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 5E6 for all animals, animals were randomized into groups; 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur010, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugates was administered by bolus tail vein injection. Animals were sacrificed when they lost more than 15% of their total body weight, the endpoint of the KMS12BM orthotopic model.FIG. 4 shows that a single dose of 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) human anti-BCMA L3.PY / P6E01 antibodies conjugated to N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101 resulted in tumor inhibition.

[0436] Thus, this study further demonstrates that treatment with BCMA-ADC induces regression and inhibits progression of multiple myeloma.

[0437] Example 7 Dose-response curves for anti-BCMA ADCs in the MM1S orthotopic model This example further illustrates the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model.

[0438] An in vivo efficacy study of the BCMA ADC was performed using the multiple myeloma cell line MM1.S expressing luciferase and GFP in an orthotopic model. Ten million MM1.S LucGFP cells were injected intravenously through the tail vein into 6-8 week old female CB17 / SCID animals. D-luciferin (Regis Technologies, Morton Grove, IL) was injected intraperitoneally (200 μL per animal at 15 mg / mL) followed by anesthesia with isofluorane, followed by whole body bioluminescence imaging (BLI) to allow monitoring of whole body tumor burden. Bioluminescent signals emitted by the interaction of luciferase and luciferin expressed by tumor cells were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 1.2E6 for all animals, animals were randomized into groups; 1) 0.1 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 2) 0.38 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 3) 0.75 mg / kg H7c / N297A / K222-amino-PEG6-C2-0131, 4) 1.5 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) 3 mg / kg control conjugate N297Q / K222R-AcLys-VC-0101. A single dose of human anti-BCMA ADC and control conjugate was administered by bolus tail vein injection. Animals were sacrificed when they exhibited hind limb paralysis, an endpoint of the MM1.S orthotopic model. Figure 5 shows that a single dose of human anti-BCMA COMBO_Rd4_0.6nM_C29 antibody conjugated to groups 1)-4) above resulted in tumor regression starting at 0.1 mg / kg and tumor inhibition up to 100 days starting at 0.75 mg / kg.

[0439] Thus, this study demonstrates that treatment with BCMA-ADC induces tumor regression and tumor inhibition in multiple myeloma.

[0440] Example 8 Generation and purification of heterodimeric antibodies This example describes the production and purification of the heterodimeric antibody of the present application.

[0441] The variable regions of a human-specific anti-CD3 antibody were cloned into human IgG1 or IgG2ΔA containing the following mutations 221R, 228R, and K409R; or 223R, 225R, 228R, and K409R, respectively, and designated hIgG1 RRR or IgG2ΔA-RRRR.

[0442] The variable regions of the anti-target antibodies were cloned into human IgG1 or IgG2ΔA containing the following mutations 221E, 228E, L368E or 223E, 225E, 228E, and L368E, respectively, and designated hIgG1EEE or hIgG2ΔA-EEEE.

[0443] Heterodimers were prepared by incubating anti-CD3 IgG1 or IgG2ΔA carrying hIgG1 RRR or IgG2ΔA-RRRR mutations with anti-target antibodies carrying hIgG1EEE or hIgG2ΔA-EEEE mutations in PBS containing 1 mM or 2 mM GSH for 24 hours at 37° C. as described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545). Heterodimers were purified by ion exchange chromatography as described below.

[0444] All heterodimers were purified by ion exchange chromatography. Briefly, analytical ion exchange separation of Fc-hetero- and Fc-homodimers was performed on an Agilent 1100 Quaternary Pump LC system (Agilent Inc, Santa Clara, CA, USA) equipped with a weak cation exchange DIONEX Propac WCX-10G (4 × 50 mm) column. Proteins in 5% buffer A (20 mM MES pH 5.4) were injected and eluted with a gradient of 25% to 75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a period of 20 min at a flow rate of 1 ml / min. Larger scale Fc-heterodimer purification was performed on an Akta Explorer (GE) equipped with a weak cation exchange DIONEX Propac WCX-10G (4 × 250 mm) column. Proteins in 5% buffer A (20 mM MES pH 5.4) were injected and eluted with a gradient of 15% to 75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a period of 60 min at a flow rate of 1 ml / min.

[0445] Example 9 Determination of the kinetics and affinity of the hCD3 / human IgG interaction at 25°C and / or 37°C This example determines the kinetics and affinity of various anti-CD3 antibodies at 25°C and 37°C.

[0446] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). Arrays of anti-CD3 antibodies were prepared using the amine coupling method on a Bio-Rad GLC sensor chip similar to that described in Abdiche et al., Anal. Biochem., 411, 139-151 (2011). The analysis temperature for immobilization was 25 °C and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) direction by injecting a mixture of 1 mM ECD and 0.25 mM NHS at a flow rate of 30 μL / min for 3 min. IgG was immobilized on the activated spots by injecting them at 20 μg / mL in 10 mM acetate pH 4.5 buffer at 30 μg / mL for 1.5 min in the ligand (perpendicular) direction. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5 at 30 μL / min for 3 min in the analyte direction.

[0447] The analysis temperature for hCD3 binding analysis was 37 °C or 25 °C in a running buffer of HBS-T+ supplemented with 1 mg / mL BSA. A kinetic titration method was used for the interaction analysis as described by Abdiche et al. The hCD3 (human CD3) analyte was injected in the analyte direction using a series of injections from low to high concentrations. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM (a 5-member series with a 5-fold dilution factor and a top concentration of 50 nM). The association time for a given analyte dilution was 2 min. Immediately after 50 nM hCD3 injection, dissociation was monitored for 2 h. Prior to the hCD3 analyte injection, buffer was injected five times using the same association and dissociation times in the hCD3 analyte cycle to prepare a buffer blank sensorgram for double referencing purposes (double referencing as described in Myszka, J. Mol. Recognit., 12, 279-284 (1999)).

[0448] Sensorgrams were double referenced and fitted to a 1:1 Langmuir using a mass transport kinetic titration model in BIAevaluation software version 4.1.1 (GE Lifesciences, Piscataway, NJ). Kinetic and affinity parameters of various anti-CD3 antibodies of the invention are shown in Table 9.

[0449] [Table 11]

[0450] Example 10 Flow cytometry of human anti-CD3 bispecific antibodies on B cells and CD8+ T cells This example demonstrates the efficacy of an anti-CD3-anti-CD20 bispecific antibody on CD20+ cells.

[0451] Cynomolgus monkey studies were performed at Charles River Laboratories, Preclinical Services Nevada in accordance with the Institutional Animal Care and Use Committee. Animals (n=2) were dosed via intravenous bolus injection with bispecific anti-CD20 / h2B4 antibodies at doses of 500 μg / kg, 100 μg / kg, 20 μg / kg, 2 μg / kg, 0.2 μg / kg, or 0.02 μg / kg. Animals were observed twice daily and at each blood collection time point. Blood for flow cytometry and cytokine analysis was collected into K2EDTA tubes from a peripheral vein not used for iv dosing.

[0452] Efficacy was determined by measuring B and T cells in peripheral blood by flow cytometry. Whole blood was collected at the indicated time points and kept at 4°C until analysis. Red blood cells were lysed with ACK buffer (Gibco) for 5 min at room temperature and white blood cells were pelleted by centrifugation. Cells were stained with a cocktail containing fluorescently labeled antibodies recognizing cynomolgus CD19 (Beckman Coulter), CD45, CD4, CD8, Ki67 (BD Biosciences) in PBS+2% FBS for 1 h at 4°C. For Ki67 analysis, cells were first stained with CD4 and CD8, then intracellularly stained for Ki67 after fixation / permeabilization with BD cyotfix / cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Acquisition of cells on a BD LSRII flow cytometer was performed immediately after staining.

[0453] The resulting B cell numbers are graphed in Figures 6A-6F as a percentage of pre-study B cell numbers. Long-term B cell depletion after a single dose was achieved at doses as low as 2 μg / kg. B cell depletion was seen at all doses. The duration of the depleting effect was dose-dependent.

[0454] The resulting CD8+ T cell numbers are graphed as a percentage of pre-study CD8+ T cell numbers in Figures 7A-7F. After the initial relocalization, T cell levels recovered to or above baseline levels for the duration of the study.

[0455] Example 11 Flow cytometry of human anti-CD3 bispecific antibodies on CD8+ T cells This example demonstrates the efficacy of monovalent anti-CD3 antibodies on T cell trafficking and activation.

[0456] Cynomolgus monkey studies were performed as described in Example 10, and efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Cynomolgus monkeys (n=2) were dosed weekly iv with anti-CD20 / h2B4 or NNC (non-specific antibody) / h2B4 at 0.2 μg / kg. In contrast to CD20-targeting bispecific antibodies, NNC / h2B4 has little to no effect on CD8+ T cell dynamics in blood as measured by flow cytometry. Ki67 was used as a marker of T cell activation.

[0457] The resulting T cell counts are graphed in Figures 8A-8B as a percentage of pre-study CD8+ T cell counts. In cynomolgus monkeys dosed with CD20 / h2B4 bispecific antibody, Ki67+ T cells increased and peaked between days 3 and 7 post-dosing, indicating T cell activation. However, in cynomolgus monkeys dosed with NNC / h2B4, there was no increase in Ki67+ T cells.

[0458] Example 12 Flow cytometry of human anti-CD3 bispecific antibodies on B cells This example demonstrates the effect of anti-CD3 arm affinity on B cell depletion.

[0459] Cynomolgus monkey studies were conducted as described in Example 10, and efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Bispecific antibodies were made using an anti-CD20 arm paired with four anti-CD3 antibody arms with different affinities. Efficacy was determined by measuring B cells in peripheral blood by flow cytometry after a single iv dose of 0.2 μg / kg.

[0460] 9A-9D, the resulting B cell numbers are graphed as a percentage of pre-study B cell numbers. The efficacy of B cell depletion correlates with anti-CD3 arm affinity.

[0461] Example 13 In vitro study of bispecific antibodies for T cell-mediated killing of BCMA-positive cells This example illustrates the in vitro cytotoxicity of anti-BCMA / CD3 hIgG2ΔA bispecific in BCMA positive cells.

[0462] Human anti-BCMA (P5A2, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C16, P5C1, C01_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C22, Combo_Rd4_0.6nM_C29, L3PY / H3TAQ, and A02_Rd4_6nM_C01), and human anti-CD3 (H2B4) antibodies were expressed as human IgG2dA engineered with EEEE for bispecific exchange as described in Example 8.

[0463] CD3+ T cells from PBMC were negatively selected using Pan T Cell Isolation kit, human (Miltenyi, San Diego, CA). Target-expressing (KMS12PE, L363, and Molp8) cells and CD3+ T cells were seeded in clear U-bottom plates at 20000 and 100000 cells / well, respectively. Cells were treated in triplicate with 10-fold serially diluted bispecific antibodies. Cell death was determined 20 hours after treatment by CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison WI). Cell cytotoxicity was determined as a percentage of untreated effector+target control wells. EC50 was calculated by Prism software. Table 10 shows that all human anti-BCMA_H2B4 bispecific antibodies exert cell killing activity in BCMA-expressing cells.

[0464] [Table 12]

[0465] Example 14 In vitro characterization of mouse hybridoma cloned anti-CD3 antibodies. This example illustrates in vitro T cell activation / proliferation of anti-CD3 cloned from a mouse hybridoma in human / cynomolgus PBMC cells for antibody screening.

[0466] Human anti-CD3 antibodies were cloned from immunized mice, expressed as mouse IgG1, and purified by protein A affinity beads. Human / cynomolgus peripheral blood mononuclear cells (hu / cyPBMCs) were prepared by Ficoll (density: 1.083 g / mL, GE) density gradient centrifugation from blood filters obtained from a local blood bank. Red blood cells were diluted with LCK buffer (155 mM NH 4 Cl, 10 mM KHCO 3 The PBMCs were then removed by incubation in 100 mM EDTA (Gibco) for 3 min at room temperature. The cells were centrifuged at 600 g for 5 min. The supernatant containing lysed red blood cells was discarded and the PBMCs were washed twice with 50 ml of 1x PBS / 1% BSA / 1 mM EDTA. The pelleted cells were cultured at 10 per ml in culture medium, X-VIVO-15, serum-free medium (Lonza). 7 Adjust the cells and plate PBMCs at 10 per well in a round-bottom 96-well tissue culture plate. 6 (100 μl). Selected Abs were serially diluted 10× from 1...

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to B-cell maturation antigen (BCMA), wherein the antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising SEQ ID NO: 150, 151, or 152, (ii) a VH CDR2 comprising SEQ ID NO: 153 or 154, and (iii) a VH CDR3 comprising SEQ ID NO: 155; and a light chain variable (VL) region comprising: (i) a VL CDR1 comprising SEQ ID NO: 209, (ii) a VL CDR2 comprising SEQ ID NO: 221, and (iii) a VL CDR3 comprising SEQ ID NO:

222.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a VH region comprising SEQ ID NO: 33 and a VL region comprising SEQ ID NO:

34.

3. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in claim 1 or 2, and a pharma- ceutically acceptable carrier.

4. 3. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

5. A vector comprising the polynucleotide of claim 4.

6. 3. An isolated host cell recombinantly producing the antibody or antigen-binding fragment of claim 1 or 2.

7. A method for producing an antibody or antigen-binding fragment, comprising culturing a host cell according to claim 6 under conditions resulting in the production of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cell or culture.

8. A treatment for a condition associated with cells expressing BCMA in a subject, comprising an effective amount of an antibody or antigen-binding fragment thereof described in claim 1 or 2, or a pharmaceutical composition described in claim 3.

9. The method of claim 8, wherein the condition is cancer.

10. Cancers include multiple myeloma, malignant plasma cell neoplasms, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), follicular lymphoma, and myelomatosis. lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenström's macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma 10. The method of claim 9, wherein the cancer is a B cell-related cancer selected from the group consisting of: intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell / histiocyte-rich large B cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B cell lymphoma (leg type), EBV-positive diffuse large B cell lymphoma of the elderly, diffuse large B cell lymphoma associated with inflammation, ALK-positive large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma arising in HHV8-associated multicentric Castremann's disease, unclassified B cell lymphoma with characteristics intermediate between diffuse large B cell lymphoma and Burkitt lymphoma, unclassified B cell lymphoma with characteristics intermediate between diffuse large B cell lymphoma and classical Hodgkin lymphoma, and other B cell-related lymphomas.

11. An inhibitor of tumor growth or progression in a subject having malignant cells expressing BCMA, comprising an effective amount of an antibody or antigen-binding fragment thereof described in claim 1 or 2, or a pharmaceutical composition described in claim 3.

12. An inhibitor of metastasis of malignant cells expressing BCMA in a subject, comprising an effective amount of an antibody or antigen-binding fragment thereof described in claim 1 or 2, or a pharmaceutical composition described in claim 3.

13. An agent for inducing tumor regression in a subject having malignant cells expressing BCMA, comprising an effective amount of an antibody or antigen-binding fragment thereof described in claim 1 or 2, or a pharmaceutical composition described in claim 3.

14. 9. The method of claim 8, wherein the condition is an autoimmune disorder.

15. The method of claim 14, wherein the autoimmune disorder is systemic lupus erythematosus or rheumatoid arthritis.

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