Therapeutic antibodies and their uses

Abstract

To provide treatment of multiple myeloma, such as using an anti-BCMA antagonist including antibodies and other immunotherapeutic agents.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 / or CD3 (Cluster of Differentiation 3). The invention also relates to antibody 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 that specifically bind to BCMA (B cell maturation antigen) and / or CD3 (cluster of differentiation 3), such as full-length antibodies or antigen-binding fragments thereof. 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 diseases), compositions comprising BCMA antibodies, and methods of using BCMA antibodies and these conjugates. The present invention further relates to hetero-multimeric antibodies that specifically bind to CD3 and tumor cell antigens (e.g., bispecific antibodies that specifically bind to CD3 and BCMA). Compositions comprising such hetero-multimeric antibodies, methods for generating and purifying such hetero-multimeric antibodies, and their use in diagnostic and therapeutic agents are also provided.

Background Art

[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. Its gene is selectively expressed in the B cell lineage and is most highly expressed in plasmablasts and plasma cells, antibody-secreting cells. BCMA binds two ligands, B cell activating factor (BAFF) (also called B lymphocyte stimulator (BLyS) and APRIL-related leukocyte-expressed ligand (TALL-1)) and a proliferation-inducing ligand (APRIL), with affinities of 1 μM and 16 nM, respectively. When APRIL or BAFF binds to BCMA, a signaling cascade involving NF-κB, Elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase is promoted, thereby generating signals for cell survival and proliferation.

[0003] BCMA is expressed in malignant B cells, as well as in several cancers with B lymphocytes, including multiple myeloma, plasmacytoma, Hodgkin lymphoma, and chronic lymphocytic leukemia. In autoimmune diseases involving blasts, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, BCMA-expressing antibody-producing cells secrete autoantibodies that attack themselves.

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the case of multiple myeloma, approximately 24,000 new cases are newly diagnosed each year in the United States, accounting for about 15% of newly diagnosed blood cancers in the United States. On average, 11,000 deaths occur each year from multiple myeloma, the average 5-year survival rate is about 44%, and the median survival period is 50-55 months. Current treatments for multiple myeloma focus on plasma cell apoptosis and / or reduced 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, and almost all patients develop resistance to these agents and ultimately relapse. Therefore, alternative treatments for multiple myeloma, such as the use of anti-BCMA antagonists, including antibodies and other immunotherapeutic agents (e.g., bispecific antibodies or antibody-drug conjugates), should provide excellent therapeutic agents.

Means for Solving the Problems

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

[0006] In one aspect, the present invention is an isolated antibody or antigen-binding fragment thereof that specifically binds to B cell maturation antigen (BCMA), wherein (a) (i) the sequence SYX 1 MX 2 (wherein 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 (wherein X 1 is G or S, X 2 is A or P, X 3 is T, N, or S) (SEQ ID NO: 303) comprising heavy chain variable (VH) complementarity determining region 1 (CDR1), (ii) the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (wherein X 1 is I, V, T, H, L, A, or C, X 2 is S, D, G, T, I, L, F, M, or V, X 3 is G, Y, L, H, D, A, S, or M, X 4 is S, Q, T, A, F, or W, X 5 is G or T, X 6 is N, S, P, Y, W, or F, X 7 is S, T, I, L, T, A, R, V, K, G, or C, X 8 is F, Y, P, W, H, or G, X 9 is V, R, or L, X 10 is G or T) (SEQ ID NO: 305), or X 1 X 2 X 3 X 4 X 5 X6 (wherein 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) the array VSPIX 1 X 2 X 3 (wherein X 1 is 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 (wherein X 1 is D, S, T, or A, and X 2 is I, S, L, P, or D)(SEQ ID NO: 308), including a VH region including a VH CDR3, and / or (b)(i) the array X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 (wherein 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, X 10 is Y or F, X 11 is L, W, or P, X 12 contains a light chain variable (VL) CDR1 that is (i) ASX (SEQ ID NO: 309), where X 1 ASX 2 RAX 3 (where X 1 is G or D, X 2 is S or I, X 3 is T or P (SEQ ID NO: 310), and (iii) a VL CDR2 that contains 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, X 2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C, X 3 is W, F, or S, X 4 is L or I (SEQ ID NO: 311), or QQYX 1 X 2 X 3 PX 4 (where X 1 is G, Q, E, L, F, A, S, M, R, K, or Y, X 2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y, X 3 is W, S, or F, X 4 is L or I (SEQ ID NO: 312), and provides an isolated antibody or antigen-binding fragment thereof that contains a VL region that contains a VL CDR3.

[0007] In another aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to BCMA, comprising a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence 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, 125, 127, 313, 314, 363, or 365, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in 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, 316, or 364, provides an isolated antibody or antigen-binding fragment thereof.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 CDR3 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 shown in SEQ ID NO: 112, or a variant having one or several conservative amino acid substitutions in residues not within the CDRs, and / or the VL region comprises the amino acid sequence shown in SEQ ID NO: 38, or a variant thereof having one or several amino acid substitutions in amino acids not within the CDRs. In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 357 and a heavy chain comprising the sequence shown in SEQ ID NO: 358. In some embodiments, the antibody comprises a VH region produced by an expression vector having ATCC accession number PTA-122094. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC accession number PTA-122093.

[0008] In another aspect, the present invention provides an isolated antibody comprising an acyl donor glutamine-containing tag engineered at a specific site of the BCMA antibody of the present invention. In some embodiments, the tag comprises an amino acid sequence 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 present 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 present invention. In some embodiments, the amino acid modification is a substitution from lysine to arginine.

[0010] In some embodiments, the BCMA antibody of the present invention further comprises a linker. In some embodiments, the linker is selected from the group consisting of 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 present 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, a contrast agent, a therapeutic protein, a biopolymer, and an oligonucleotide. In some embodiments, the agent is a cytotoxic agent including, but not limited to, anthracycline, auristatin, camptothecin, combretastatin, dolastatin, duocarmycin, enediyne, geldanamycin, indolobenzodiazepine dimer, maytansine, puromycin, pyrrolobenzodiazepine dimer, taxane, vinca alkaloid, tubulysin, hemiasterlin, spicamycin, pladienolide, and stereoisomers, isoters, analogs, or derivatives thereof.For example, 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-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-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 present 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 sequences 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, provided is a method of generating a BCMA antibody 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 present invention provides the use of a BCMA antibody or BCMA antibody conjugate described herein in the manufacture of a medicament for treating a condition associated with BCMA expression (e.g., cancer or autoimmune disorder). In some embodiments, the use of a BCMA antibody or BCMA antibody conjugate described herein in the manufacture of a medicament for inhibiting tumor growth or progression is provided. In some embodiments, the use of a BCMA antibody or BCMA antibody conjugate described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing BCMA is provided. In some embodiments, the use of a BCMA antibody or BCMA antibody conjugate described herein in the manufacture of a medicament for inducing tumor regression is provided.

[0015] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising a VH region that includes VH CDR1, VH CDR2, and VH CDR3 of the VH sequences 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, and / or a VL region that includes VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequences 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 antibody comprises a VH region that includes VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388, and / or a VL region that includes VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (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 shown 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 shown 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 CDR3 comprising the sequence shown 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 shown in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341, 433, 452, or 436, and (iii) a VL CDR3 comprising the sequence shown 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 shown in SEQ ID NO: 331, 332, 333, 401, 407, or 408, (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336, 404, 405, or 417, and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335 or 406, and / or the VL region comprises (i) a light chain variable (VL) CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441, (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or 439. In some embodiments, the antibody comprises a VH region produced by an expression vector having ATCC accession number PTA-122513. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC accession number PTA-122512.

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

[0017] In another aspect, the present invention provides a bispecific antibody that is a full-length human antibody comprising a first antibody variable domain of the bispecific antibody capable of mobilizing 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 bispecific antibody capable of specifically binding to a target antigen, wherein the first antibody variable domain comprises a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable (VH) sequence shown 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 region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence shown 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 first antibody variable domain comprises a VH region comprising (i) a VH CDR1 of the heavy chain variable (VH) sequence shown 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 shown 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 CDR3 comprising the sequence shown in SEQ ID NO: 335, 406, 423, 429, or 451, and / or (i) a light chain variable (VL) CDR1 comprising the sequence shown in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341, 433, 452, or 436, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453. In some embodiments, the first antibody variable domain comprises a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable (VH) sequence shown in SEQ ID NO: 324 or 388, and / or a VL region comprising a VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence shown in SEQ ID NO: 323 or 387, and the second antibody variable domain comprises a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable (VH) sequence shown in SEQ ID NO: 112, and / or a VL region comprising a VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence shown in SEQ ID NO: 38.

[0018] In some embodiments, the second antibody variable domain comprises (a) (i) the sequence SYX 1 MX 2 (wherein 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 (wherein X 1 is G or S, and X 2 is A or P, and X 3 is T, N, or S)(SEQ ID NO: 303) and includes a heavy chain variable (VH) complementarity determining region 1 (CDR1), (ii) the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (wherein 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 (wherein 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 6a VH CDR2 containing N, S, P, Y, or W (SEQ ID NO: 306), and (iii) the sequence VSPIX 1 X 2 X 3 (wherein X 1 is A or Y, X 2 is A or S, X 3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMX 1 X 2 (wherein X 1 is D, S, T, or A, X 2 is I, S, L, P, or D (SEQ ID NO: 308) and includes a VH region containing a VH CDR3, and / or (b) (i) the 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 (wherein X 1 is R, G, W, A, or C, X 2 is A, P, G, L, C, or S, X 3 is S, G, or R, X 4 is Q, C, E, V, or I, X 5 is S, L, P, G, A, R, or D, X 6 is V, G, or I, X 7 is S, E, D, or P, X 8 is S, P, F, A, M, E, V, N, D, or Y, X 9 is I, T, V, E, S, A, M, Q, Y, H, or R, X 10 is Y or F, X 11 is L, W, or P, X 12 is A, S, or G (SEQ ID NO: 309) and includes a light chain variable (VL) CDR1, (ii) the sequence X 1 ASX 2 RAX 3 (wherein X1 is G or D, X 2 is S or I, X 3 contains a VL CDR2 comprising (SEQ ID NO: 310) and (iii) 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, X 2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C, X 3 is W, F, or S, X 4 is L or I (SEQ ID NO: 311), or QQYX 1 X 2 X 3 PX 4 (where X 1 is G, Q, E, L, F, A, S, M, R, K, or Y, X 2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y, X 3 is W, S, or F, X 4It includes a VL region that includes a VL CDR3 that includes L or I (SEQ ID NO: 312). In some embodiments, the second antibody variable domain includes a heavy chain variable (VH) CDR1 that includes the sequence shown in SEQ ID NO: 150, 151, 152, 156, 157, 348, 349, 353, 354, or 355, (ii) a VH CDR2 that includes the sequence shown 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 CDR3 that includes the sequence shown in SEQ ID NO: 155, 161, 197, 205, 164, or 352, or 358, and / or (i) a VL CDR1 that includes the sequence shown 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 that includes the sequence shown in SEQ ID NO: 221, 359, or 362, and (iii) a VL CDR3 that includes the sequence shown 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, both the first and second antibody variable domains of the bispecific antibody comprise amino acid modifications at positions 223, 225, and 228 within the hinge region and at position 409 or 368 within 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) described herein or conjugates thereof (e.g., BCMA antibody-drug conjugate).

[0022] In another aspect, the present invention also provides a cell line that recombinantly produces any of the antibodies described herein (e.g., BCMA, CD3, or bispecific) or their conjugates (e.g., BCMA antibody-drug conjugate).

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

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

[0025] The present invention provides a method for 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), the method comprising preparing an isolated antibody (e.g., BCMA) or binding fragment, bispecific antibody (BCMA-CD3 bispecific), or their conjugates (e.g., BCMA antibody-drug conjugate) described herein, and administering the antibody or conjugate to the subject.

[0026] A method of treating a condition associated with malignant cells expressing a tumor antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of the present invention is also provided. In some embodiments, the condition is cancer. In some embodiments, the cancer is multiple myeloma, malignant plasma cell neoplasm, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Castleman 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 myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenström macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, mediastinal (thymic) primary 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 in the elderly, diffuse large B-cell lymphoma associated with inflammation, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma having intermediate characteristics between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma having intermediate characteristics 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 include a constant region. In some embodiments, the antibodies described herein are of the human IgG1, IgG2 or IgG2Δa, IgG3, or IgG4 subclass. In some embodiments, the antibodies described herein include a glycosylated constant region. In some embodiments, the antibodies described herein include a constant region with increased binding affinity to one or more human Fc gamma receptors.

Brief Description of the Drawings

[0028]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 7F

Figure 8A

Figure 8B

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 10A

Figure 10B

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 12

Figure 13

Figure 14

Figure 15A

Figure 15B

Figure 15C

Figure 16

Mode for Carrying Out the Invention

[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. Further, 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 conditions associated with malignant BCMA expression in a subject, such as cancer or an autoimmune disease, using the antibodies (e.g., BCMA, CD3, or bispecific antibody) or their conjugates (BCMA antibody-drug conjugate) described herein.

[0030] General techniques The practice of the present invention, unless otherwise indicated, will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the skill of the art. Such techniques are described in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989), Cold Spring Harbor Press; Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J.E. Cellis, 1998), Academic Press; Animal Cell Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (eds. 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 (eds. D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (eds. J.M. Miller and M.P. Calos, 1987); Current Protocols in Molecular Biology (eds. F.M. Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (eds. Mullis et al., 1994); Current Protocols in Immunology (eds. J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.It is fully described in Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and J. D. Capra, Harwood Academic Publishers, 1995), etc.

[0031] Definition "Antibody" is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen - recognition site located within the variable region of the immunoglobulin molecule. In this specification, the term refers not only to intact polyclonal or monoclonal antibodies, but also to fragments thereof (Fab, Fab’, F(ab’) 2, such as Fv, single-chain (ScFv), and domain antibodies (including shark and camel antibodies), as well as fusion proteins containing antibodies, and any other modified configurations of immunoglobulin molecules containing antigen recognition sites are also included. Antibodies include antibodies of any class, such as IgG, IgA, or IgM, etc. (or their subclasses), and the antibody does not have to be of any specific class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional arrangements of 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 can be performed by fragments of the intact antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody are Fab, Fab’, F(ab’) 2 , the Fd fragment consisting of the VH and CH1 domains, the Fv fragment consisting of the VL and VH domains of a single arm of the antibody, the single-domain antibody (dAb) fragment (Ward et al., Nature, 341:544-546, 1989), and the isolated complementarity-determining region (CDR).

[0033] An antibody, antibody conjugate, or polypeptide that "binds preferentially to" or "binds specifically to" (used interchangeably herein) a target (e.g., BCMA protein or CD3 protein) is a term 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 a greater affinity than it reacts or associates with an alternative cell or substance. An antibody "binds specifically" or "binds preferentially" if it binds to the target with a greater affinity, binding activity, more readily, and / or for a greater duration than it binds to other substances. For example, an antibody that binds specifically or preferentially to a BCMA epitope or a CD3 epitope is an antibody that binds to this epitope with a greater affinity, binding activity, more readily, and / or for a 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 understood that, for example, an antibody (or portion or epitope) that binds specifically or preferentially to a first target may or may not bind specifically or preferentially to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it can include exclusive binding). Generally, but not always, references to binding mean 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 proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) a method based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol., 273:927-948). In this specification, CDR can refer to CDRs defined by either method or by a combination of both methods.

[0035] The "CDR" of a variable domain is an amino acid residue within a variable region identified according to the accumulation of both Kabat, Chothia, Kabat and Chothia, AbM, contact, and / or conformation definitions, or the definition of any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs can also be identified as the structural loop structures originally described by Chothia et al. See, for example, Chothia et al., Nature, 342:877-883, 1989. Other methods of CDR identification include the "AbM definition" derived using the AbM antibody modeling software of Oxford Molecular (now Accelrys®), which is a compromise between Kabat and Chothia, or the "contact definition" of CDRs based on observed antigen contacts shown in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another method, herein referred to as the "conformation definition" of CDRs, the positions of the CDRs can be identified as residues that make an enthalpic contribution to antigen binding. See, for example, 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 nevertheless, they can be made shorter or longer based on predictions or experimental findings that a particular residue or group of residues, or even the entire CDR, does not significantly impact antigen binding, and will overlap at least in part with the Kabat CDR. As used herein, CDR can refer to CDRs defined by any method known in the art, including combinations of methods. The methods used herein can utilize CDRs defined by any of these methods.For any given embodiment containing more than one CDR, the CDRs can be defined according to any of Kabat, Chothia, extended, AbM, contact, and / or conformation definitions.

[0036] As used herein, a "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site and are highly specific. Further, in contrast to polyclonal antibody preparations, which 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 should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495, 1975, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries made using, for example, the techniques described in McCafferty et al., Nature 348:552-554, 1990.

[0037] As used herein, a "humanized" antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab’, F(ab’) 2Refers to the form of a non-human (e.g., mouse) antibody, such as an antibody or other antigen-binding portion sequence. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody) having the desired specificity, affinity, and capacity, such as a mouse, rat, or rabbit. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Further, a humanized antibody may contain residues that are not found within the transferred CDRs or framework sequences in the recipient antibody but are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of the immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies having an Fc region modified 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 have been altered with respect to the original antibody, and these CDRs are also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0038] As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody produced using any of the techniques known to those of skill in the art or disclosed herein for making human antibodies. This definition of a human antibody includes antibodies that contain at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that contains 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, a human antibody is selected from a phage library, where the phage library expresses 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 made by immunizing an animal in which the human immunoglobulin locus has been introduced by gene transfer in place of the endogenous locus, e.g., a mouse in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. 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 recovered from an individual or from single cell cloning of cDNA, or can be immunized in vitro). See, e.g., 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. Pat. No. 5,750,373.

[0039] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is 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 to 100 amino acids). The chain can be linear or branched and can contain modified amino acids and / or can be interrupted by non-amino acids. The term also encompasses amino acid chains that are naturally or artificially modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included within the definition. It is understood that polypeptides can exist as single chains or as 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 antigen sites per molecule (e.g., IgG), such that the two binding sites bind to 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] "Bispecific" or "dual specificity" refers to a hybrid antibody having 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] "Bifunctional" refers to an antibody having the same antigen-binding site (i.e., the same amino acid sequence) in two arms, but each binding site can recognize two different antigens.

[0046] "Heteromultimer", "heteromultimeric complex", or "heteromultimeric polypeptide" refers to a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide has an amino acid sequence different from that of 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 higher-order tertiary structure in which polypeptides are present in addition to the first and second polypeptides.

[0047] "Heterodimer", "heterodimeric protein", "heterodimeric complex", or "heteromultimeric polypeptide" refers to a molecule comprising a first polypeptide and a second polypeptide, wherein the second polypeptide has an amino acid sequence different from that of the first polypeptide by at least one amino acid residue.

[0048] "Hinge region", "hinge sequence", and variations thereof are herein defined to include the meanings known in the art as exemplified, for example, in 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] The terms "immunoglobulin-like hinge region", "immunoglobulin-like hinge sequence", and variations thereof, as used herein, refer to the hinge regions and hinge sequences of immunoglobulin-like or antibody-like molecules (e.g., immunoadhesins). In some embodiments, the immunoglobulin-like hinge region can be or be derived from any IgG1, IgG2, IgG3, or IgG4 subtype, including its chimeric forms, or can be derived from IgA, IgE, IgD, or IgM, e.g., 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 target cells. The viability of target cells can include cell survival, proliferation, and / or the ability to interact with other cells.

[0051] The antibodies of the present invention can be generated using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of such techniques, or other techniques readily discernible in the art (see, e.g., Jayasena, S.D., Clin. Chem., 45:1628-50, 1999, and Fellouse, F.A. et al., J. Mol. Biol., 373(4):924-40, 2007).

[0052] As is known in the art, as used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a chain of nucleotides of any length, including DNA and RNA. 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. Modifications to the nucleotide structure, if present, can be imparted before or after the strands are assembled. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications, such as those having uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and those having charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendent moieties, such as those containing proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those having modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and the unmodified form of the polynucleotide. Furthermore, any of the hydroxyl groups normally present in the sugar can be replaced, for example, by a phosphonate group, phosphorylated, protected by standard protecting groups, or activated to effect additional linkage to an additional nucleotide, or conjugated to a solid support. The 5' and 3' terminal OHs can be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups.Polynucleotides can also contain analogs of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs 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 being replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR. 2 (“amidate”), P(O)R, P(O)OR’, CO, or CH 2 (“formacetal”) (wherein each R or R’ is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl that may contain an ether (-O-) linkage). There are embodiments where all linkages in the polynucleotide are not the same. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

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

[0054] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of 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 a recipient of a vector for taking up a polynucleotide insert. Host cells include progeny of a single host cell, which progeny may not necessarily be identical to the original parent cell due to natural, accidental, or deliberate mutations (in form or in genomic DNA complement). Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0056] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is usually defined to extend from the amino acid residue at position Cys226 or 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 includes two constant regions, CH2 and CH3.

[0057] As used in the art, the terms "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include 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 ("inhibitory receptor"), which have similar amino acid sequences and differ mainly 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. The term "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] As used herein with respect to antibodies, the term "competes" means that a first antibody or an antigen-binding fragment (or portion) thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or its antigen-binding portion such that the result of the binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody as compared to the binding of the first antibody in the absence of the second antibody. The remaining option that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may apply, but need not be so. 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, antibodies are said to "cross-compete" with each other for the binding of these respective epitopes 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. 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 changes, or binding to a common epitope or portion thereof), one of ordinary skill in the art should 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; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assayed using a variety of assays known in the art for evaluating such antibody effector functions.

[0060] The "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The "variant Fc region" differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification and still comprises an amino acid sequence that 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 the parent polypeptide, e.g., in the native sequence Fc region or the Fc region of the parent polypeptide, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions. The variant Fc regions herein 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 with these, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with these.

[0061] The term "effector function" refers to the biological activities 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 downregulation of cell surface receptors (e.g., B cell receptor; BCR). See, e.g., U.S. Patent No. 6,737,056. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., the antibody variable domain) and can be assayed using various assays known in the art for evaluating such antibody effector functions. Exemplary measurements of effector function are 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 receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The ADCC activity of a molecule of interest can be assayed using an in vitro ADCC assay such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assayed 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 lysis of a target in the presence of complement. The complement activation pathway is initiated by binding to a molecule (e.g., an antibody) that has complexed with the homologous antigen of the first component (C1q) of the complement system. To assay for complement activation, for example, a CDC assay such as that described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996) can be performed.

[0064] As used herein, "treatment" is a procedure for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the following: reduction (or destruction) of the growth of neoplastic or cancerous cells, inhibition of metastasis of neoplastic cells, remission of BCMA-related diseases (e.g., cancer or autoimmune diseases), reduction of symptoms resulting from BCMA-related diseases (e.g., cancer or autoimmune diseases), increase in the quality of life of a person suffering from a BCMA-related disease (e.g., cancer or autoimmune diseases), reduction in the dosage of other drug therapies required to treat a BCMA-related disease (e.g., cancer or autoimmune diseases), delay in the progression of a BCMA-related disease (e.g., cancer or autoimmune diseases), cure of a BCMA-related disease (e.g., cancer or autoimmune diseases), and / or extension of the survival period of a patient having a BCMA-related disease (e.g., cancer or autoimmune diseases), one or more of which are included.

[0065] "Remitting" means the reduction or improvement of one or more symptoms as compared to not administering a BCMA antibody or BCMA antibody conjugate. "Remitting" also includes shortening or reducing the duration of 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 elimination or reduction of the risk of a disease, its complications, and biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the disease, reduction in severity, or delay in onset. For therapeutic use, beneficial or desired results include clinical outcomes such as reduction in the incidence of various BCMA-related diseases or conditions (such as multiple myeloma) or remission of one or more symptoms, reduction in the dosage of other drug therapies required to treat the disease, enhancement of the effect of another drug therapy, and / or delay in the progression of the patient's BCMA-related disease. The effective dosage can be administered in one or more administrations. For the purposes of the present invention, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical context, the effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" can be considered in relation to the administration of one or more therapeutic agents, and a single agent, when combined with one or more other agents, can be considered to be administered in an effective amount if a desired result can or is achieved.

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

[0068] As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing 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" means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

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

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

[0072] The term "k on " or "k a " as used herein refers to the rate constant for the association of an antibody with an antigen. Specifically, the rate constants (k on / k a and k off / k d ) as well as 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 the dissociation of an antibody from an antibody / antigen complex.

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

[0075] References herein to values or parameters with the term "about" include (and describe) embodiments directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X". Numerical ranges include the numbers defining the range.

[0076] It is understood that whenever an embodiment is described herein in terms of the recitation "comprising", other similar embodiments are also provided from the perspective of "consisting of" and / or "consisting essentially of".

[0077] When aspects or embodiments of the present invention are described from the perspective of a Markush group of alternatives or other classifications, the present invention includes not only the entire group recited as a whole, in addition to 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 present invention also contemplates any one or more explicit exclusions of group members in the claimed invention.

[0078] Unless defined otherwise, 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 the claims, words such as "comprise", "comprises" or "comprising" and variations thereof imply the inclusion of the stated integer or group of integers, but do not imply the exclusion of any other integer or group of integers. Unless the context requires otherwise, terms in the singular form shall include the plural and terms in the plural form shall include the singular.

[0079] Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also 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 antibody and method for producing the same The present invention provides an antibody that binds to BCMA (e.g., human BCMA (e.g., SEQ ID NO: 353 or accession number: Q02223-2)) and is characterized by any one or more of the following properties: (a) treating, preventing, or remitting one or more symptoms of a condition associated with malignant cells expressing BCMA in a subject (e.g., B cell-related cancers such as multiple myeloma); (b) inhibiting tumor growth or progression in a subject (having a malignant tumor expressing BCMA); (c) inhibiting the metastasis of cancer (malignant) cells expressing BCMA in a subject (having one or more malignant cells expressing BCMA); (f) inducing regression (e.g., long-term regression) of a tumor expressing BCMA; (d) exerting cytotoxic activity in malignant cells expressing BCMA; and (e) blocking 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), wherein (a) (i) the heavy chain variable (VH) complementarity determining region 1 (CDR1) comprises the sequence SYX 1 MX 2 (wherein 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 (wherein X 1 is G or S, X 2 is A or P, and X 3 is T, N, or S) (SEQ ID NO: 303), (ii) the sequence AX 1 X 2 X 3 X 4 GX 5 X 6 X 7 X 8 YADX 9 X 10 KG (wherein X 1is I, V, T, H, L, A, or C, X 2 is S, D, G, T, I, L, F, M, or V, X 3 is G, Y, L, H, D, A, S, or M, X 4 is S, Q, T, A, F, or W, X 5 is G or T, X 6 is N, S, P, Y, W, or F, X 7 is S, T, I, L, T, A, R, V, K, G, or C, X 8 is F, Y, P, W, H, or G, X 9 is V, R, or L, X 10 is G or T (SEQ ID NO: 305), or X 1 X 2 X 3 X 4 X 5 X 6 (wherein X 1 is S, V, I, D, G, T, L, F, or M, X 2 is G, Y, L, H, D, A, S, or M, X 3 is S, G, F, or W, X 4 is G or S, X 5 is G or T, X 6 is N, S, P, Y, or W (SEQ ID NO: 306) - containing VH CDR2, and (iii) sequence VSPIX 1 X 2 X 3 (wherein X 1 is A or Y, X 2 is A or S, X 3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMX 1 X 2 (wherein 、 X 1 is D, S, T, or A, X 2 is I, S, L, P, or D (SEQ ID NO: 308) - containing VH CDR3 - containing VH region, and / or (i) sequence X 1 X 2 X3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 (wherein, 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 is A, S, or G) (SEQ ID NO: 309) - containing light chain variable (VL) CDR1, (ii) the sequence X 1 ASX 2 RAX 3 (wherein, X 1 is G or D, and X 2 is S or I, and X 3 is T or P) (SEQ ID NO: 310) - containing VL CDR2, and (iii) the sequence QQYX 1 X 2 X 3 PX 4 T (wherein, 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 (wherein 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) and provides an isolated antibody or antigen-binding fragment thereof comprising a VL region comprising a VL CDR3.

[0082] In another aspect, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to BCMA, wherein the antibody comprises a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequences shown in SEQ ID NOs: 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 a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequences shown in 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, 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 is provided that has 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.

[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, except for the heavy chain CDR2 sequence where the Chothia CDR sequence is underlined and the Kabat CDR sequence is in bold, the underlined sequences are the CDR sequences according to Kabat, and the bold sequences are the CDR sequences according to Chothia.

[0118] The present invention also provides the CDR portions (including Chothia, Kabat CDR, and CDR contact regions) of antibodies against BCMA. The determination of the CDR regions is within the well - known scope of the art. It is understood that in some embodiments, the CDR can be a combination of Kabat and Chothia CDRs (also referred to as "combined CDR" or "extended CDR"). In some embodiments, the CDR is the Kabat CDR. In other embodiments, the CDR is the Chothia CDR. In other words, in embodiments having more than one CDR, the CDR can be any of Kabat, Chothia, combined CDR, or combinations thereof. Table 2 provides examples of the 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 Provided are antibodies that compete with the antibodies described herein, including 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_C09, 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, 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.

[0158] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to BCMA and comprises a VH region comprising the sequence set forth in SEQ ID NO: 112 and / or 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 heavy chain comprising the sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 358).

[0159] In some embodiments, the invention provides an antibody or antigen-binding fragment that specifically binds to BCMA and 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 invention also provides the CDR portion of an antibody against a BCMA antibody based on the CDR contact region. The CDR contact region is the region of the antibody that imparts specificity for the antigen to the antibody. Generally, the CDR contact region includes residue positions within the CDRs and the framework zones that are constrained to maintain the proper loop structure of the antibody for binding to the specific antigen. See, e.g., Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of the CDR contact region is within the ordinary skill in the art.

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

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

[0163] The present invention also provides a method for producing any of these antibodies. The antibodies of the present invention can be produced by procedures known in the art. The polypeptide can be generated by proteolysis or other degradation of the antibody, by the recombinant methods described above (i.e., single or fusion polypeptides), or by chemical synthesis. The polypeptide of the antibody, particularly a shorter polypeptide of up to about 50 amino acids, is conveniently produced by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, the antibody can be generated by an automated polypeptide synthesizer 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 a fusion protein comprising one or more fragments or regions derived from the antibody of the present invention. In one embodiment, at least 10 consecutive amino acids of the variable light chain region shown in 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, 80, 315, 36, or 364, 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 are provided. 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 consecutive 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 consecutive amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide is SEQ ID NO: 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 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 363and contains a light chain variable region and / or a heavy chain variable region as shown in any of the array pairs selected from 364 and 365. In another embodiment, the fusion polypeptide comprises one or more CDRs. In still other embodiments, 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 not attached to a natural molecule, e.g., a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags" such as, for example, a FLAG tag or a 6His tag. Tags are well known in the art.,

[0165] The present invention also provides an isolated polypeptide encoding an antibody of the present invention, as well as a vector and a host cell containing the polynucleotide.

[0166] In one embodiment, the polynucleotide is antibody 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 It includes an array encoding the heavy and / or light chain variable regions of 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_C09, 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, 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. The array encoding the antibody of interest may be maintained in a vector within a host cell, and then the host cell can be expanded 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 produced by linking the light and / or heavy chain variable regions by using a short linker peptide (Bird et al., Science, 242:423-426, 1988). An example of the linker peptide is (GGGGS) 3(Accession No. 498), which crosslinks 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 and preferably should be composed of less than about 20 amino acid residues. The linker can, in turn, 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 the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, an appropriate plasmid containing the polynucleotide encoding scFv can be introduced into a suitable host cell of a eukaryote such as yeast, plant, insect, or mammalian cells, or a prokaryote such as Escherichia coli (E. coli). The polynucleotide encoding the scFv of interest can be made by conventional manipulations 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, such as diabodies or minibodies, are also included. A diabody is a bivalent bispecific antibody expressed using a linker where 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 the complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993; Poljak, R. J. et al., Structure, 2:1121-1123, 1994). A minibody contains 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 present invention provides a composition (such as a pharmaceutical composition) comprising any of the polynucleotides of the present invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein. In still other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 486 and SEQ ID NO: 485 below: COMBO_Rd4_0.6nM_C29 heavy chain variable region GAAGTCCAACTCCTCGAATCCGGTGGCGGCCTTGTCCAGCCTGGAGGTTCCTTGCGCCTGTCATGTGCCGCCAGCGGATTCACCTTCTCGTCCTACCCGATGTCGTGGGTCCGCCAGGCTCCGGGAAAGGGCCTGGAATGGGTGTCAGCCATCGGAGGATCGGGGGGCTCCCTGCCCTACGCCGATATCGTGAAGGGAAGGTTCACCATTAGCCGGGACAACTCCAAGAACACTCTGTACCTCCAAATGAACAGCCTGAGAGCGGAGGACACCGCAGTGTACTATTGCGCCCGGTACTGGCCAATGGACATCTGGGGCCAGGGGACTCTGGTCACCGTCTCCTCA (SEQ ID NO: 486) COMBO_Rd4_0.6nM_C29 light chain variable region GAGATCGTGCTGACTCAGTCCCCTGGAACCCTGTCCCTGTCACCTGGCGAAAGAGCTACCTTGTCCTGTCGCGCATCACAATCCGTGTCGTCGAGCTATCTCGCGTGGTACCAGCAGAAGCCCGGACAGGCCCCAAGGCTGCTTATGTACGACGCCTCCATCCGGGCCACTGGTATCCCCGACCGCTTCTCGGGCTCCGGAAGCGGCACCGACTTCACCCTGACTATTTCCCGGCTCGAACCGGAGGATTTCGCCGTGTACTACTGCCAACAGTACCAGAGCTGGCCGCTGACGTTTGGGCAGGGGACCAAGGTCGAAATCAAA (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 below: L3.PY / H3TAQ heavy chain variable region GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCGGCAGCTACGCTATGACCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAATACCTTCTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACACGGGTGTCCCCTATCGCCGCGCAGATGGATTATTGGGGCCAGGGCACTCTGGTCACCGTCTCCTCA (SEQ ID NO: 488) L3.PY / H3TAQ heavy chain variable region GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGAGCGTGTCCAGCAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTATGGCGCCTCTTCTAGAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCACTACCCTTATCCCCCCAGCTTCACATTTGGCCAGGGCACCAAGGTGGAGATCAAA (SEQ ID NO: 487)

[0171] In still other embodiments, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO: 490 and SEQ ID NO: 489 below: Variable region of the heavy chain of A02_Rd4_0.6nM_C01 GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTACTGCGTCTGGTGGTTCTACTTACTATGCCGATGTGGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGTCGTTGTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA (SEQ ID NO: 490) Variable region of the light chain of A02_Rd4_0.6nM_C01 GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGCGTATTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATGAGCGTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG (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: Variable region of the heavy chain of A02_Rd4_0.6nM_C16 GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTTTGGTGGTTCTACTTACTATGCCGATATCGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA (SEQ ID NO: 492) Variable light chain of A02_Rd4_0.6nM_C16 GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGATCTGTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATCAGACTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG (SEQ ID NO: 491)

[0173] The administration of the expression vector and the polynucleotide composition is further described herein.

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

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

[0176] The polynucleotides can include natural sequences (i.e., endogenous sequences encoding an antibody or a portion thereof), or can include variants of such sequences. Polynucleotide variants contain one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not decreased compared to the natural immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assayed as described herein. Variants preferably exhibit 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 the polynucleotide sequence encoding a natural antibody or a 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 with respect to maximum correspondence as described below. Comparison of 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" as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which the sequences 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 the arrays 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, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships; Dayhoff, M.O. (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, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS, 4:11-17; Robinson, E.D., 1971, Comb. Theor., 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol., 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 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, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include from 20 percent or less, usually from 5 to 15 percent, or from 10 to 12 percent addition or deletion (i.e., gaps) as 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 at which the identical nucleic acid base or amino acid residue is 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., 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 the native gene or portions or complements thereof. Such polynucleotide variants can hybridize to the naturally occurring DNA sequence (or complementary sequence) encoding the native antibody under moderately stringent conditions.

[0181] Suitable "moderately stringent conditions" include prewashing in a solution of 5× SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing overnight at 50° C. to 65° C. in 5× SSC; and washing twice for 20 minutes at 65° C. with 2×, 0.5×, and 0.2× SSC, each containing 0.1% SDS.

[0182] As used herein, "highly stringent conditions" or "high stringency conditions" means (1) using low ionic strength and high temperature for washing, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, together with 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) washing at 42°C in 0.2× SSC (sodium chloride / sodium citrate) and in 50% formamide at 55°C, followed by high stringency washing consisting of 0.1× SSC containing EDTA at 55°C, using at 42°C, 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. Those skilled in the art will recognize how to adjust the temperature, ionic strength, etc. as needed to account for factors such as probe length.

[0183] As a result of the degeneracy of the genetic code, those skilled in the art will understand that there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides carry minimal homology to the nucleotide sequences of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated by the present invention. Furthermore, alleles of the genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have been altered as a result of one or more mutations such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and protein may or may 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 polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can generate the desired DNA sequence using the sequences provided herein and commercially available DNA synthesizers.

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

[0186] Alternatively, PCR enables the replication of DNA sequences. The PCR technique 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, as well as PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkhauswer Press, Boston, 1994.

[0187] RNA can be obtained by using isolated DNA in an appropriate vector and inserting it into an appropriate 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, as shown above in Sambrook et al., 1989.

[0188] Suitable cloning vectors can be constructed according to standard techniques or selected from among the numerous cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended to be used, but useful cloning vectors generally will have the ability to self-replicate, can have a single target for a particular restriction endonuclease, and / or can carry a gene for a marker that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacteriophage 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, among others.

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

[0190] Vectors containing the polynucleotide of interest can be introduced into host cells 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., if the vector is an infectious agent such as vaccinia virus). The choice of means for introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0191] The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an 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. subtilis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis). Preferably, the host cell expresses the cDNA at a level more than about 5-fold, more preferably more than 10-fold, even more preferably more than 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cell. Screening of host cells for specific binding to BCMA or a BCMA domain (e.g., domains 1-4) is performed by immunoassay or FACS. Cells overexpressing 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 with ATCC accession number PTA-122094 is a polynucleotide encoding a humanized BCMA antibody heavy chain variable region, and the vector with ATCC accession number 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). This guarantees the maintenance of viable cultures of the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and the ATCC, which agreement guarantees the permanent and unrestricted availability of progeny of the deposited cultures to the public after the issuance of the relevant U.S. patent or after the earliest public disclosure of any U.S. or foreign patent application, and guarantees the availability of progeny to those determined by the Commissioner of the U.S. Patent and Trademark Office to be entitled thereto in accordance with 35 U.S.C. § 122 and the Commissioner's rules thereunder (including 37 C.F.R. § 1.14, with particular reference to 886 OG 638).

[0193] The agent of the present application agrees to immediately replace the material with another identical one upon notification if the culture of the material on deposit dies, is lost, or is destroyed when cultured under appropriate conditions. The availability of the deposited material should not be construed as a license to practice the invention in violation of rights granted under the patent laws of any government authority.

[0194] BCMA antibody conjugate The present invention also provides a conjugate (or immune complex) of a BCMA antibody or an antigen-binding fragment thereof described herein, wherein the antibody or antigen-binding fragment is conjugated directly or indirectly via a linker to an agent for targeted immunotherapy (e.g., a cytotoxic agent) (e.g., an antibody-drug conjugate). For example, a cytotoxic agent can be linked or conjugated to a BCMA antibody or an antigen-binding fragment thereof described herein for targeted local delivery to a tumor (e.g., a BCMA-expressing tumor) of the cytotoxic agent moiety.

[0195] Methods for conjugating a cytotoxic agent or other therapeutic agent to an antibody have been described in various publications. For example, chemical modifications can be made in an antibody through lysine side-chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds to perform a conjugation reaction. See, e.g., Tanaka et al., FEBS Letters, 579:2092-2096, 2005, and Gentle et al., Bioconjugate Chem., 15:658-663, 2004. Reactive cysteine residues engineered at specific sites of an antibody for specific drug conjugation at defined stoichiometric ratios have also been described. See, e.g., Junutula et al., Nature Biotechnology, 26:925-932, 2008. Conjugation using an acyl donor glutamine-containing tag or endogenous glutamine (i.e., the ability to form a covalent bond as an acyl donor) made reactive by manipulating a polypeptide in the presence of transglutaminase and an amine (e.g., a cytotoxic agent containing or attached to a reactive amine) has also been described in International Applications WO2012 / 059882 and WO2015015448.

[0196] In some embodiments, the BCMA antibodies or conjugates described herein include an acyl donor glutamine-containing tag engineered at 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 is 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), 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 antibodies or conjugates described herein include an acyl donor glutamine-containing tag engineered at a specific site of the antibody, and the tag includes 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 antibodies or conjugates described herein include an acyl donor glutamine-containing tag engineered at a specific site of the antibody, and the tag includes 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 antibodies or conjugates described herein include an acyl donor glutamine-containing tag engineered at a specific site of the antibody, and the tag includes 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, and the lysine residue at the carboxyl terminus of the heavy chain is deleted. In some embodiments, the BCMA antibodies or conjugates described herein include an amino acid substitution at position 297 (EU numbering scheme) of the BCMA antibody. For example, the amino acid asparagine (N) can be substituted with glutamine (Q) or alanine (A) at position 297 of the BCMA antibody.

[0198] Isolated antibodies are also provided that contain an acyl donor glutamine-containing tag and an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody, the modification being 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)) conjugated at a specific site of the BCMA antibody (e.g., the carboxyl terminus of the heavy or light chain, residue T135 in the antibody heavy chain, or another site), and the BCMA antibodies or conjugates described herein that contain an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody are provided. In some embodiments, the amino acid modification is a substitution from lysine to arginine (e.g., K222R, K340R, or K370R).

[0199] In some embodiments, the BCMA antibodies or conjugates described herein comprise 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 from lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, the BCMA antibodies or conjugates described herein comprise 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 from lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, the BCMA antibodies or conjugates described herein comprise 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 from lysine to arginine at position 222 (EU numbering scheme) of the antibody, wherein the lysine residue at the heavy chain carboxy terminus is deleted. In some embodiments, the BCMA antibodies or conjugates described herein comprise 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 from lysine to arginine at position 222 (EU numbering scheme) of the antibody.

[0200] In some embodiments, the BCMA antibodies or conjugates described herein include glutamine engineered at position 297 in the BCMA antibody, or an acyl donor glutamine-containing tag comprising 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 antibodies or conjugates described herein include 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 antibodies or conjugates described herein include 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 amino acid substitution of asparagine (N) to alanine (A) 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.

[0201] Agents that can be conjugated to the BCMA antibodies or antigen-binding fragments of the invention include, but are not limited to, cytotoxic agents, immunomodulatory agents, imaging agents, therapeutic proteins, biopolymers, or oligonucleotides.

[0202] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatin, combretastatin, duocarmycin, pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, camptothecin, tubulysin, hemiasterlin, spirostatin, pladienolide, and stereoisomers, isoters, analogs, or derivatives thereof.

[0203] Anthracyclines are derived from the bacterium Streptomyces and are used to treat a wide range of cancers, such as 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] Dolastatin and its peptide analogs and derivatives, auristatin, are highly potent antimicrotubule agents that have been shown to have anticancer and antifungal activities. 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 (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (such as those described in U.S. Published Patent Application No. 2013 / 0129753). In some embodiments, 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) having the following structure:

[0205]

Chemical formula

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

[0207]

Chemical formula

[0208] In some embodiments, the auristatin is 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) having the following structure:

[0209]

Chemical formula

[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) having the following structure:

[0211]

Chemical formula

[0212] In other embodiments, auristatin has the structure of 0121 (2-methyl-L-prolyl-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):

[0213]

Chem.

[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] Combretastatin is a natural phenol that has vaso-disruptive properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

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

[0217]

Chem.

[0218]

Chem.

[0219]

Chem.

[0220] Enediynes are a class of antitumor bacterial products characterized by the presence of 9- and 10-membered rings, or a conjugated triple-double-triple bond ring system. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamycin, 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 agent. 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 (e.g., HTI-286) bind to tubulin, disrupt normal microtubule dynamics, and depolymerize microtubules in stoichiometric amounts.

[0223] Maytansine or its 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, maytansine (DM1) and its derivatives, and ansamitocin.

[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. PBD and IGN molecules are based on the natural product anthramycin and interact with DNA in a sequence-selective manner preferentially for the purine-guanine-purine sequence. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

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

[0226]

Chemical formula

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

[0228] Tubulysin is a natural product isolated from a strain of myxobacterium that has 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 anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0230] Thus, in some embodiments, the cytotoxic agent is MMAF (monomethyl auristatin F), 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-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-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-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), 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.It is selected from the group consisting of [[ID=]],

[0231] In some embodiments, the agent is an immunomodulatory agent. Examples of immunomodulatory agents include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, pimecrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, 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), a stem cell growth factor called "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0232] In some embodiments, the agent moiety is a contrast agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide phosphor, 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, erythrosin, Alexa Fluor® (e.g., Alexa350, 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, erythrosin, Alexa Fluor® (e.g., Alexa350, 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, a therapeutic or diagnostic radioisotope, or other label (e.g., a PET or SPECT label), can be incorporated into an agent for conjugation to a BCMA antibody or antigen-binding fragment 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 Ac is present.

[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 exotoxin and endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii protein, dianthin protein, 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, crocin, sapaonaria officinalis inhibitor, mitogelin, restrictocin, phenomycin, enomycin, trichothecene, inhibitor cystine knot (ICK) peptides (e.g., serratotoxin), and conotoxins (e.g., 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 the in vivo half-life. Examples of biocompatible polymers include water-soluble polymers such as polyethylene glycol (PEG) or its derivatives, 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 is engineered at a specific site of 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), the tag is conjugated to a linker (e.g., a linker containing one or more reactive amines (e.g., primary amine NH 2 )), and the linker is conjugated to a cytotoxic agent (e.g., MMAF or other auristatins such as 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-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, 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, including N297Q; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101, including N297Q; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131, including N297A; 5) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377, including N297A; 6) antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101. In some embodiments, for example, the acyl donor glutamine-containing tag containing GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475) is engineered at the C-terminus of the light chain of the antibody. In other embodiments, the 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 heavy chain of the antibody, and the C-terminal lysine residue is deleted. In some embodiments, for example, the acyl donor glutamine-containing tag containing LLQG (SEQ ID NO: 454) is engineered after residue T135 in the heavy chain of the antibody, or replaces the amino acid residues E294-N297 in the heavy chain of the antibody.Examples of antibodies include, but are not limited to, 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 There is 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_C09, 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, 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.

[0242] In one variation, the conjugate further comprises an amino acid substitution from lysine to arginine at position 222. Thus, for example, the conjugate is 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101, containing K222R; 2) antibody-AcLys-VC-PABC-0101, containing N297Q and K222R; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101, containing N297Q and K222R; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131, containing N297A and K222R; 5) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377, containing N297A and K222R; 6) antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101, containing K222R. In some embodiments, for example, the acyl donor glutamine-containing tag comprising GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475) is engineered at the C-terminus of the light chain of the antibody. In other embodiments, the 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 heavy chain of the antibody, and the C-terminal lysine residue is deleted. In some embodiments, for example, the acyl donor glutamine-containing tag comprising LLQG (SEQ ID NO: 454) is engineered after residue T135 in the heavy chain of the antibody, or replaces the amino acid residues E294-N297 in the heavy chain of the antibody.Examples of antibodies include, but are not limited to, 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_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, 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, or P4G4, or P1A11 is present.

[0243] CD3 antibody and method for producing the same The present invention further provides an antibody that binds to CD3 (e.g., human CD3 (SEQ ID NO: 502; or accession number: NM_000733.3)).

[0244] In one aspect, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, the isolated antibody or antigen-binding fragment thereof comprising a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequences shown 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 region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequences shown 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 aspect, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, the VH region of which comprises (i) VH complementarity determining region 1 (CDR1) comprising the sequences shown 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) VH CDR2 comprising the sequences shown 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) VH CDR3 comprising the sequences shown in SEQ ID NO: 335, 406, 423, 429, or 451, and / or a VL region comprising (i) light chain variable (VL) CDR1 comprising the sequences shown in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441, (ii) VL CDR2 comprising the sequences shown in SEQ ID NO: 341, 433, 452, or 436, and (iii) VL CDR3 comprising the sequences shown in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453.

[0246] In some embodiments, an antibody is provided that has 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.

[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 bold ones are the CDR sequences according to Chothia.

[0253] The present invention also provides the CDR portions of antibodies against CD3 (including Chothia, Kabat CDR, and CDR contact regions). Determination of the CDR regions is within the well - known scope of the art. In some embodiments, it is understood that the CDR can be a combination of Kabat and Chothia CDRs (also referred to as "combined CDR" or "extended CDR"). In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other words, in embodiments having more than one CDR, the CDR can be any of Kabat, Chothia, combined CDR, 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 present invention also provides an isolated polynucleotide encoding an antibody of the present invention, as well as a vector and a host cell comprising the polynucleotide.

[0263] In one embodiment, the polynucleotide comprises a sequence encoding the heavy and / or light chain variable regions of the 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 VL_TK-S55Y / S105Q, 2B4, h2B4-11, 1C10, 1A4, 7A3, 25A8, 16G7, h25A8-B5, h25A8-B8, h25A8-B12, h25A8-B13, h25A8-C5, h25A8-C8, h25A8-D13, h25A8-E13, h25A8-F13, or h25A8-G13. The sequence encoding the antibody of interest may be maintained in a vector within a host cell and then the host cell can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0264] The present invention also encompasses fusion proteins comprising one or more fragments or regions derived 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 shown 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 shown 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 shown in any of the sequence pairs selected from SEQ ID NO: 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).With respect to the object of the present invention, the fusion protein contains one or more antibodies and another amino acid sequence not attached to a natural molecule, for example, a heterologous sequence or a homologous sequence derived 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] The fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly. Typically, the fusion proteins of the present invention are made by preparing and expressing the polynucleotides encoding them using the recombinant methods described herein, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[0266] Representative materials of the CD3 antibody in the present invention were deposited at the American Type Culture Collection (ATCC) on September 11, 2015. The vector with ATCC accession number PTA - 122513 is a polynucleotide encoding the humanized CD3 antibody heavy chain variable region, and the vector with ATCC accession number PTA - 122512 is a polynucleotide encoding the humanized CD3 antibody light chain variable region. The deposit was made under the terms 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). This guarantees the maintenance of viable cultures of the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and the ATCC, which agreement guarantees the permanent and unrestricted availability of progeny of the deposit culture to the public after the issuance of the relevant U.S. patent or, at the earliest, after any U.S. or foreign patent application related thereto has been published to the public, and guarantees the availability of progeny to those determined by the Commissioner of the U.S. Patent and Trademark Office to be entitled thereto in accordance with 35 U.S.C. § 122 and the Commissioner's regulations thereunder (including 37 CFR § 1.14, particularly referring to 886 OG 638).

[0267] The agent of the present application agrees to immediately replace the material with another identical one if the culture of the deposited material dies, is lost, or is destroyed when cultured under appropriate conditions. The availability of the deposited material should not be construed as a license to practice the invention in violation of the rights granted under the patent laws of any government.

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

[0269] According to one approach for making bispecific antibodies, antibody variable domains having 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 that includes at least a portion of the hinge, CH2, and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. The 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 provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in embodiments where the unequal ratios of the three polypeptides used in the construct result in optimal yields. However, when the expression of at least two polypeptide chains in equimolar amounts results in high yields, or when the ratio is not particularly critical, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

[0270] In one approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (resulting in a second binding specificity) in the other arm. This asymmetric structure, having immunoglobulin light chains only in one half of the bispecific molecule, facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication No. WO94 / 04690.

[0271] In another approach, the bispecific antibody is composed of amino acid modifications within a first hinge region in one arm, where the substituted / replaced amino acids within the first hinge region have an opposite charge to the corresponding amino acids within a 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 altering or engineering the interface between a first and a second immunoglobulin-like Fc region (e.g., hinge region and / or CH3 region). In this approach, the bispecific antibody can be composed of CH3 regions, which include a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 interface, and one or more amino acids within the CH3 interface destabilize homodimer formation and are electrostatically unfavorable for 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 an antibody engineered with a glutamine-containing peptide tag directed against an epitope (e.g., BCMA) in one arm, and another peptide tag engineered into a second antibody directed against a second epitope in the other arm (e.g., a Lys-containing peptide tag or a reactive endogenous Lys) 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 proteins (e.g., bispecific antibodies) described herein comprise full-length human antibodies, wherein the first antibody variable domain of the heterodimeric protein can recruit the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells, and the second antibody variable domain of the heterodimeric protein can specifically bind 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 cells can be any of a variety of immune effector cells known in the art. For example, the immune effector cells can be members 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 cells can also be members of the human myeloid lineage including, but not limited to, monocytes, neutrophils, and dendritic cells. Such immune effector cells can have a cytotoxic or apoptotic effect on target cells, or other desired effects, when activated by binding of the effector antigen.

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

[0277] A target cell can be a cell that is natural or foreign to a human. In the case of 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 the case of a foreign target cell, the cell is an invading pathogen, such as a bacterium, a malaria parasite, or a virus.

[0278] The target antigen is expressed on target cells 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). The 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, polysialic acid, GD2, carbonic anhydrase 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 (Müllerian inhibiting substance) receptor type II, sTn (sialylated Tn antigen; tag-72), FAP (fibroblast activation antigen), endothelin, EGFRvIII, LG, SAS, and CD63.

[0279] In some embodiments, the heterodimeric proteins (e.g., bispecific antibodies) described herein include full-length human antibodies, wherein the first antibody variable domain of the heterodimeric protein can recruit 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, the second antibody variable domain of the heterodimeric protein can specifically bind to a target antigen (e.g., CD20 antigen or EpCAM), and the first and second antibody variable domains of the heterodimeric protein include amino acid modifications at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225R), and (P228E or P228R)) within the hinge region of human IgG2 (SEQ ID NO: 493), and at position 409 or 368 within the CH3 region ((e.g., K409R or L368E (EU numbering scheme)).

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

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

[0282] In another embodiment, the first antibody variable domain of the heterodimeric protein comprises a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequences shown 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, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequences shown 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 second antibody variable domain of the heterodimeric protein comprises a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequences shown in SEQ ID NOs: 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 a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequences shown in 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, 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 VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable (VH) sequence shown in SEQ ID NO: 324 or 388, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence shown in SEQ ID NO: 323 or 387, and the second antibody variable domain comprises a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable (VH) sequence shown in SEQ ID NO: 112, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the light chain variable (VL) sequence shown in SEQ ID NO: 38.

[0284] Antibodies useful in the present invention 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 comprising antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified constructs of immunoglobulin molecules comprising an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies can be of mouse, 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. For example, the BCMA or CD3 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

[0286] In some embodiments, the antibody comprises a modified constant region, e.g., without limitation, a constant region with increased potential to induce an immune response. For example, the constant region may be modified to have increased affinity for Fc gamma receptors, e.g., 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 a reduced potential to induce 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 the mutation 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 containing 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) having the deletion G236. In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb, or IgG4Δc) containing the hinge stabilization 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 non-glycosylated by mutating the oligosaccharide attachment residue (such as Asn297) and / or the flanking residues that are part of the glycosylation recognition sequence within the constant region. In some embodiments, the constant region is enzymatically non-glycosylated for N-linked glycosylation. The constant region may be non-glycosylated enzymatically or by expression in a glycosylation-deficient host cell for N-linked glycosylation.

[0289] In some embodiments, the constant region has a modified constant region that removes 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: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK has a modified constant region.

[0290] In some embodiments, the constant region has the sequence shown in SEQ ID NO: 497: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK has a modified constant region.

[0291] One way to determine the binding affinity of an antibody to BCMA or CD3 is by measuring the binding affinity of the monovalent Fab fragment of the antibody. To obtain the monovalent Fab fragment, the antibody (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of the BCMA Fab fragment of the antibody can be determined using a surface plasmon resonance (Biacore™ 3000™ surface plasmon resonance (SPR) system, Biacore™, INC, Piscataway NJ) equipped with a pre-immobilized streptavidin sensor chip (SA), or with anti-mouse Fc or anti-human Fc using HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v / v surfactant P20). Biotinylated or Fc-fused human BCMA is diluted in HBS-EP buffer to a concentration of less than 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. The regeneration study showed that 25 mM NaOH in 25% v / v ethanol effectively removes the bound Fab while maintaining the activity of BCMA on the chip over 200 injections. Typically, serial dilutions of the purified Fab sample (K was estimated by spanning concentration of 0.1 - 10×) are injected at 100 μL / min for 1 minute, with a dissociation time of up to 2 hours allowed. The concentration of the Fab protein is determined by ELISA and / or SDS-PAGE electrophoresis using a known concentration (determined by amino acid analysis) Fab as a standard. The kinetic association rate (k D was estimated) and dissociation rate (k on ) and dissociation rate (k off) is obtained simultaneously by comprehensively 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. The equilibrium dissociation constant (K D ) value is calculated as k off / k on . This protocol is suitable for determining the binding affinity of an antibody to any BCMA, including human BCMA, BCMA of another mammal (such as mouse BCMA, rat BCMA, or primate BCMA), and different forms of BCMA (e.g., glycosylated BCMA). The binding affinity of the antibody is generally measured at 25°C, but can also be measured at 37°C.

[0292] The antibodies described herein can be made by any method known in the art. With respect to the production of hybridoma cell lines, the route and schedule of immunization of the host animal generally conform to established conventional techniques for 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 are described herein.

[0293] It is contemplated that any mammalian subject, including humans, or antibody - producing cells derived therefrom can be manipulated to function as a basis for the production of mammals, including humans, and hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intradermally, and / or plantarly with an amount of 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, D. W. et al., In Vitro, 18:377-381, 1982. Available myeloma strains, 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. Generally, in this technique, a fusogen such as polyethylene glycol is used, or myeloma cells and lymphoid cells are fused 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 unfused parental cells. Any of the media described herein, supplemented with serum or serum-free, can be used to culture hybridomas that secrete monoclonal antibodies. As another option for cell fusion techniques, EBV-immortalized B cells can be used to produce the monoclonal antibodies of the present invention. Hybridomas are expanded and subcloned as needed, and the supernatant is assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0295] Hybridomas that can be used as a source of antibodies include all derivatives and progeny cells of the parental hybridomas 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 the culture medium or body fluids, if desired, by conventional immunoglobulin purification procedures such as ammonium sulfate fractionation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Unwanted activities, if present, can be removed, for example, by running the preparation over an adsorbent made from the immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. Bifunctional agents or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (for conjugation by cysteine residues), N-hydroxysuccinimide (for lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N=C=NR (wherein R and R 1 are different alkyl groups) are used to conjugate human BCMA or CD3, or a fragment containing the target amino acid sequence, to a protein that is immunogenic in the immunized species, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Immunizing the host animal with such conjugates can result in a population of antibodies (e.g., monoclonal antibodies).

[0297] If desired, 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 may be maintained in a vector within a host cell and then the host cell can be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be carried out by cloning the antibody gene from B cells by means known in the art. See, for example, Tiller et al., J. Immunol. Methods, 329, 112, 2008; U.S. Patent No. 7,314,622.

[0298] In an alternative, the polynucleotide sequence can be used for genetic manipulations to "humanize" an antibody or to improve the affinity or other properties of the antibody. For example, when an antibody is used in clinical trials and treatment in humans, the constant region can be engineered to more closely resemble the human constant region to avoid an immune response. There may be a desire to genetically manipulate the antibody sequence to obtain greater affinity for BCMA or CD3, and greater potency in the inhibition of BCMA.

[0299] There are four general steps in humanizing a monoclonal antibody. These are: (1) determination of the nucleotide and predicted amino acid sequences of the starting antibody light and heavy variable domains, (2) design of the humanized antibody, i.e., determination of which antibody framework regions to use during the humanization process, (3) 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 having rodent or modified rodent V regions fused to human constant regions and their associated CDRs. See, e.g., 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 support framework regions (FRs) prior to fusion with 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. Another reference describes rodent CDRs supported by recombinant engineered rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize the unwanted immune response directed against rodent anti-human antibody molecules that limits the duration and effectiveness of the therapeutic use of these portions 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 of humanizing antibodies that can also be utilized have been disclosed by Daugherty et al., Nucl. Acids Res., 19:2471-2476, 1991, as well as 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. WO01 / 27160.

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

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

[0303] In an alternative, the antibody may be recombinantly produced and expressed using any method known in the art. In another alternative, the antibody can 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, humanized antibodies and antibody fragments can be generated in vitro from an immunoglobulin variable (V) domain gene repertoire from non-immunized donors using phage display technology (McCafferty et al., Nature, 348:552-553, 1990). According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous bacteriophages such as M13 or fd and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that exhibit these properties. Thus, phage mimics some of the properties of B cells. Phage display can be carried out in various formats. For reviews, see, for example, 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 non-immunized human donors can be constructed, and antibodies against diverse arrays of antigens (including autoantigens) can be isolated essentially according to 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 the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes will confer higher affinity, and B cells displaying high-affinity surface immunoglobulins are preferentially replicated and differentiated during subsequent antigen challenges. This natural process can be mimicked by using techniques known as "chain shuffling" (Marks et al., Bio / Technol., 10:779-783, 1992). In this method, the affinity of the "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with the repertoire of naturally occurring variants of the V domain genes obtained from non-immunized donors. This technique enables the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for generating a very large phage antibody repertoire (also known as the "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, in which case the human antibodies have similar affinity and specificity to the starting rodent antibodies. According to this method, also known as "epitope imprinting", the heavy or light chain V domain genes of rodent antibodies obtained by phage display techniques are replaced with the repertoire of human V domain genes to create rodent-human chimeras. Antigen selection results in the isolation of human variable regions capable of restoring a functional antigen-binding site, i.e., the epitope dictates (imprints) the selection of partners. When the process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication No. WO93 / 06213). Unlike the traditional humanization of rodent antibodies by CDR grafting, this technique results in fully human antibodies that do not have framework or CDR residues of rodent origin.

[0304] Antibodies can be recombinantly produced by first isolating the antibody and antibody-producing cells from a host animal, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in a host cell (e.g., CHO cells). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or in transgenic milk. Methods for recombinantly expressing antibodies in plants or milk are 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, such as humanization, single-chain, etc., are known in the art.

[0305] Flow cytometry sorting techniques such as immunoassays and fluorescence-activated cell sorting (FACS) can also be used to isolate antibodies specific for BCMA, CD3, or the tumor antigen of interest.

[0306] The antibodies described herein can be conjugated to many different carriers. The carrier can be active and / or inactive. 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 with respect to the purposes of the present invention. One of ordinary skill in the art will know or be able to identify other carriers suitable for conjugating antibodies or can confirm such by routine experimentation. In some embodiments, the carrier includes a moiety that targets the myocardium.

[0307] DNA encoding a monoclonal antibody can be readily 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 vectors disclosed in PCT Publication No. WO87 / 04462), and they can then be transfected into host cells that would otherwise not produce immunoglobulin protein, e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, etc., to obtain synthesis of the monoclonal antibody in the recombinant host cells. See, e.g., PCT Publication No. WO87 / 04462. The DNA may also be modified, e.g., by substituting the coding sequences of human heavy and light chain constant regions for the homologous mouse sequences, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence, in the manner described by Morrison et al., Proc. Nat. Acad. Sci., 81:6851, 1984. In that way, "chimeric" or "hybrid" antibodies having the binding specificity of the monoclonal antibodies herein are prepared.

[0308] The BCMA or tumor antigen antibody described herein can be identified or characterized using methods known in the art, whereby a reduction in BCMA or other tumor antigen expression levels is detected and / or measured. In some embodiments, the BCMA antibody is identified by incubating a candidate agent with BCMA and monitoring binding and / or an attendant reduction in BCMA expression levels. The binding assay can be performed using a purified BCMA polypeptide or cells that naturally express or are transfected to express the BCMA polypeptide. In one embodiment, the binding assay is a competitive binding assay, in which case the ability of the candidate antibody to compete with a known BCMA antibody for BCMA binding is evaluated. The assay can be performed in a variety of formats, including an ELISA format.

[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 the 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 well known in the art. For example, one method is to identify the epitope to which it binds, or "epitope mapping". For example, described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, 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 the antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In additional examples, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various suppliers, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, Netherlands). Epitopes can be linear epitopes, i.e., those contained within a single stretch of amino acids, or conformational epitopes formed by three-dimensional interactions of amino acids that need not necessarily be contained within a single stretch. Peptides of various lengths (e.g., at least 4-6 amino acids in length) 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 a BCMA, CD3, or other tumor antigen antibody binds can be determined by systematic screening 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 the gene fragment expression assay, the open reading frame encoding BCMA, CD3, or other tumor antigen is fragmented randomly or by a specific gene construct, and the reactivity of the expressed fragments of BCMA, CD3, or other tumor antigen with the antibody being tested is determined. The gene fragments can be generated, for example, in vitro by PCR, then transcribed and translated in the presence of radiolabeled amino acids to make proteins.Next, the binding of the antibody to radiolabeled BCMA, CD3, or other tumor antigen fragments is determined by immunoprecipitation and gel electrophoresis. A particular epitope can also be identified by using a large library of random peptide sequences (phage library) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, mutagenesis of the antigen-binding domain, domain-exchange experiments, and alanine-scanning mutagenesis can be performed to identify residues sufficient and / or necessary for epitope binding. For example, domain-exchange 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 (exchanged) with sequences from BCMA from another species (e.g., mouse), or a closely related but antigenically different protein (e.g., Trop-1). By assaying the binding of the antibody to the mutant BCMA, CD3, or other tumor antigen, the importance of a particular BCMA, CD3, or other tumor antigen fragment for antibody binding can be assayed.

[0311] Yet another method that can be used to characterize BCMA, CD3, or other tumor antigen antibodies is to use a competition assay 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 antibodies. Competition assays are well known to those of skill in the art.

[0312] The expression vector can be used to directly express BCMA, CD3, or other tumor antigen antibodies. Those skilled in the art are proficient in 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. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun, or catheter insertion administration, and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglia, or into the 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, ed. J.A. 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 within the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. Concentration ranges of 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 of DNA 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 within a desired cell are well known in the art. Exemplary viral vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publications WO90 / 07936; WO94 / 03622; WO93 / 25698; WO93 / 25234; WO93 / 11230; WO93 / 10218; WO91 / 02805; U.S. Patents 5,219,740 and 4,777,127; British Patent 2,200,651; and European Patent 0345242), alphavirus vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publications WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984, and WO95 / 00655). Administration of DNA linked to a killed adenovirus, as described by Curiel, Hum. Gene Ther., 1992, 3:147, can also be used.

[0315] Not limited to just that, polycation condensed DNA (see, e.g., Curiel, Hum. Gene Ther., 3:147, 1992) linked or not linked to inactivated adenovirus alone; ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264:16985, 1989); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO95 / 07994; WO96 / 17072; WO95 / 30763; and WO97 / 42338), as well as non-viral delivery vehicles and methods including nuclear charge neutralization or fusion with cell membranes can also be used. Naked DNA can also be used. Exemplary methods of introducing naked DNA are described in PCT Publication No. WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO95 / 13796; WO94 / 23697; WO91 / 14445; and EP0524968. 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 includes compositions including pharmaceutical compositions containing antibodies having the properties described herein and made using the methods described herein. As used herein, the compositions include one or more antibodies that bind to CD3 and a tumor antigen (e.g., BCMA), and / or one or more polynucleotides encoding one or more of these antibodies. These compositions can further include suitable excipients, such as pharmaceutically acceptable excipients including buffers, well known in the art.

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

[0318] Heteroconjugate antibodies, which contain two antibodies linked by a covalent bond, are also within the scope of the present invention. Such antibodies have been used to direct immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and to treat HIV infection (PCT Publications WO91 / 00360 and WO92 / 200373; EP03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are described in U.S. Patent 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 cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutylimidate.

[0320] In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction with Fcγ receptors as well as complement and the immune system. Techniques for preparing such antibodies are described in WO99 / 58572. For example, when an antibody is used in clinical trials and treatments in humans, the constant region can be engineered to be more similar to the human constant region to avoid an 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, the amino acid sequence can be mutated to obtain an antibody having a desired binding affinity for BCMA and / or CD3. Modification of polypeptides is routine in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitutions of amino acid residues, deletions or additions of one or more amino acids that do not significantly and detrimentally alter the functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides having the use of chemical analogs.

[0322] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from 1 residue to 100 or more residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of the antibody molecule include fusions of an enzyme or a polypeptide that increases the half-life of the antibody in the bloodstream to the N or C terminus of the antibody.

[0323] A substitution variant has at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Of most interest for substitution mutagenesis are the hypervariable regions, although FR changes are also contemplated. Conservative substitutions are shown in Table 5 under the heading “Conservative Substitutions”. If such substitutions change the biological activity, more substantial changes can be introduced as named in Table 5 “Exemplary Substitutions” or further described below in reference to amino acid classes, and the products can be screened.

[0324]

Table 5

[0325] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that (a) are significantly different in their effect on maintaining the structure of the polypeptide backbone in the area of substitution, for example as a sheet or helical conformation, (b) change the charge or hydrophobicity of the molecule at the target site, or (c) change the bulk of the side chain. 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 influence 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 can generally be replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, especially when the antibody is an antibody fragment such as an Fv fragment, cysteine bonds can be added to the antibody to improve its stability.

[0328] Amino acid modifications can range from the alteration or modification of one or more amino acids to the complete redesign of regions such as the variable region. Changes within the variable region can alter binding affinity and / or specificity. In some embodiments, at most 1 to 5 conservative amino acid substitutions are made within the CDR domain. In other embodiments, at most 1 to 3 conservative amino acid substitutions are made within 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 having other post-translational modifications such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions within these 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 portions of the glycoprotein that can affect the conformation and presented three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech., 7:409-416, 1996). Oligosaccharides can also function to direct a given glycoprotein to a particular molecule based on a specific recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, CHO cells having tetracycline-regulated expression of a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), have been reported to have improved ADCC activity (Umana et al., Mature Biotech., 17:176-180, 1999).

[0330] Antibody glycosylation is typically either 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 carbohydrate moieties 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 one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0331] The addition of glycosylation sites to an antibody is conveniently achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be done by the addition or substitution of one or more serine or threonine residues relative to the sequence of the original antibody (for O-linked glycosylation sites).

[0332] The glycosylation pattern of an antibody can also be altered without changing the underlying nucleotide sequence. Glycosylation generally depends on the host cell used to express the antibody. Since the cell types used for the expression of recombinant glycoproteins, such as antibodies, as potential therapeutic agents are rarely native cells, modification of the glycosylation pattern of an antibody can be expected (see, for example, Hse et al., J. Biol. Chem., 272:9062-9070, 1997).

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

[0334] Other methods of modification include, but are not limited to, the use of coupling techniques known in the art, including enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, for the attachment of labels for immunoassays. Modified polypeptides are performed using procedures established in the art and can be 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 for human Fc gamma receptors, and is immunologically inert or partially inert, e.g., does not induce complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate macrophages, or has reduced activity (compared to an unmodified antibody) in any one or more of the following: induction of complement-mediated lysis, stimulation of antibody-dependent cell-mediated cytotoxicity (ADCC), or activation of microglia. Different modifications of the constant region can be used to achieve an optimal level and / or combination 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 mutation: 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 non-glycosylated instead of N-linked glycosylated. In some embodiments, the constant region is non-glycosylated instead of N-linked glycosylated by mutating a glycosylated amino acid residue or flanking residue that is part of the N-glycosylation recognition sequence within the constant region. For example, the 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 non - glycosylated instead of N - linked glycosylated. The constant region can be non - glycosylated enzymatically (such as removal of carbohydrates by the enzyme PNGase) or by expression in a glycosylation - defective host cell instead of N - linked glycosylation.

[0336] Other antibody modifications include antibodies modified as described in PCT Publication No. WO99 / 58572. These antibodies contain an effector domain having an amino acid sequence substantially homologous to all or part of the constant region of the human immunoglobulin heavy chain in addition to the binding domain directed to the target molecule. These antibodies can bind to the target molecule without inducing significant complement - dependent lysis or cell - mediated destruction of the target. In some embodiments, the effector domain can specifically bind to FcRn and / or FcγRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain C H 2 domains. Antibodies modified in this way are particularly suitable for use in long - term antibody therapy to avoid inflammatory and other adverse reactions to conventional antibody therapies.

[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 modulate the affinity of an antibody and to characterize the CDRs. One way to characterize the CDRs of an antibody and / or to alter (such as improve) the binding affinity of a polypeptide such as an antibody is called "library scanning mutagenesis". Generally, library scanning mutagenesis works as follows. One or more amino acid positions within a CDR are replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 etc.) amino acids using methods approved in the art. This generates a small library of clones (in some embodiments, one for each amino acid position being analyzed), each having a complexity of two or more members (when two or more amino acids are substituted per position). Generally, the library includes clones containing native (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 a target polypeptide (or other binding target), and candidates with increased, the same, decreased, or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore™ surface plasmon resonance analysis, which can detect differences in binding affinity of about two-fold or more. Biacore™ is particularly useful when the starting antibody already binds with a relatively high affinity, for example, with a K D of about 10 nM or less. Screening using Biacore™ surface plasmon resonance is described in the Examples herein.

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

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

[0341] In some embodiments, the library being screened contains substitutions within two or more positions, which may be within the same CDR or within two or more CDRs. Thus, the library can contain substitutions at two or more positions within one CDR. The library can contain substitutions at two or more positions within two or more CDRs. The library can contain substitutions at 3, 4, 5, or more positions, which are found within 2, 3, 4, 5, or 6 CDRs. The substitutions can be prepared using low redundancy codons. See, for example, Table 2 of Balint et al., Gene, 137(1):109-18, 1993.

[0342] The CDR can be CDRH3 and / or CDRL3. The CDR can be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and / or CDRH3. The CDR can be a Kabat CDR, a Chothia CDR, or an extended CDR.

[0343] Candidate(s) with improved binding can be sequenced to identify CDR substitution mutants (also referred to as "improved" substitutions) that improve affinity. Candidates that bind can also be sequenced to identify CDR substitutions that retain binding.

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

[0345] Library scanning mutagenesis also provides a means for characterizing CDRs insofar as frequent clones with improved binding, same binding, decreased binding, or no binding also provide information on the importance of each amino acid position with respect to the stability of the antibody - antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, that position is identified as a position not required for antigen binding. Conversely, if a CDR position retains binding in only a small percentage of substitutions, that position is identified as a position important for CDR function. Thus, the library scanning mutagenesis method generates information on positions within the CDR that can be changed to many different amino acids (including all 20 amino acids), and positions within the CDR that cannot be changed or can be changed to only 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 include additional substitutions at that position depending on the desired screening or selection method and the desired or acceptable library complexity. Additionally, if desired, adjacent amino acid positions can be randomized to at least two or more amino acids. Randomization of adjacent amino acids can allow for additional conformational flexibility within the mutant CDR, which in turn can allow or facilitate the introduction of a larger number of improved mutations. The library can also include substitutions at positions that did not show an improvement in 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, including selection using phage display, yeast display, and ribosome display.

[0348] The present invention also provides compositions comprising an antibody conjugated (e.g., linked) to an agent (such as biotin or avidin) that promotes coupling to a solid support. For simplicity, reference is generally made to the antibody 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 described herein. Linking (which generally involves immobilizing these components in proximity for at least administration) can be achieved in any number of ways. For example, a direct reaction between the agent and the antibody is possible if each has substituents that can react with the other. For example, one nucleophilic group, such as an amino or sulfhydryl group, can react with the other carbonyl-containing group, such as an anhydride or acid halide, or an alkyl group containing a good leaving group (e.g., a halide).

[0349] In another aspect, the present 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. The polynucleotide can be single-stranded (coding or antisense) or double-stranded and can be a DNA (genomic, cDNA, or synthetic) or RNA molecule. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one fashion, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can be present within the polynucleotides of the present invention, but are not required, and the polynucleotide may or may not be linked to other molecules and / or supports.

[0351] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a 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 decreased as compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assayed as described herein. The 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 the polynucleotide sequence encoding the native antibody or a 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. The comparison of 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" as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which the sequences 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 the arrays 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, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships; Dayhoff, M.O. (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, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS, 4:11-17; Robinson, E.D., 1971, Comb. Theor., 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol., 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 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, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include from 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent addition or deletion (i.e., gaps) as 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 at which the identical nucleic acid base or amino acid residue is 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., 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 the native gene or a portion or complement thereof. Such polynucleotide variants can hybridize to a naturally occurring DNA sequence (or complementary sequence) encoding a native antibody under moderately stringent conditions.

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

[0357] As used herein, "highly stringent conditions" or "high stringency conditions" refer to: (1) using low ionic strength and high temperature for washing, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) during hybridization, using a denaturing agent such as formamide, e.g., 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, together with 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) washing at 42°C in 0.2× SSC (sodium chloride / sodium citrate) and in 50% formamide at 55°C, followed by high stringency washing consisting of 0.1× SSC containing EDTA at 55°C, using at 42°C 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. One of ordinary skill in the art will recognize how to adjust the temperature, ionic strength, etc. as needed to account for factors such as probe length.

[0358] As a result of the degeneracy of the genetic code, those skilled in the art will appreciate that there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides carry minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated by the present invention. Furthermore, alleles of the genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have been altered as a result of one or more mutations such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and protein may or may 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 polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can generate the desired DNA sequence using the sequences provided herein and commercially available DNA synthesizers.

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

[0361] Alternatively, PCR enables the replication of DNA sequences. The PCR technique 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, as well as in PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0362] RNA can be obtained by using 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, as shown above in Sambrook et al., 1989.

[0363] Suitable cloning vectors can be constructed according to standard techniques or selected from a number of cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended to be used, but useful cloning vectors will generally have the ability to self-replicate, can have a single target for a particular restriction endonuclease, and / or can carry a gene for a marker that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacteriophage 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, etc.

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

[0365] Vectors containing the polynucleotide of interest can be introduced into host cells 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., if the vector is an infectious agent such as vaccinia virus). The choice of means for introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0366] The present invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an 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. subtilis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis). Preferably, the host cell expresses the cDNA at a level greater than about 5-fold, more preferably greater than 10-fold, even more preferably greater than 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, within the host cell. Screening of host cells for specific binding to BCMA or BCMA domains (e.g., domains 1-4) is performed by immunoassay or FACS. Cells overexpressing the antibody or protein of interest can be identified.

[0367] Methods of using bispecific antibodies [Therapeutic uses] The antibodies (e.g., BCMA, CD3, or bispecific) and antibody conjugates (e.g., BCMA antibody-drug conjugate) of the present invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods 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 a BCMA antibody conjugate described herein. Conditions associated with BCMA expression include, but are not limited to, abnormal 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 a method for treating a B cell-related cancer or malignant cells expressing a tumor antigen. In some embodiments, a method for treating a B cell-related cancer in a subject in need thereof is provided, comprising a) providing a bispecific antibody described herein, and b) administering the bispecific antibody to the patient. In some embodiments, a method for treating a condition associated with malignant cells expressing a tumor antigen in a subject is provided, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bispecific antibody described herein.

[0370] Accordingly, in some embodiments, provided is a method of treating cancer in a subject, the method comprising administering to a subject in need thereof, an effective amount of a composition comprising an antibody described herein (e.g., a BCMA or CD3-BCMA bispecific antibody) or a BCMA antibody conjugate. As used herein, cancer can be, but is not limited to, multiple myeloma, malignant plasma cell neoplasms, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Castleman 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 myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenström macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, mediastinal (thymic) primary 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 in the elderly, diffuse large B-cell lymphoma associated with inflammation, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell related cancers including B-cell related lymphomas.

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

[0372] In some embodiments, 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, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate described herein.

[0373] In this specification, autoimmune disorders include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, diabetes (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, anti-synthetase 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, Behcet's disease, Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, hidradenitis suppurativa, 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 present invention provides an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody described herein (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate for treating a condition associated with BCMA expression in a subject in need thereof (e.g., cancer or an autoimmune disorder). In some embodiments, there is provided an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody described herein (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate for inhibiting tumor growth or progression in a subject having malignant cells that express BCMA. In some embodiments, there is provided an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody described herein (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate for inhibiting metastasis of malignant cells that express BCMA in a subject in need thereof. In some embodiments, there is provided an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody described herein (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate for inducing tumor regression in a subject having malignant cells that express BCMA.

[0375] In another aspect, the present 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 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 is provided for inhibiting tumor growth or progression in a subject having malignant cells that express BCMA. In some embodiments, an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein is provided for inhibiting metastasis of malignant cells that express BCMA in a subject in need thereof. In some embodiments, an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate described herein is provided for inducing tumor regression in a subject having malignant cells that express BCMA.

[0376] In another aspect, the present 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 autoimmune disorder). In some embodiments, 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 is provided. In some embodiments, 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 that express BCMA is provided. In some embodiments, 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 is provided.

[0377] In another aspect, methods are provided for detecting, diagnosing, and / or monitoring a condition associated with BCMA expression. For example, the antibodies described herein (e.g., BCMA or CD3-BCMA bispecific) can be labeled with detectable moieties such as contrast 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 as staining reagents.

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

[0379] In some embodiments, the additional form of therapy comprises administering one or more therapeutic agents in addition to the antibodies described herein (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates. 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®)), an angiogenesis inhibitor, a cytotoxic agent (e.g., anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone), taxanes (e.g., paclitaxel and docetaxel), dolastatin, duocarmycin, enediyne, geldanamycin, maytansine, puromycin, vinca alkaloids (e.g., vincristine), topoisomerase inhibitors (e.g., etoposide), tubulysin, pyrimidine analogs (e.g., fluorouracil), platinum-containing agents (e.g., cisplatin, carboplatin, and oxaliplatin), alkylating agents (e.g., melphalan, cyclophosphamide, or carmustine), and hemiasterlin), an immunomodulatory agent (e.g., prednisone and lenalidomide (REVLIMID®)), an anti-inflammatory agent (e.g., dexamethasone), an aromatase inhibitor (e.g., anastrozole, exemestane, letrozole, vorozole, formestane, or testolactone), a proteasome inhibitor (e.g., bortezomib such as [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid, or carfilzomib), and other agents such as tamoxifen, and can be chemotherapeutic agents.

[0380] For example, in some embodiments, a method of treating multiple myeloma comprises administering to a patient in need thereof an effective amount of a composition comprising an antibody described herein (e.g., a BCMA or CD3-BCMA bispecific antibody) or a BCMA antibody conjugate, 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. Accordingly, provided is a method of treating multiple myeloma comprising administering to a patient in need thereof an effective amount of a composition comprising an antibody described herein (e.g., a BCMA or CD3-BCMA bispecific antibody) or a BCMA antibody conjugate, and one or more other therapeutic agents selected from the group consisting of bortezomib, lenalidomide, carfilzomib, and doxorubicin. In some embodiments, the patient is refractory or relapsed to a previous multiple myeloma therapy.

[0381] An 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 limit the available techniques, but rather are intended to be illustrative. Thus, in some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate is administered to the individual by known methods such as intravenous administration, e.g., as a bolus or by continuous infusion over time, intramuscular, intraperitoneal, intrathecal, intracranial, transdermal, subcutaneous, intra-articular, sublingual, intra-synovial, insufflation, intrathecal, oral, inhalation, or via a topical route. Administration can be systemic, e.g., intravenous administration, or can be local. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. The liquid formulation can be sprayed directly or can be sprayed after reconstitution of a lyophilized powder. 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 lyophilized and inhaled as a micronized 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, e.g., infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, outer membrane 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] A variety of formulations of antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates 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 a pharmaceutically acceptable excipient can be present in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically active substances. For example, an excipient can provide form or viscosity, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for various osmolarities, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and nonparenteral drug delivery are shown in Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005.

[0384] In some embodiments, these agents are formulated for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). Thus, these agents can be combined with a pharmaceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, etc. The specific dosing 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., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). The antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can also be administered via inhalation, as described herein. Generally, with respect to the administration of antibodies (e.g., BCMA or CD3-BCMA bispecific) and BCMA antibody conjugates, the initial candidate dosage can be about 2 mg / kg. For the purposes of the present invention, typical daily dosages can 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 described 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 can be used. For repeated administration over several days or more, depending on the condition, the treatment is continued until the desired suppression of symptoms occurs or a sufficient therapeutic level is achieved, e.g., until tumor growth / progression or cancer cell metastasis is inhibited or delayed. Exemplary dosing regimens include administration of an initial dose of about 2 mg / kg of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate, followed by a maintenance dose of about 1 mg / kg weekly, or a maintenance dose of about 1 mg / kg every other week thereafter. Other exemplary dosing regimens include administration of escalating doses (e.g., an initial dose of 1 mg / kg and stepwise increases to higher doses one or more times every week or longer). Other dosing regimens can also be useful depending on the pattern of pharmacokinetic decay desired as realized by an expert. For example, in some embodiments, dosing once to four times per week is contemplated. In other embodiments, dosing once per month or once every other month or every three months is contemplated. The progress of this therapy is readily monitored by conventional techniques and assays. The dosing regimen (including the antibody (e.g., BCMA or CD3-BCMA bispecific used) or BCMA antibody conjugate) can vary over time.

[0386] Regarding the object of the present invention, the appropriate dosage of an antibody (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate will depend on the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate (or its composition) used, the type and severity of the condition being treated, whether the agent is administered for therapeutic purposes, previous therapies, the patient's medical history and response to the agent, the clearance rate of the agent administered to the patient, 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 that achieves the desired result is reached. The dosage and / or frequency can vary over the course of treatment. Empirical considerations such as half-life generally contribute to the determination of dosage. For example, an antibody that is compatible with the human immune system, such as a humanized antibody or a fully human antibody, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted over the course of therapy and generally, but not necessarily, is based on the treatment and / or suppression and / or remission and / or delay of the condition, such as inhibition or delay of tumor growth. 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 can be determined empirically in an individual who has received one or more administrations of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. The individual is given a gradually increasing dosage of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. Disease indicators can be employed to assess efficacy.

[0388] Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate by the methods of the present invention can be continuous or intermittent, e.g., depending on the recipient's physiological condition, whether the purpose of administration is therapeutic or prophylactic, and other factors known to skilled professionals. Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be essentially continuous over a preselected period of time or can be a series of spaced dosages.

[0389] In some embodiments, more than one antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be present. 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 can be present. Generally, these antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can have complementary activities that do not adversely affect each other. For example, one or more of the following antibodies can be used: a first BCMA or CD3 antibody directed to one epitope on BCMA or CD3 and a second BCMA or CD3 antibody directed to a different epitope on BCMA or CD3.

[0390] The therapeutic formulations of the antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates used by the present invention are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington, The Science and Practice of Pharmacy, 21st Edition, Mack Publishing, 2005). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used, and include buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (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, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins, etc.; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, etc.; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol, etc.; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0391] Liposomes containing an antibody (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate 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). The liposomes are extruded through a filter with a defined pore size so as to yield liposomes having the desired diameter.

[0392] The active ingredient can also be encapsulated, for example, by coacervation techniques or by interfacial polymerization, in microcapsules such as those prepared by colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, in hydroxyethylmethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, 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 antibodies, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent 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] Formulations for in vivo administration must be sterile. This can be readily accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibodies (such as BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate compositions are generally placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle.

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

[0396] To prepare solid compositions such as tablets, the principal active ingredient is admixed with a pharmaceutical carrier 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 homogeneous mixture of the compounds of the invention or non-toxic pharmaceutically acceptable salts thereof. When referring to these preformulation compositions as homogeneous, it means that the active ingredient is evenly dispersed 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 above type containing from 0.1 to about 500 mg of the active ingredient of the invention. Tablets or pills of the novel composition can be coated or otherwise compounded to provide dosage forms that afford the advantage of sustained action. For example, a tablet or pill can comprise inner and outer administration components, the latter being in the form of an outer coating over the former. The two components can be separated by an enteric layer that functions to withstand disintegration in the stomach and permits the inner component to pass intact or with delayed release into the duodenum. A variety of materials can be used for such enteric layers or coatings, such materials including several polymeric acids and mixtures of such polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0397] Suitable surfactants include, inter alia, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween (trademark) 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span (trademark) 20, 40, 60, 80, or 85). Compositions containing surfactants will advantageously contain from 0.05 to 5% surfactant, and can be between 0.1 and 2.5%. It will be understood that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, can be added if desired.

[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 the premixed emulsion composition or, alternatively, it may be dissolved in an emulsion formed by mixing 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 understood that other components such as glycerol or glucose may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g., between 5 and 20% oil. The fat emulsion may contain fat droplets between 0.1 and 1.0 μm, particularly between 0.1 and 0.5 μm, and may have a pH in the range of 5.5 - 8.0.

[0399] The emulsion composition can be one prepared by mixing an antibody (e.g., BCMA or CD3 - BCMA bispecific) or 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, and powders, in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as indicated above. In some embodiments, the composition is administered via the oral or nasal respiratory route for local or systemic effects. Preferably, the composition in a sterile pharmaceutically acceptable solvent can be nebulized by using a gas. The nebulized solution may be breathed directly from the nebulizer or the nebulizer may be attached to a face mask, tent, or intermittent positive pressure breathing machine. The solution, suspension, or powder composition can be preferably administered orally or nasally from a device that delivers the formulation in a suitable manner.

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

[0402] It is understood that the composition can comprise more than one antibody (e.g., BCMA or CD3 - BCMA bispecific) or a 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 include more than one BCMA antibody, CD3 - BCMA antibody, or BCMA antibody conjugate that recognize the same epitope, or different species of BCMA antibodies, CD3 - BCMA bispecific antibodies, or BCMA antibody conjugates 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 contain pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy, 21st Edition, 2005, Lippincott Williams and Wilkins, edited by K.E. Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the intended dosage and concentrations, and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins 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 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 a kit for use in the present method. The kit of the present invention comprises one or more containers containing a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate as described herein, and instructions for use according to any of the methods of the present invention described herein. Generally, these instructions include a description of the administration of the BCMA antibody, the CD3-BCMA bispecific antibody, or the BCMA antibody conjugate for the therapeutic treatment described above.

[0405] Instructions regarding the use of the BCMA antibody, the CD3-BCMA bispecific antibody, or the BCMA antibody conjugate described herein generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (e.g., a multiple-dose package), or a subunit dose. The instructions supplied with the kit of the present invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable.

[0406] The kit of the present invention is present in a suitable package. Suitable packages include, but are 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 minipumps, are also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable 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 can further contain a second pharmaceutically active agent.

[0407] The kit may provide additional components, such as buffers and interpretive information. Usually, the kit includes a container and a label or an accompanying document on or attached to the container.

[0408] The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the invention. In fact, various modifications of the invention will become apparent to those skilled in the art from the above description and fall within the scope of the appended claims.

Example

[0409] (Example 1) Determination of 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). An array of anti-BCMA antibodies was prepared using the amine coupling method on a Bio-Rad GLC sensor chip in the same manner as described by 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 channels were 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 minutes. 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 minutes in the ligand (vertical) direction. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 minutes in the analyte direction.

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

[0412] The sensorgrams were double referenced and fit to a 1:1 Langmuir using the 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 for various anti-BCMA antibodies of the present 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 antibody on BCMA-positive tumor cells This example demonstrates the binding of various BCMA antibodies of the present invention to BCMA-positive tumor cells.

[0421] The binding of human anti-hBCMA expressed in mouse IgG2a was assayed by flow cytometry in BCMA-expressing cells (KMS12BM, L363, MM1S, and KMS12PE). 250,000 cells were incubated with 0.5 μg of antibody in 100 μL of binding buffer (PBS (phosphate buffered saline) + 0.2% BSA (bovine serum albumin)) and then incubated 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_0.6 nM_C29, A02_Rd4_6 nM_C01, A02_Rd4_6 nM_C16, and P6E01 / H3TAQ).

[0422]

Table 9

[0423] (Example 3) Cytotoxicity of anti-BCMA ADC in BCMA-positive cells This example illustrates the efficacy of anti-BCMA ADC 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 subtypes engineered with a glutamine-containing transglutaminase (the "Q") tag (e.g., LCQ05, H7c, N297A, N297Q, N297A / H7c, N297Q / LCQ05) for drug-antibody ratios (DARs) of 2, 4, and 6. Respectively, TG17 corresponds to SEQ ID NO: 472 (LLQGPP), LCQ05 corresponds to SEQ ID NO: 474 (GGLLQGPP), H7c corresponds to SEQ ID NO: 454 (LLQG), and were 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 example, the transglutaminase tag can be engineered in the light chain, heavy chain, or a combination of the light and heavy chains. In other examples, the transglutaminase tag (e.g., Q) is engineered at 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 substituted with glutamine or alanine at position 297 of the BCMA antibodies of the present invention (N297Q or N297A). Next, anti-BCMA antibody conjugation to Aur0101, Aur3377, and Aur0131 was achieved via a microbial transglutaminase-catalyzed amidotransfer reaction between an anti-BCMA antibody having a glutamine or glutamine tag targeted at a specific site (e.g., the carboxyl or amino terminus of the heavy or light chain, position 297, or another site of the antibody) and an amine-containing derivative of the payload (e.g., MMAD, Aur0101, Aur3377, or Aur0131). In some examples, 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 has been found to have the unexpected effect of resulting in more homogeneous antibodies and payload conjugates, better intermolecular cross-linking between the antibody and the payload, and / or a significant decrease in intermolecular cross-linking with the glutamine tag on the C-terminus of the antibody light chain.

[0425] In the amidotransfer reaction, glutamine on the antibody acted as the acyl donor, and the amine-containing compound acted as the acyl acceptor (amine donor). A purified anti-BCMA antibody at a concentration of 1 - 150 μM was incubated with a 5 - 100 molar excess of acyl acceptor in the range of 5 μM - 15 mM in 10 - 1000 mM NaCl, pH range 6.2 - 8.8, and 25 mM MES, HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] or Tris HCl buffer, in the presence of 0.23 - 0.55% (w / v) Streptoverticillium mobaraense transglutaminase (ACTIVA™, Ajinomoto, Japan). The reaction conditions were adjusted for each acyl acceptor derivative, and optimal efficiency and specificity were typically observed with 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 at 20 - 37 °C for 1 - 24 hours, the antibody was purified using a standard chromatography method known to those skilled in the art, such as commercially available hydrophobic interaction chromatography from GE Healthcare, on a butyl Sepharose high performance (butyl HP) resin (GE Healthcare, Waukesha, WI).

[0426] Next, target-expressing (MM1.S, KMS12BM, and L363) cells were seeded in clear-bottom plates at 3000 cells / well. The cells were treated in triplicate with 4-fold serial dilution 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 the percentage of untreated controls. EC50 was calculated by Prism software. Table 8 shows that all human anti-BCMA antibodies of the invention conjugated to cytotoxic agents 0101, 3377, and 0131 by transglutaminase tag and linker exhibit potent cell killing activity in BCMA-expressing cells.

[0427]

Table 10

[0428] (Example 4) Anti-BCMA ADC induces tumor regression in an orthotopic multiple myeloma model This example illustrates the in vivo efficacy of anti-BCMA ADC in the MM1S orthotopic multiple myeloma model.

[0429] An in vivo efficacy study of BCMA ADC was conducted using the multiple myeloma cell line MM1.S expressing luciferase and GFP (green fluorescent protein) in an orthotopic model. Ten million MM1.S LucGFP cells were intravenously injected via the tail vein into female CB17 / SCID animals 6 - 8 weeks old. D-Luciferin (Regis Technologies, Morton Grove, IL) was intraperitoneally injected (200 μL per animal at 15 mg / mL), and then the animals were anesthetized with isoflurane, followed by whole-body bioluminescence imaging (BLI) to enable monitoring of the total tumor tissue mass. The bioluminescence signal emitted by the interaction of luciferase expressed by the tumor cells with luciferin was 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 - 3E6 for all animals, the animals were randomized and a single dose of a human anti-BCMA antibody conjugated with LCQ05 / K222R-vc0101 at the C-terminus of the antibody light chain and a control conjugate were administered by bolus tail vein injection. The animals were sacrificed when they presented with hind limb paralysis, an endpoint of the MM1.S orthotopic model. Figure 2 shows that various single-dose human anti-BCMA ADCs at 3 mg / kg, 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, inhibit tumor progression compared to a negative control (NNC).

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

[0431] (Example 5) The anti-BCMA ADC induces tumor regression and inhibition in an orthotopic multiple myeloma model This example also illustrates the in vivo efficacy of the anti-BCMA ADC in the MM1.S orthotopic multiple myeloma model.

[0432] An in vivo efficacy study of the BCMA ADC was conducted using the multiple myeloma cell line MM1.S expressing luciferase and GFP in an orthotopic model. Ten million MM1.S LucGFP cells were intravenously injected via the tail vein into female CB17 / SCID animals at 6 - 8 weeks of age. D - Luciferin (Regis Technologies, Morton Grove, IL) was injected intraperitoneally (200 μL per animal at 15 mg / mL), and then the animals were anesthetized with isoflurane, followed by whole - body bioluminescence imaging (BLI) to enable monitoring of the total tumor tissue mass. The bioluminescence signal emitted by the interaction of luciferase expressed by the tumor cells with luciferin was captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / second) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 1 - 3E6 for all animals, the 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. Single - dose human anti - BCMA ADC and control conjugate were administered by bolus tail vein injection. The animals were killed when they presented the endpoint of the MM1.S orthotopic model, hind - limb paralysis. Figure 3 shows that single - dose 1) H7c / N297A / K222R - amino - PEG6 - C2 - 0131 and 2) H7c / N297A / K222R - amino - PEG6 - C2 - 3377 conjugated human anti - BCMA L3.PY / P6E01 antibodies resulted in tumor regression.Human anti-BCMA L3.PY / P6E01 antibodies conjugated with single doses of 1) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 2) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, and 3) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101 resulted in tumor inhibition.

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

[0434] (Example 6) Anti-BCMA ADC induces tumor inhibition in an orthotopic multiple myeloma model This example also illustrates the in vivo efficacy of anti-BCMA ADC in the KMS12BM orthotopic multiple myeloma model.

[0435] An in vivo efficacy study of BCMA ADC was conducted using the multiple myeloma cell line KMS12BM expressing luciferase and GFP in an orthotopic model. Female NSG animals 6 - 8 weeks old were irradiated with 100 cGy, and 24 hours after irradiation, 10 million KMS12BM LucGFP cells were intravenously injected through the tail vein. D-Luciferin (Regis Technologies, Morton Grove, IL) was intraperitoneally injected (200 μL per animal at 15 mg / mL), and then the animals were anesthetized with isoflurane, followed by whole-body bioluminescence imaging (BLI) to enable monitoring of the total tumor tissue burden. The bioluminescence signal emitted by the interaction of luciferase expressed by the tumor cells and luciferin was captured by imaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / second) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 5E6 for all animals, the 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. Single-dose human anti-BCMA ADC and control conjugate were administered by bolus tail vein injection. The animals were killed when they lost more than 15% of their total body weight, which is the endpoint of the KMS12BM orthotopic model.Figure 4 shows that human anti-BCMA L3.PY / P6E01 antibodies 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 at a single dose resulted in tumor inhibition.

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

[0437] (Example 7) Dose-response curve of anti-BCMA ADC in the MM1S orthotopic model This example further illustrates the in vivo efficacy of anti-BCMA ADC 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 intravenously injected via the tail vein into female CB17 / SCID animals 6 - 8 weeks old. D-luciferin (Regis Technologies, Morton Grove, IL) was intraperitoneally injected (200 μL per animal at 15 mg / mL), and then the animals were anesthetized with isoflurane, followed by whole-body bioluminescence imaging (BLI) to enable monitoring of the total tumor tissue mass. The bioluminescence signal emitted by the interaction of luciferase expressed by the tumor cells with luciferin was 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, the 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 of the control conjugate N297Q / K222R-AcLys-VC-0101. Single-dose human anti-BCMA ADC and control conjugate were administered by bolus tail vein injection. Animals were sacrificed when they presented the endpoint of the MM1.S orthotopic model, hind limb paralysis. Figure 5 shows that the single-dose human anti-BCMA COMBO_Rd4_0.6 nM_C29 antibody conjugated to the above groups 1)-4) resulted in tumor regression starting at 0.1 mg / kg and tumor inhibition of up to 100 days starting at 0.75 mg / kg.

[0439] Therefore, 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 generation and purification of the heterodimeric antibodies of the present application.

[0441] The variable regions of human-specific anti-CD3 antibodies were cloned into human IgG1 or IgG2ΔA containing the following mutations 221R, 228R, and K409R; or 223R, 225R, 228R, and K409R, respectively, and named 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 named hIgG1EEE or hIgG2ΔA-EEEE.

[0443] The heterodimer was prepared by incubating anti-CD3 IgG1 or IgG2ΔA with the hIgG1 RRR or IgG2ΔA-RRRR mutation together with the anti-target antibody with the hIgG1EEE or hIgG2ΔA-EEEE mutation in PBS containing 1 mM or 2 mM GSH at 37 °C for 24 hours as described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545). The heterodimer was 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-homo dimers 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 at a flow rate of 1 ml / min with a gradient of 25% - 75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a period of 20 minutes. Larger scale purification of Fc-heterodimers was carried out 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 at a flow rate of 1 ml / min with a gradient of 15% - 75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a period of 60 minutes.

[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). An array of anti-CD3 antibodies was prepared on a Bio-Rad GLC sensor chip using the amine coupling method as described by 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 channels were 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 minutes. IgG was immobilized on the activated spots at 20 μg / mL in 10 mM acetate pH 4.5 buffer and at 30 μg / mL in the ligand (vertical) direction by injecting these for 1.5 minutes. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 minutes in the analyte direction.

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

[0448] The sensorgrams were double-referenced and fitted to a 1:1 Langmuir using the mass transport kinetic titration model in BIAevaluation software version 4.1.1 (GE Lifesciences, Piscataway, NJ). The kinetic and affinity parameters of various anti-CD3 antibodies of the present 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 in CD20+ cells.

[0451] Cynomolgus monkey studies were conducted at Charles River Laboratories, Preclinical Services Nevada, in accordance with the Institutional Animal Care and Use Committee. Animals (n = 2) were dosed with a bispecific anti-CD20 / h2B4 antibody at doses of 500 μg / kg, 100 μg / kg, 20 μg / kg, 2 μg / kg, 0.2 μg / kg, or 0.02 μg / kg via intravenous bolus injection. 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 peripheral vessels not used for i.v. dosing.

[0452] The 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 minutes at room temperature, and white blood cells were pelleted by centrifugation. Cells were stained for 1 hour at 4°C with a cocktail containing fluorescently labeled antibodies recognizing cynomolgus CD19 (Beckman Coulter), CD45, CD4, CD8, and Ki67 (BD Biosciences) in PBS + 2% FBS. For Ki67 analysis, cells were first stained with CD4 and CD8, then fixed / permeabilized with the BD cyotfix / cytoperm kit (BD Biosciences) according to the manufacturer's instructions, and then Ki67 was stained intracellularly. Acquisition of cells on a BD LSRII flow cytometer was performed immediately after staining.

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

[0454] The number of CD8+ T cells obtained was graphed in Figures 7A - 7F as a percentage of the pre-study CD8+ T cell number. After the initial relocalization, T cell levels recovered to or above baseline levels over the duration of the study.

[0455] (Example 11) Flow Cytometry of Human Anti-CD3 Bispecific Antibody on CD8+ T Cells This example demonstrates the efficacy of a monovalent anti-CD3 antibody on T cell kinetics and activation.

[0456] As described in Example 10, a cynomolgus monkey study was conducted, and the efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Cynomolgus monkeys (n = 2) were dosed i.v. weekly with anti-CD20 / h2B4 or NNC (non-specific antibody) / h2B4 at 0.2 μg / kg. In contrast to the CD20-targeted bispecific antibody, NNC / h2B4 has no or near-no effect on the kinetics of CD8+ T cells in the blood as measured by flow cytometry. Ki67 was used as a marker of T cell activation.

[0457] The obtained T cell numbers were graphed in FIGS. 8A - 8B as a percentage of the pre-study CD8+ T cell numbers. In cynomolgus monkeys dosed with the CD20 / h2B4 bispecific antibody, Ki67+ T cells increased and peaked between 3 and 7 days after 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] As described in Example 10, a cynomolgus monkey study was conducted, and the efficacy was determined by measuring T cells in peripheral blood by flow cytometry. The bispecific antibodies were generated 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 i.v. dose at 0.2 μg / kg.

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

[0461] (Example 13) In vitro study of bispecific antibodies on T cell-mediated killing of BCMA-positive cells This example illustrates the in vitro cytotoxicity of anti-BCMA / CD3 hIgG2ΔA bispecificity 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), as well as 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 derived from PBMC were negatively selected using the Pan T Cell Isolation Kit, human (Miltenyi, San Diego, CA). Target-expressing (KMS12PE, L363, and Molp8) cells and CD3+ T cells were seeded into clear U-bottom plates at 20,000 and 100,000 cells / well, respectively. Cells were treated in triplicate with 10-fold serial dilution of bispecific antibodies. Cell death was determined 20 hours after treatment by the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison WI). The cytotoxicity of the cells was determined as a percentage of the untreated effector + target control wells. EC50 was calculated by Prism software. Table 10 shows that all human anti-BCMA_H2B4 bispecific antibodies exhibit cytotoxic activity in BCMA-expressing cells.

[0464]

Table 12

[0465] (Example 14) In vitro characterization of mouse hybridoma-cloned anti-CD3 antibodies T...

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: 151, 156, or 157, (ii) a VH CDR2 comprising SEQ ID NO: 158 or 159, 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:

225.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising SEQ ID NO: 151, (ii) a VH CDR2 comprising SEQ ID NO: 159, 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:

225.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a VH region comprising SEQ ID NO: 112 and a VL region comprising SEQ ID NO:

38.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable (VH) region having an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:486, and a light chain variable (VL) region having an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:

485.

5. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. 5. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. A vector comprising the polynucleotide of claim 6.

8. 5. An isolated host cell recombinantly producing the antibody or antigen-binding fragment of any one of claims 1 to 4.

9. 10. A method for producing an antibody or antigen-binding fragment, comprising culturing a host cell of claim 8 under conditions that result in the production of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cell or culture.

10. 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 any one of claims 1 to 4 or a pharmaceutical composition described in claim 5.

11. The method of claim 10, wherein the condition is cancer.

12. 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, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, disease, 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, 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 in the elderly 12. The method of claim 11, wherein the cancer is a B cell-related cancer selected from the group consisting of diffuse large B cell lymphoma, inflammation-associated diffuse large B cell lymphoma, 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's lymphoma, unclassified B cell lymphoma with characteristics intermediate between diffuse large B cell lymphoma and classical Hodgkin's lymphoma, and other B cell-related lymphomas.

13. 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 any one of claims 1 to 4 or a pharmaceutical composition described in claim 5.

14. 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 any one of claims 1 to 4 or a pharmaceutical composition described in claim 5.

15. 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 any one of claims 1 to 4 or a pharmaceutical composition described in claim 5.

16. The method of claim 10, wherein the condition is an autoimmune disorder.

17. 17. The method of claim 16, wherein the autoimmune disorder is systemic lupus erythematosus or rheumatoid arthritis.

Images