Therapeutic antibodies and their uses

Abstract

The present invention relates to antibodies, such as full-length antibodies or antigen-binding fragments thereof, that specifically bind to BCMA (B-cell maturation antigen) and CD3 (cluster of differentiation 3). The present invention also relates to antibody conjugates (e.g., antigen-drug-conjugates) comprising BCMA antibodies, compositions comprising BCMA antibodies, and methods of using BCMA antibodies and their conjugates to treat disease conditions associated with cells expressing BCMA (e.g., cancer or autoimmune diseases). The present invention further relates to heteromultimeric antibodies that specifically bind to CD3 and tumor cell antigens (e.g., bispecific antibodies that specifically bind to CD3 and BCMA). Compositions comprising such heteromultimeric antibodies, methods for preparing and purifying such heterodimeric antibodies, and their use in diagnosis and treatment are also provided.

Description

Field of the Invention

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

[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 harboring the t(4;16) translocation. This gene is selectively expressed in the B-cell lineage, with highest expression in antibody-secreting plasmablasts and plasma cells. BCMA binds two ligands, B-cell activating factor (BAFF) (also known as B-lymphocyte stimulator (BLyS) and APOL-related leukocyte-expressed ligand (TALL-1)) and proliferation-inducing ligand (APRIL), with affinities of 1 μM and 16 nM, respectively. Binding of APRIL or BAFF to BCMA promotes a signaling cascade involving NF-κB, Elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, which generates signals for cell survival and proliferation.

[0003] BCMA is also expressed on malignant B cells and several cancers involving B lymphocytes, including multiple myeloma, plasmacytoma, Hodgkin lymphoma, and chronic lymphocytic leukemia. In autoimmune diseases involving plasmablasts, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, BCMA-expressing antibody-producing cells secrete self-attacking autoantibodies.

[0004] In the case of multiple myeloma, approximately 24,000 new cases are diagnosed in the United States each year, and this number accounts for approximately 15% of all newly diagnosed blood cancers in the United States. Multiple myeloma causes an average of 11,000 deaths each year, and the average 5-year survival rate is approximately 44%, with a median survival of 50-55 months. Current treatments for multiple myeloma focus on inducing plasma cell apoptosis and / or reducing osteoclast activity (e.g., chemotherapy, thalidomide, lenalidomide, bisphosphonates, and / or proteasome inhibitors such as bortezomib). However, multiple myeloma remains an incurable disease, and nearly all patients develop resistance to these agents and eventually relapse. Therefore, alternative treatments for multiple myeloma, such as the use of anti-BCMA antagonists, including antibodies and other immunotherapeutics (e.g., bispecific antibodies or antibody-drug conjugates), are promising. SUMMARY OF THE INVENTION

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

[0006] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to B-cell maturation antigen (BCMA), wherein the antibody comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region 1 (CDR1) comprising the sequence SYX1MX2, wherein X1 is A or P; and X2 is T, N, or S (SEQ ID NO: 301), GFTFX1SY, wherein X1 is G or S (SEQ ID NO: 302), or GFTFX1SYX2MX3, wherein X1 is G or S, X2 is A or P; and X3 is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequence AX1X2X3X4GX5X6X7X8YADX9X 10 KG, wherein X1 is I, V, T, H, L, A, or C; X2 is S, D, G, T, I, L, F, M, or V; X3 is G, Y, L, H, D, A, S, or M; X4 is S, Q, T, A, F, or W; X5 is G or T; X6 is N, S, P, Y, W, or F; X7 is S, T, I, L, T, A, R, V, K, G, or C; X8 is F, Y, P, W, H, or G; X9 is V, R, or L; and X 10is G or T (SEQ ID NO: 305), or XXXXXXXXXXXXXXX, wherein X1 is S, V, I, D, G, T, L, F, or M; X2 is G, Y, L, H, D, A, S, or M; X3 is S, G, F, or W; X4 is G or S; X5 is G or T; and X6 is N, S, P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprising the sequence VSPIXXXXXX, wherein X1 is A or Y; X2 is A or S; and X3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMXXXX, wherein X1 is D, S, T, or A; and X2 is I, S, L, P, or D (SEQ ID NO: 308); and / or (b) a light chain variable (VL) region comprising (i) a VL CDR1, containing the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 , wherein X1 is R, G, W, A or C; X2 is A, P, G, L, C or S; X3 is S, G or R; X4 is Q, C, E, V or I; X5 is S, P, G, A, R or D; X6 is V, G, I or L; X7 is S, E, D, P or G; X8 is S, P, F, A, M, E, V, N, D or Y; X9 is I, T, V, E, S, A, M, Q, Y, H, R or F; X 10 Y or F; X 11 is L, W or P; and X 12 is A, S or G (SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X1ASX2RAX3, wherein X1 is G or D; X2 is S or I; and X3 is T or P (SEQ ID NO: 310); and (iii) a VL CDR3 comprising the sequence QQYX1X2X3PX4T, wherein X1 is G, Q, E, L, F, A, S, M, K, R or Y; X2 is S, R, T, G, V, F, Y, D, A, H, V, E, K or C; X3 is W, F or S; and X4 is L or I (SEQ ID NO: 311). NO:311), or QQYX1X2X3PX4, wherein X1 is G, Q, E, L, F, A, S, M, R, K or Y; X2 is S, R, T, G, R, V, D, A, H, E, K, C, F or Y; X3 is W, S or F; and X4 is L or I (SEQ ID NO:312).

[0007] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BCMA, wherein the antibody comprises: a VH region comprising a VH CDR1, a VH CDR2, and a VH sequence set forth in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 125, 127, 313, 314, 363, or 365. CDR3; and / or VL region comprising 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, 8 1, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, 316, or 364.In some embodiments, the VH region comprises (i) a VH CDR1 comprising SEQ ID NO: 150, 151, 152, 156, or 157; (ii) a VH CDR2 comprising SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 203, or 204; and (iii) a VH CDR3 comprising SEQ ID NO: 155, 161, 197, 205, or 164; and / or wherein the VL region comprises (i) a VL CDR1 comprising SEQ ID NO: 1 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 more 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 more 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 No. PTA-122094. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC Accession No. PTA-122093.

[0008] In another aspect, the present invention provides an isolated antibody comprising a tag containing the acyl donor glutamine 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). NO:469), LLQLLQ (SEQ ID NO:470), LLQGR (SEQ ID NO:471), LLQGPP (SEQ ID NO:472), LLQGPA (SEQ ID NO:473), GGLLQGPP (SEQ ID NO:474), GGLLQGA (SEQ ID NO:475), LLQGPGK (SEQ ID NO:476), LLQGPG (SEQ ID NO:477), LLQGP (SEQ ID NO:478), LLQP (SEQ ID NO:479), LLQPGK (SEQ ID NO:480), LLQAPGK (SEQ ID NO:481), LLQGAPG (SEQ ID NO:482), LLQGAP (SEQ ID NO:483) and LLQLQG (SEQ ID NO:484).

[0009] In one variation, the invention provides an isolated antibody comprising a tag containing an acyl donor glutamine and an amino acid modification at position 222, 340, or 370 of a BCMA antibody of the 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 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, aminohexanoyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}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 as described herein, wherein the antibody or antigen-binding fragment is conjugated to an agent, wherein the agent is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide. In some embodiments, the agent is a cytotoxic agent, including but not limited to anthracyclines, auristatins, camptothecins, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indoline-benzodiazepines, dimer, maytansine, puromycin, pyrrolobenzodiazepine dimer, taxane, vinca alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide and stereoisomers, isosteres, 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-oxoheptyl alkyl-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-prolyl-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-oxoheptane-4-yl]-N-methyl-L-valinamide ) or 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).

[0012] In some embodiments, the present invention provides a conjugate comprising the formula: antibody-(tag containing an acyl donor glutamine)-(linker)-(cytotoxic agent). In some embodiments, the tag containing an acyl donor glutamine comprises the amino acid sequence LLQG (SEQ ID NO: 319) and / or GGLLQGPP (SEQ ID NO: 339), and wherein the linker comprises acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl or amino-PEG6-C2. In some embodiments, the conjugate is selected from 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 preparing a BCMA antibody as described herein, the method comprising culturing a host cell under conditions that result in the production of the BCMA antibody, and isolating the BCMA antibody from the host cell or culture.

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

[0015] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the 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 comprises a VH CDR2, a VH CDR3, and a VH CDR4 of 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; NO: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399. In some embodiments, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388; and / or a light chain variable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387. In some embodiments, the VH region comprises: (i) a VH complementarity determining region 1 (CDR1) comprising the sequence 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: NO: 335, 406, 423, 429 or 451; and / or a light chain variable (VL) region comprising (i) a 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 set forth in SEQ ID NO: 331, 332, 333, 401, 407, or 408; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 336, 404, 405, or 417; and iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 335 or 406; and / or a light chain variable (VL) region comprising (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO: 343 or 441; (ii) a VL CDR2 comprising the sequence set forth in SEQ ID NO: 341 or 436; and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 342 or 439. In some embodiments, the antibody comprises a VH region produced by an expression vector having ATCC Accession No. PTA-122513. In some embodiments, the antibody comprises a VL region produced by an expression vector having ATCC Accession No. PTA-122512.

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

[0017] In another aspect, the present invention provides a bispecific antibody, wherein the bispecific antibody is a fully human antibody, comprising a first antibody variable domain of the bispecific antibody capable of recruiting human immune effector cell activity by specifically binding to an effector antigen located on human immune effector cells, and comprising 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 heavy chain variable (VH) region comprising VH CDR1, VH CDR2 and VH CDR3 of the VH sequence shown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384386, 388, 390, 392, 394, 396, 398 or 400; and / or a light chain variable (VL) region comprising SEQ ID NO: 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 heavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the VH sequence shown in SEQ ID NO: 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: NO: 335, 406, 423, 429 or 451; and / or a light chain variable (VL) region comprising (i) a 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 heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388; and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387; and the second antibody variable domain comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 112; and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 38.

[0018] In some embodiments, the second antibody variable domain comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region 1 (CDR1) comprising the sequence SYX1MX2, wherein X1 is A or P; and X2 is T, N, or S (SEQ ID NO: 301), GFTFX1SY, wherein X1 is G or S (SEQ ID NO: 302), or GFTFX1SYX2MX3, wherein X1 is G or S, X2 is A or P; and X3 is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequence AX1X2X3X4GX5X6X7X8YADX9X 10 KG, wherein X1 is I, V, T, H, L, A, or C; X2 is S, D, G, T, I, L, F, M, or V; X3 is G, Y, L, H, D, A, S, or M; X4 is S, Q, T, A, F, or W; X5 is G or T; X6 is N, S, P, Y, W, or F; X7 is S, T, I, L, T, A, R, V, K, G, or C; X8 is F, Y, P, W, H, or G; X9 is V, R, or L; and X 10is G or T (SEQ ID NO: 305), or XXXXXXXXXXXXXXX, wherein X1 is S, V, I, D, G, T, L, F, or M; X2 is G, Y, L, H, D, A, S, or M; X3 is S, G, F, or W; X4 is G or S; X5 is G or T; and X6 is N, S, P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprising the sequence VSPIXXXXXX, wherein X1 is A or Y; X2 is A or S; and X3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMXXXX, wherein X1 is D, S, T, or A; and X2 is I, S, L, P, or D (SEQ ID NO: 308); and / or (b) a light chain variable (VL) region comprising (i) a VL CDR1, containing the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 , wherein X1 is R, G, W, A or C; X2 is A, P, G, L, C or S; X3 is S, G or R; X4 is Q, C, E, V or I; X5 is S, L, P, G, A, R or D; X6 is V, G or I; X7 is S, E, D or P; X8 is S, P, F, A, M, E, V, N, D or Y; X9 is I, T, V, E, S, A, M, Q, Y, H or R; X 10 Y or F; X 11 is L, W or P; and X 12is A, S or G (SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X1ASX2RAX3, wherein X1 is G or D; X2 is S or I; and X3 is T or P (SEQ ID NO: 310); and (iii) a VL CDR3 comprising the sequence QQYX1X2X3PX4T, wherein X1 is G, Q, E, L, F, A, S, M, K, R or Y; X2 is S, R, T, G, V, F, Y, D, A, H, V, E, K or C; X3 is W, F or S; and X4 is L or I (SEQ ID NO: 311). NO:311), or QQYX1X2X3PX4, wherein X1 is G, Q, E, L, F, A, S, M, R, K or Y; X2 is S, R, T, G, R, V, D, A, H, E, K, C, F or Y; X3 is W, S or F; and X4 is L or I (SEQ ID NO:312). In some embodiments, the second antibody variable domain comprises a heavy chain variable (VH) region comprising (i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 150, 151, 152, 156, 157, 348, 349, 353, 354, or 355; (ii) a VH CDR2 comprising 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, 203, 204, 350, 351, 356, or 357; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: NO: 155, 161, 197, 205, 164 or 352 or 358; and / or wherein the light chain variable (VL) region comprises (i) a VL CDR1 comprising 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 comprising the sequence shown in SEQ ID NO: 221, 359 or 362; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 231, 359 or 362. 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 heavy chain variable (VH) region comprising (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, 417, 404 or 405; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335 or 406; and / or a light chain variable (VL) region comprising (i) a 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: NO: 342 or 439; and (b) the second antibody variable domain comprises a heavy chain VH region comprising a heavy chain variable (VH) region comprising (i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156 or 157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or 159; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 155; and / or wherein the light chain variable (VL) region comprises (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 225.

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

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

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

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

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

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

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

[0027] In some embodiments, the antibodies described herein comprise a constant region. In some embodiments, the antibodies described herein are of human IgG1, IgG2, or IgG2Δa, IgG3, or IgG4 subclass. In some embodiments, the antibodies described herein comprise a glycosylated constant region. In some embodiments, the antibodies described herein comprise a constant region that has increased binding affinity for one or more human Fcγ receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A-Figure 1DDual-referenced sensorgrams with fitted curves for the interaction between selected anti-BCMA antibodies of the invention and human BCMA are shown.

[0029] Figure 2 Figure 2 shows in vivo efficacy studies of various anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model, including P6E01_VHVL-AcLys-Val-Cit-PABC-Aur0101; P5A2_VHVL-AcLys-Val-Cit-PABC-Aur0101; P5C1_VHVL-AcLys-Val-Cit-PABC-Aur0101; P4G4-AcLys-Val-Cit-PABC-Aur0101; and P1A11-AcLys-Val-Cit-PABC-Aur0101. NNC is a negative control non-BCMA antibody. "LCQ05" and "LCQ04" correspond to glutamine-containing transglutaminase tags, SEQ ID NOs: 474 and 475, respectively.

[0030] Figure 3 Figure 2 shows in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model, including L3.PY / P6E01 antibody conjugated to 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMA antibody. "LCQ05" and H7c correspond to the glutamine-containing transglutaminase tags SEQ ID NO: 474 and SEQ ID NO: 454, respectively.

[0031] Figure 4Also shown are in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model, including L3.PY / P6E01 antibodies conjugated to 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMA antibody (Antibody-N297Q / K222R-AcLys-VC-PABC-0101). "LCQ05" and H7c correspond to glutamine-containing transglutaminase tags SEQ ID NO: 474 and SEQ ID NO: 454, respectively.

[0032] Figure 5 Also shown is the in vivo efficacy of an anti-BCMA ADC in the MM1S orthotopic multiple myeloma model. The anti-BCMA antibody COMBO_Rd4_0.6nM-C29 ("Combo C29 DI") was conjugated to H7c / N297A / K222R-amino-PEG6-C2-131 at doses ranging from 0.1 mg / kg, 0.38 mg / kg, 0.75 mg / kg, and 1.5 mg / kg, compared to 3 mg / kg for the control non-BCMA antibody NNC (antibody-N297Q / K222R-AcLys-VC-PABC-0101). H7c corresponds to the transglutaminase tag containing glutamine, SEQ ID NO: 454.

[0033] Figures 6A-6F In vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys is shown. B cell depletion after a single dose of the bispecific antibody is shown as a percentage of pre-study counts.

[0034] Figures 7A-7F In vivo efficacy of an anti-CD3 / anti-CD20 bispecific antibody in cynomolgus monkeys is shown. CD8+ T cell kinetics were tracked after a single dose of the bispecific antibody.

[0035] Figure 8A and Figure 8B In vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys is shown. The effects of monovalent CD3 antibodies on T cell kinetics and proliferation were analyzed.

[0036] Figures 9A-9DIn vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys is shown. The effect of anti-CD3 arm affinity on B cell depletion was analyzed.

[0037] Figure 10A and 10B Selected anti-CD3 antibodies are shown with thymidine incorporation readout on human and cynomolgus monkey PBMCs.

[0038] Figures 11A-11D All human anti-EpCam_h2B4 bispecific antibodies were shown to have cell-killing activity in vitro.

[0039] Figure 12 We show that a single dose of a human anti-BCMA / CD3 bispecific antibody leads to tumor regression in an orthotopic MM1.S myeloma model in a dose-dependent manner.

[0040] Figure 13 We show that two doses of a human anti-BCMA / CD3 bispecific antibody result in increased tumor regression in an orthotopic Molp8 myeloma model.

[0041] Figure 14 showed that in an orthotopic Molp8 tumor model, the anti-BCMA / CD3 bispecific antibody, alone or in combination with the standard of care for multiple myeloma (lenalidomide or bortezomib), was more effective than the combined lenalidomide and bortezomib.

[0042] Figures 15A-15C Carfilzomib, lenalidomide, and doxorubicin, respectively, showed that anti-BCMA / CD3 bispecific antibodies had no negative impact on the function of OPM2 cells compared with anti-BCMA / CD3 bispecific antibodies alone.

[0043] Figure 16 A synergistic effect was shown on the anti-BCMA / CD3 bispecific antibody function when combined with carfilzomib and lenalidomide compared to each molecule alone. Detailed Description of the Invention

[0044] The invention disclosed herein provides antibodies and antibody conjugates (e.g., antibody-drug conjugates) that specifically bind to BCMA (e.g., human BCMA). The present invention also provides polynucleotides encoding these antibodies and conjugates, compositions comprising these antibodies and conjugates, and methods for preparing these antibodies and conjugates. In addition, 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 tumor antigens (e.g., BCMA). The present invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods for preparing and using these antibodies. The present invention further provides methods for treating disease conditions associated with malignant BCMA expression in individuals, such as cancer or autoimmune diseases, using antibodies (e.g., BCMA, CD3, or bispecific antibodies) or conjugates thereof (BCMA antibody-drug conjugates) as described herein.

[0045] General techniques

[0046] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are well described in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, HμMana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) PlenμM Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths, and DG Newell, eds., 1993-1998) J.Wiley and Sons; Methods in Enzymology (Academic) Press, Inc.); Handbook of Experimental Immunology (DMWeirand CCBlackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMillerand MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubelet al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: alaboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0047] definition

[0048] "Antibody" is an immunoglobulin molecule that is capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies (including, for example, shark and camel antibodies), and fusion proteins comprising antibodies, as well as any other modified configurations of immunoglobulin molecules comprising antigen recognition sites. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or their subclasses), and the antibodies do not necessarily have to be of any particular class. Immunoglobulins can be divided into different classes based on the amino acid sequence of the constant region of the antibody heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into several subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0049] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retains 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 a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab; Fab'; F(ab')2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0050] "Preferentially binding" or "specifically binding" (used interchangeably herein) to an antibody, antibody conjugate, or polypeptide to a target (e.g., BCMA protein or CD3 protein) are terms well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular molecule or substance more frequently, more rapidly, for a greater duration, and / or with a greater affinity than with an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more rapidly, and / or for a greater duration than the antibody binds to other substances. For example, an antibody that specifically or preferentially binds to a BCMA epitope or CD3 epitope is an antibody that binds to that epitope with greater affinity, avidity, more rapidly, and / or for a greater duration than it binds to other BCMA epitopes, non-BCMA epitopes, CD3 epitopes, or non-CD3 epitopes. By reading this definition, it should also be understood that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0051] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are tightly held together by the FRs, as well as with those from other chains, to help form the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDRs can refer to CDRs defined by either method or by a combination of the two methods.

[0052] The "CDRs" of a variable domain are amino acid residues within the variable region identified according to the definitions of Kabat, Chothia, the accumulation of Kabat and Chothia, AbM, contact and / or conformational definitions, or any method of CDR determination known in the art. Antibody CDRs can 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 DC. The positions of CDRs can also be identified as the structural loop structures originally described by Chothia and others. See, for example, Chothia et al., Nature 342:877-883, 1989. Other methods of CDR identification include the "AbM definition," which is a compromise between Kabat and Chothia and utilizes the Oxford Molecular AbM antibody modeling software (now ) derived from, or based on observed antigen contacts, the "contact definition" of CDRs is shown in MacCall μM et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the "conformational definition" of CDRs, the positions of CDRs can be identified as residues that make enthalpic contributions 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 overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art, including combinations of methods. The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact and / or conformational definitions.

[0053] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody comprising the population is identical, except for the possible naturally occurring mutations that may exist in small quantities. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is only directed against a single determinant on the antigen. The modifier "monoclonal" represents that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring the preparation of antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, Nature 256:495,1975, or can be prepared by the recombinant DNA method described in US Pat. No. 4,816,567. Monoclonal antibodies can also be separated from the phage library produced by the technology described in, for example, McCafferty et al., Nature 348:552-554,1990.

[0054] As used herein, "humanized" antibody refers to a form of non-human (e.g., mouse) antibody, which is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) comprising a minimal sequence derived from a non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the complementary determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) such as a mouse, rat, or rabbit with desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not found in the receptor antibody or the introduced CDR or framework sequences, but include the residues to further refine and optimize antibody performance. In general, humanized antibodies comprise substantially all at least one, usually two variable domains, wherein all or substantially all CDR regions correspond to those regions of non-human immunoglobulins, and all or substantially all FR regions are those regions of human immunoglobulin consensus sequences. Humanized antibodies most preferably also comprise at least a portion of immunoglobulin constant region or domain (Fc), typically the constant region of human immunoglobulin. Preferably there is an antibody in the Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDRL3, CDR H1, CDR H2 or CDR H3) that the original antibody is changed, which is also referred to as "deriving from" one or more CDRs from the one or more CDRs of the original antibody.

[0055] As used herein, "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or an antibody prepared using any technique for preparing human antibodies known to those skilled in the art or disclosed herein. This definition of human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a mouse light chain and a human heavy chain polypeptide. Human antibodies can be prepared using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein 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 prepared by immunizing animals into which human immunoglobulin loci have been transgenically introduced in place of endogenous loci, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be recovered from individuals or single-cell clones of cDNA, or can be immunized in vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1): 86-95, 1991; and US Pat. No. 5,750,373.

[0056] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0057] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid chains of any length, preferably relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, may contain modified amino acids, and / or may be interrupted by non-amino acids. The term also encompasses amino acid chains that are natural or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that a polypeptide can exist as a single chain or related chains.

[0058] A "monovalent antibody" contains one antigen binding site (eg, IgG or Fab) per molecule. In certain cases, a monovalent antibody may have more than one antigen binding site, but the binding sites are from different antigens.

[0059] "Monospecific antibodies" contain two identical antigen-binding sites per molecule (e.g., IgG), whereby the two binding sites bind to the same epitope on the antigen. Therefore, they compete with each other for binding to a single antigen molecule. Most antibodies found in nature are monospecific. In some cases, monospecific antibodies can also be monovalent antibodies (e.g., Fab).

[0060] 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.

[0061] "Bispecific" or "dual specificity" is a hybrid antibody with two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which can be located on the same or different protein targets.

[0062] "Bifunctional" antibodies are antibodies that have identical antigen-binding sites (ie, identical amino acid sequences) in both arms, but each binding site can recognize two different antigens.

[0063] A "heteromultimer," "heteromultimeric complex," or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the amino acid sequence of the second polypeptide differs from that of the first polypeptide by at least one amino acid residue. A heteromultimer may comprise a "heterodimer" formed by the first and second polypeptides, or may form a higher order tertiary structure in which polypeptides other than the first and second polypeptides are present.

[0064] A "heterodimer," "heterodimer protein," "heterodimer complex," or "heterodimer polypeptide" is a molecule comprising a first polypeptide and a second polypeptide, wherein the amino acid sequence of the second polypeptide differs from that of the first polypeptide by at least one amino acid residue.

[0065] As used herein, "hinge region," "hinge sequence," and variations thereof include those known in the art and described in, for example, 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; HμMphreys et al., J. Immunol. Methods (1997), 209:193-202.

[0066] As used herein, "immunoglobulin-like hinge region," "immunoglobulin-like hinge sequence," and variations thereof refer to the hinge region and hinge sequence of an immunoglobulin-like or antibody-like molecule (e.g., an immunoadhesin). In some embodiments, the immunoglobulin-like hinge region can be from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD, or IgM, including chimeric forms thereof, such as a chimeric IgG1 / 2 hinge region.

[0067] As used herein, the term "immune effector cell" or "effector cell" refers to a cell within the natural cell pool of the human immune system that can be activated to affect the viability of a target cell. The viability of a target cell can include the ability of the cell to survive, proliferate, and / or interact with other cells.

[0068] The antibodies of the present invention can be prepared using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination of such technologies or other technologies readily known in the art (see, for example, Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA, et al, J. MoI. Biol., 373(4): 924-40, 2007).

[0069] As is known in the art, "polynucleotide" or "nucleic acid" used interchangeably herein refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase. The polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If modifications are present, modifications to the nucleotide structure can be made before or after chain assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications include, for example, "capping"; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as having uncharged bonds (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged bonds (e.g., phosphorothioate, phosphorodithioate, etc.), containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), having intercalators (e.g., acridine, psoralen, etc.), containing chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, having modified bonds (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. In addition, any hydroxyl group normally present in the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected or activated by standard protecting groups to provide additional bonds for additional nucleotides, or can be conjugated to a solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1-20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose well known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl ribonucleoside. One or more phosphodiester bonds can be replaced with optional linking groups. These optional linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thio"), P(S)S ("dithio"), (O)NR2 ("amide"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), and optionally contains an ether (-O-) linkage, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl, or an araldyl. All linkages in a polynucleotide need not be identical.The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0070] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination.

[0071] As used herein, "substantially pure" means that the material is at least 50% pure (i.e., free of contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, more preferably, at least 98% pure, and most preferably, at least 99% pure.

[0072] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include progeny of a single host cell, which may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0073] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl-terminus. The numbering of residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.

[0074] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR that binds to an IgG antibody (gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0075] As used herein with respect to antibodies, the term "compete" means that a first antibody or its antigen-binding fragment (or portion) binds to an epitope in a manner very similar to the binding of a second antibody or its antigen-binding portion, such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Another option is possible, but not necessarily so, in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its epitope while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are considered to "cross-compete" with each other for binding to their respective epitopes. The present invention encompasses competing and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), based on the teachings provided herein, a skilled artisan will understand that such competing and / or cross-competing antibodies are encompassed and can be used in the methods disclosed herein.

[0076] A "functional Fc region" possesses 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 an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art for evaluating such antibody effector functions.

[0077] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but still 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, for example, about 1 to about 10 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, and preferably, about 1 to about 5 amino acid substitutions. The variant Fc region herein preferably has at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of the parent polypeptide, and most preferably, at least about 90% sequence identity therewith, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[0078] The term "effector function" refers to the biological activity attributable 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, for example, US Pat No. 6,737,056. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be evaluated using various assays known in the art for evaluating such antibody effector functions. An exemplary measurement of effector function is by Fcγ3 and / or C1q binding.

[0079] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells, subsequently causing the lysis of target cells. The ADCC activity of the molecule of interest can be evaluated using an in vitro ADCC assay, as described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[0080] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), can be performed.

[0081] As used herein, "treatment" is a method of obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of tumors or cancer cells (or destroying tumors or cancer cells), inhibiting the metastasis of tumor cells, alleviating a BCMA-associated disease (e.g., cancer or autoimmune disease), reducing the symptoms caused by a BCMA-associated disease (e.g., cancer or autoimmune disease), improving the quality of life of patients with a BCMA-associated disease (e.g., cancer or autoimmune disease), reducing the dose of other drugs required to treat a BCMA-associated disease (e.g., cancer or autoimmune disease), delaying the progression of a BCMA-associated disease (e.g., cancer or autoimmune disease), curing a BCMA-associated disease (e.g., cancer or autoimmune disease), and / or prolonging the survival of patients with a BCMA-associated disease (e.g., cancer or autoimmune disease).

[0082] "Improvement" refers to a reduction or improvement in one or more symptoms compared to when no BCMA antibody or BCMA antibody conjugate was administered. "Improvement" also includes shortening or reducing the duration of symptoms.

[0083] As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes present in the progression of disease. For therapeutic use, beneficial or desired results include clinical results, such as reducing the occurrence or improvement of one or more symptoms of various BCMA-related diseases or disease conditions (such as multiple myeloma), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, and / or delaying the progression of a BCMA-related disease in a patient. An effective dose can be given by one or more administrations. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve, directly or indirectly, a prophylactic or therapeutic treatment. As understood in the clinical context, an effective dose of a drug, compound, or pharmaceutical composition can be achieved with or without combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" can be considered in terms of administering one or more therapeutic agents, and if used in combination with one or more other drugs, whether a single agent administered in an effective amount can achieve the desired result.

[0084] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

[0085] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences 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 coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0086] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to a nucleic acid sequence to be transcribed.

[0087] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to maintain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions (such as oil / water emulsions), and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or normal saline (0.9%). 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 Ed. Mack Publishing, 2005).

[0088] As used herein, the term "tag containing the acyl donor glutamine" or "glutamine tag" refers to a polypeptide or protein comprising one or more Gln residues that act as an amine acceptor for transglutaminase. See, for example, WO2012059882 and WO2015015448.

[0089] As used herein, the term "k on ” or “k a ” refers to the rate constant for antibody binding to antigen. Specifically, the rate constant (k) was measured using intact antibody (i.e., bivalent) and monomeric BCMA protein. on / k a and k off / k d ) and the equilibrium dissociation constant.

[0090] As used herein, the term "k off ” or “k d ” refers to the rate constant for the dissociation of an antibody from the antibody / antigen complex.

[0091] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0092] Reference herein to "about" a value or parameter includes (and describes) embodiments involving that value or parameter itself. For example, a description referring to "about X" includes a description of "X." Numerical ranges include the numbers defining the range.

[0093] It should be understood that wherever an embodiment is described herein using the language "comprising," other similar embodiments described according to "consisting of" and / or "consisting essentially of" are also provided.

[0094] When aspects or embodiments of the invention are described in terms of Markush groups or other groupings of alternatives, the invention encompasses not only the entire group listed as a whole, but also each member of the individual groups and all possible subgroups of the main group, and also encompasses the main group without one or more of the group members. The invention also contemplates the specific exclusion of one or more of any group members from the claimed invention.

[0095] 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 the specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0096] Exemplary methods and materials are described herein, although 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 not intended to be limiting.

[0097] BCMA antibodies and preparation methods thereof

[0098] The present invention provides an antibody that binds to BCMA (e.g., human BCMA (e.g., SEQ ID NO: 353 or Accession No.: Q02223-2) and is characterized by any one or more of the following characteristics: (a) treats, prevents, or ameliorates one or more symptoms of a disease condition associated with malignant cells expressing BCMA (e.g., B-cell-related cancers such as multiple myeloma) in an individual; (b) inhibits tumor growth or progression in an individual having a malignant tumor expressing BCMA; (c) inhibits metastasis of cancer (malignant) cells expressing BCMA in an individual having one or more malignant cells expressing BCMA; (f) induces regression (e.g., long-term regression) of a tumor expressing BCMA; (d) exerts cytotoxic activity in malignant cells expressing BCMA; and (e) blocks the interaction of BCMA with other factors yet to be identified.

[0099] In one aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to B-cell maturation antigen (BCMA), wherein the antibody comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region 1 (CDR1) comprising the sequence SYX1MX2, wherein X1 is A or P; and X2 is T, N, or S (SEQ ID NO: 301), GFTFX1SY, wherein X1 is G or S (SEQ ID NO: 302), or GFTFX1SYX2MX3, wherein X1 is G or S, X2 is A or P; and X3 is T, N, or S (SEQ ID NO: 303); and (ii) a VH CDR2 comprising the sequence AX1X2X3X4GX5X6X7X8YADX9X 10 KG, wherein X1 is I, V, T, H, L, A, or C; X2 is S, D, G, T, I, L, F, M, or V; X3 is G, Y, L, H, D, A, S, or M; X4 is S, Q, T, A, F, or W; X5 is G or T; X6 is N, S, P, Y, W, or F; X7 is S, T, I, L, T, A, R, V, K, G, or C; X8 is F, Y, P, W, H, or G; X9 is V, R, or L; and X 10 is G or T (SEQ ID NO: 305), or XXXXXXXXXXXXXXX, wherein X1 is S, V, I, D, G, T, L, F, or M; X2 is G, Y, L, H, D, A, S, or M; X3 is S, G, F, or W; X4 is G or S; X5 is G or T; and X6 is N, S, P, Y, or W (SEQ ID NO: 306); and iii) VHCDR3 comprising the sequence VSPIXXXXXXXXX, wherein X1 is A or Y; X2 is A or S; and X3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMXXXXXXX, wherein X1 is D, S, T, or A; and X2 is I, S, L, P, or D (SEQ ID NO: 308); and / or a light chain variable (VL) region comprising (i) VL CDR1, containing the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 , wherein X1 is R, G, W, A or C; X2 is A, P, G, L, C or S; X3 is S, G or R; X4 is Q, C, E, V or I; X5 is S, P, G, A, R or D; X6 is V, G, I or L; X7 is S, E, D, P or G; X8 is S, P, F, A, M, E, V, N, D or Y; X9 is I, T, V, E, S, A, M, Q, Y, H, R or F; X 10 Y or F; X 11 is L, W or P; and X 12is A, S or G (SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X1ASX2RAX3, wherein X1 is G or D; X2 is S or I; and X3 is T or P (SEQ ID NO: 310); and (iii) a VL CDR3 comprising the sequence QQYX1X2X3PX4T, wherein X1 is G, Q, E, L, F, A, S, M, K, R or Y; X2 is S, R, T, G, V, F, Y, D, A, H, V, E, K or C; X3 is W, F or S; and X4 is L or I (SEQ ID NO: 311). NO:311), or QQYX1X2X3PX4, wherein X1 is G, Q, E, L, F, A, S, M, R, K or Y; X2 is S, R, T, G, R, V, D, A, H, E, K, C, F or Y; X3 is W, S or F; and X4 is L or I (SEQ ID NO:312).

[0100] In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to BCMA, wherein the antibody comprises: a VH region comprising a VH CDR1, a VH CDR2, and a VH sequence set forth in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 112, 125, 127, 313, 314, 363, or 365. CDR3; and / or VL region comprising SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 80 , 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, 316, or 364.

[0101] In some embodiments, an antibody is provided that has any partial light chain sequence listed in Table 1 and / or any partial heavy chain sequence listed in Table 1.

[0102] Table 1

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] In Table 1, the underlined sequences are CDR sequences according to Kabat, while the boldfaced sequences are according to Chothia, except for the following heavy chain CDR2 sequences, where the Chothia CDR sequences are underlined and the Kabat CDR sequences are boldfaced. CDR sequences are in bold: P5A2_VHVL, A02_Rd4_0.6nM_C06, A02_Rd4_0.6nM_C09, A02_Rd4_6nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6nM_C01, A02_Rd4_6nM_C26, A02_Rd4_6nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6nM_C19, A02_Rd4_0.6nM_C03, A02_Rd4_6nM_C07, A02_Rd4_6nM_C23, _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_C2 5. 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_C 19. 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_R d4_0.6nM_C05, COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, and COMBO_Rd4_0.6nM_C11. .

[0126] The present invention also provides CDR portions of BCMA antibodies (including Chothia, Kabat CDRs, and CDR contact regions). The determination of CDR regions is within the skill of the art. It should be understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments having more than one CDR, the CDRs may be any Kabat, Chothia, combined CDRs, or combinations thereof. Table 2 provides examples of CDR sequences provided herein.

[0127] Table 2

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] In some embodiments, the present invention provides an antibody that binds BCMA and competes with the antibodies described herein, including 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 / H<h2 style=";text-align:left;direction:ltr">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.K F / 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、A0 2_Rd4_0.6nM_C03、A02_Rd4_6nM_C07、A02_Rd4_6nM_C23、A02_Rd4_0.6 nM_C18、A02_Rd4_6nM_C10、A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A0 2_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, P4G4 or P1A11.

[0145] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to BCMA, wherein the antibody comprises a VH region comprising the sequence shown in SEQ ID NO: 112; and / or a VL region comprising the sequence shown in SEQ ID NO: 38. In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain comprising the sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:357), the heavy chain includes the sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYW PMDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:358).

[0146] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to BCMA, wherein the antibody 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.

[0147] In some embodiments, the present invention also provides antibody CDR portions of BCMA antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that impart specificity to an antigen. Generally, CDR contact regions include residue positions in the CDRs and Vernier regions that are constrained to maintain appropriate loop structure for the antibody to bind to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of CDR contact regions is within the skill of the art.

[0148] The binding affinity (K) of a BCMA antibody as described herein to BCMA, such as human BCMA (e.g., (SEQ ID NO: 353) D ) can be from about 0.002 nM to about 6500 nM. In some embodiments, the binding affinity is about any of 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 about 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.

[0149] In some embodiments, the present invention encompasses compositions, including pharmaceutical compositions, comprising the antibodies described herein or prepared by the methods described herein and having the characteristics described herein. As used herein, compositions comprise one or more antibodies that bind to BCMA, and / or one or more polynucleotides comprising sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

[0150] The present invention also provides methods for preparing any of these antibodies. The antibodies of the present invention can be prepared by methods known in the art. The polypeptides can be prepared by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Polypeptides of the antibody, particularly shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, the antibodies can be prepared by an automated polypeptide synthesizer using a solid phase method. See also, US Pat. No. 5,807,715; 4,816,567; and 6,331,415.

[0151] The present invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody of the present invention. In one embodiment, a fusion polypeptide is provided comprising 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, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 5, 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 SEQ ID NO: 1 122, 112, 125, 127, 313, 314, 363, or 365. In other embodiments, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 consecutive amino acids of a 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 a variable heavy chain region.In another embodiment, the fusion polypeptide comprises a light chain variable region and / or a heavy chain variable region as represented by any sequence pair selected from the group consisting of SEQ ID NOs: 1 and 2, 1 and 3, 4 and 2, 5 and 2, 6 and 2, 4 and 7, 4 and 8, 4 and 3, 9 and 8, 9 and 3, 10 and 7, 10 and 8, 10 and 3, 11 and 7, 11 and 8, 11 and 3, 12 and 3, 13 and 7, 13 and 8, 14 and 3, 15 and 2, 16 and 2, 17 and 2, 18 and 2, 19 and 2, 20 and 2, 21 and 2, 22 and 2, 23 and 2, 16 and 24, 16 and 25, 16 and 26, 16 and 27, 16 and 28, 16 and 29, 16 and 30, 16 and 31, 16 and 3, 16 and 32, 16 and 2, 18 and 24, 18 and 25, 18 and 2 6, 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, 2 2 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, 5 1 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, 122, 124 and 112, 126 and 125, 128 and 127, 80 and 363, or 364 and 365. In another embodiment, the fusion polypeptide comprises one or more CDRs.In other embodiments, the fusion polypeptide comprises a CDRH3 (VH CDR3) and / or a CDR L3 (VL CDR3). For the purposes of the present invention, a fusion protein comprises one or more antibodies and another amino acid sequence that is not connected in the native molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags" such as FLAG tags or 6His tags. Tags are well known in the art.

[0152] The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides.

[0153] In one embodiment, the polynucleotide comprises a sequence encoding the variable region of the heavy and / or light chain of an 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 / H$<h2 style=";text-align:left;direction:ltr">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 / H 2.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.P Y / 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_6 nM_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_0.6n M_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_C 09. C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM_C06, C01_Rd4_6nM_C04, COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C2 1. 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, COM 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 sequences encoding the antibodies of interest can be maintained in vectors in host cells, which can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0154] The present invention also encompasses scFvs of the antibodies of the present invention. Single-chain variable region fragments are prepared by connecting the light chain and / or heavy chain variable regions using a short connecting peptide (Bird et al., Science 242:423-426, 1988). An example of a connecting peptide is (GGGGS)3 (SEQ ID NO:498), which bridges approximately 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). The linker should be a short, flexible polypeptide and preferably contains less than about 20 amino acid residues. The linker can, in turn, be modified for additional functions, such as the connection of a drug or connection to a solid support. 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, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, a eukaryotic organism such as a yeast, plant, insect or mammalian cell, or a prokaryotic organism such as Escherichia coli (E. coli). The polynucleotide encoding the scFv of interest can be prepared by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0155] Other forms of single-chain antibodies are also encompassed, such as diabodies or minibodies. Diabodies are bivalent bispecific antibodies in which the heavy chain variable (VH) and light chain variable (VL) domains are expressed on a single polypeptide chain, but a very short linker is used to prevent pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and produce two antigen-binding sites (see, for example, Holliger, P., et al., Proc. Natl. Acad Sci. USA 90: 6444-6448, 1993; Poljak, RJ, et al., Structure 2: 1121-1123, 1994). Minibodies include the VL and VH domains of natural antibodies fused to the hinge region and CH3 domains of an immunoglobulin molecule. See, for example, US5,837,821.

[0156] In another aspect, the present invention provides compositions (e.g., pharmaceutical compositions) 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 other embodiments, the composition comprises any one or both of the polynucleotides shown in SEQ ID NO: 486 and SEQ ID NO: 485:

[0157] COMBO_Rd4_0.6nM_C29 heavy chain variable region

[0158] gaagtccaactcctcgaatccggtggcggccttgtccagcctggaggttccttgcgcctgtcatgtgccgccagcggattcaccttctcgtcctacccgatgtcgtgggtccgccaggctccgggaaagggcctggaatgggtgtcagccatcggaggatcggggggctccctg ccctacgccgatatcgtgaagggaaggttcaccattagccgggacaactccaagaacactctgtacctccaaatgaacagcctgagagcggaggacaccgcagtgtactattgcgcccggtactggccaatggacatctggggccaggggactctggtcaccgtctcctca(SEQ ID NO:486)

[0159] COMBO_Rd4_0.6nM_C29 light chain variable region

[0160] Gagatcgtgctgactcagtcccctggaaccctgtccctgtcacctggcgaaagagctaccttgtcctgtcgcgcatcacaatccgtgtcgtcgagctatctcgcgtggtaccagcagaagcccggacaggccccaaggctgcttatgtacgacgcctccatc cgggccactggtatccccgaccgcttctcgggctccggaagcggcaccgacttcaccctgactatttcccggctcgaaccggaggatttcgccgtgtactactgccaacagtaccagagctggccgctgacgtttgggcaggggaccaaggtcgaaatcaaa

[0161] (SEQ ID NO:485)

[0162] In other embodiments, the composition comprises either or both of the following polynucleotides set forth in SEQ ID NO: 488 and SEQ ID NO: 487:

[0163] L3.PY / H3TAQ heavy chain variable region

[0164] gaagtgcagctgctggaatctggcggaggactggtgcagcctggcggctctctgagactgtcttgtgccgccagcggcttcaccttcggcagctacgctatgacctgggtgcgccaggcccctggcaaaggactggaatgggtgtccgccatctctggcagcggcggcaataccttct acgccgagagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactattgtacacgggtgtcccctatcgccgcgcagatggattattggggccagggcactctggtcaccgtctcctca

[0165] (SEQ ID NO:488)

[0166] L3.PY / H3TAQ heavy chain variable region

[0167] Gagatcgtgctgacacagagccctggcaccctgagcctgtctccaggcgaaagagccaccctgtcctgcagagccagccagagcgtgtccagcagctacctggcctggtatcagcagaagcccggccaggctccccggctgctgatctatggcgcctcttcta gagccaccggcatccccgatagattcagcggctctggcagcggcaccgacttcaccctgaccatcagcagactggaacccgaggacttcgccgtgtactactgccagcactacccttatccccccagcttcacatttggccagggcaccaaggtggagatcaaa

[0168] (SEQ ID NO:487)

[0169] In other embodiments, the composition comprises either or both of the following polynucleotides set forth in SEQ ID NO: 490 and SEQ ID NO: 489:

[0170] A02_Rd4_0.6nM_C01 heavy chain variable region

[0171] GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTACTGCGTCTGGTGGTTCTA CTTACTATGCCGATGTGGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTTGTTTATTACTGTGCTAGATACTGGCCAATGTCGTTGTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

[0172] (SEQ ID NO:490)

[0173] A02_Rd4_0.6nM_C01 light chain variable region

[0174] GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGCGTATTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCGTTTACTACTGTCAACAATATGAGCGTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG

[0175] (SEQ ID NO:489)

[0176] In other embodiments, the composition comprises either or both of the following polynucleotides set forth in SEQ ID NO: 492 and SEQ ID NO: 491:

[0177] A02_Rd4_0.6nM_C16 heavy chain variable region

[0178] GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTTTGGTGGTTCTA CTTACTATGCCGATATCGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

[0179] (SEQ ID NO:492)

[0180] A02_Rd4_0.6nM_C16 light chain variable region

[0181] GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGATCTGTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCGTTTACTACTGTCAACAATATCAGACTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG

[0182] (SEQ ID NO:491).

[0183] Expression vectors and administration of polynucleotide compositions are further described herein.

[0184] In another aspect, the invention provides methods of making any of the polynucleotides described herein.

[0185] The present invention also encompasses polynucleotides complementary to any such sequences. 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, which contain introns and correspond to DNA molecules in a one-to-one manner; and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present invention, and polynucleotides may, but need not, be linked to other molecules and / or support materials.

[0186] 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. The polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be evaluated as described herein. The variant preferably exhibits at least about 70% identity to the polynucleotide sequence encoding the native antibody or a portion thereof, more preferably, at least about 80% identity, more preferably, at least about 90% identity, and most preferably, at least about 95% identity.

[0187] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically 30 to about 75, or 40 to about 50, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned.

[0188] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program includes several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0189] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less additions or deletions (i.e., gaps), typically 5-15%, or 10-12%, when compared to a reference sequence (which does not contain 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 occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence and multiplying the result by 100 to produce the percentage of sequence identity.

[0190] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0191] Suitable "moderately stringent conditions" include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C-65°C, 5X SSC; and then washing twice at 65°C for 20 minutes, each time with 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[0192] As used herein, "high stringency conditions" or "high stringency conditions" are: (1) using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, during hybridization, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 at 42°C with 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate ... Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate, washes in 0.2× SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1× SSC containing EDTA at 55° C. The skilled artisan will recognize how to adjust temperature, ionic strength, etc., and, if necessary, factors such as probe length.

[0193] Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that differ due to differences in codon usage. Moreover, alleles of genes comprising 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 deletions, additions, and / or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0194] Polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. The method for chemical polynucleotide synthesis is well known in the art and need not be described in detail herein. Those skilled in the art can use the sequence provided by this paper and commercial DNA synthesizer to prepare the desired DNA sequence.

[0195] In order to utilize recombinant methods to prepare polynucleotides, as further discussed herein, the polynucleotide comprising the desired sequence can be inserted into a suitable vector, and then the vector is introduced into a suitable host cell for replication and amplification. Polynucleotides can be inserted into host cells by any method known in the art. Exogenous polynucleotides are introduced into cells by direct absorption, endocytosis, transfection, F-conjugation or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as a non-integrated vector (e.g., plasmid) or integrated into the host cell genome. The polynucleotides amplified in this way can be separated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0196] Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in the art and are described in, for example, US Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

[0197] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, such as shown in Sambrook et al., 1989, supra.

[0198] Suitable cloning vectors can be built according to standard techniques, or can be selected from a large amount of cloning vectors available in this area. Although selected cloning vectors can vary according to the host cell used in expectation, useful cloning vectors generally have the ability of self-replication, can have the single target of specific restriction endonuclease, and / or can carry the gene of the marker that can be used for selecting the clone containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (such as pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers, such as BioRad, Strategene and Invitrogen.

[0199] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide of the present invention. It is implied that the expression vector must be replicable in the host cell as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components generally may include, but are not limited to, one or more of the following components: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are generally also required.

[0200] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, when the vector is an infectious agent, such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0201] 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 genes encoding antibodies, polypeptides, or proteins 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. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli (E. coli) or Bacillus subtilis (B. subtillis)) and yeast (e.g., Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe); or Kluyveromyces lactis (K. lactis)). Preferably, the host cell expresses the cDNA at a level that is about 5-fold higher, more preferably 10-fold higher, and even more preferably 20-fold higher than the corresponding endogenous antibody or protein of interest (if present) in the host cell. Screening for host cells that specifically bind to BCMA or a BCMA domain (e.g., domains 1-4) is performed by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

[0202] Representative materials of the present invention were deposited with the American Type Culture Collection (ATCC) on April 15, 2015. The vector with ATCC accession number PTA-122094 is a polynucleotide encoding the heavy chain variable region of a humanized BCMA antibody, while the vector with ATCC accession number PTA-122093 is a polynucleotide encoding the light chain variable region of a humanized BCMA antibody. Deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure and Regulations (Budapest Treaty). This ensures that viable cultures of the deposited material will be maintained for 30 years from the date of deposit. ATCC will make the deposit available under the terms of the Budapest Treaty, and an agreement has been entered into between Pfizer, Inc. and ATCC which ensures the perpetual and non-restricted availability of progeny of cultures of the deposit to the public upon the issuance of a related U.S. patent or disclosure of any U.S. or foreign patent application, whichever occurs first, and to such persons as the Commissioner of the United States Patent and Trademark determines to be authorized pursuant to 35 U.S.C. § 122 and the Commissioner's rules thereunder (including 37 CFR § 1.14, specifically referring to 886 OG 638).

[0203] The assignee of this application has agreed that if a culture of the stored material should die or be lost or destroyed when grown under suitable conditions, the material will be promptly replaced with another of the same material upon notice. The availability of the stored material should not be construed as a violation of any government's right to practice the invention in accordance with rights granted under its patent laws.

[0204] BCMA antibody conjugate

[0205] The present invention also provides conjugates (or immunoconjugates) of BCMA antibodies or antigen-binding fragments thereof as described herein, wherein the antibody or antigen-binding fragment is conjugated directly or indirectly through a linker to an agent (e.g., a cytotoxic agent) for targeted immunotherapy (e.g., an antibody-drug conjugate). For example, a cytotoxic agent can be linked or conjugated to a BCMA antibody or antigen-binding fragment thereof as described herein for targeted local delivery of the cytotoxic agent moiety to a tumor (e.g., a BCMA-expressing tumor).

[0206] Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies have been described in various publications. For example, chemical modification of cysteine ​​sulfhydryl groups activated by lysine side chain amines or by reduction of interchain disulfide bonds can be performed in antibodies for the conjugation reaction to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, 2005, and Gentle et al., Bioconjugate Chem. 15:658-663, 2004. Engineering reactive cysteine ​​residues with defined stoichiometric amounts for specific drug conjugation at specific sites of antibodies has also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. International applications WO2012 / 059882 and WO2015015448 also describe the conjugation of endogenous glutamine using a tag containing the acyl donor glutamine or by engineering the polypeptide to make it reactive (i.e., capable of forming a covalent bond as an acyl donor) in the presence of a transglutaminase and an amine (e.g., a cytotoxic agent containing or linked to a reactive amine).

[0207] In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing the acyl donor glutamine engineered at a specific site of the antibody (e.g., the carboxyl terminus, the amino terminus, or at another site in the BCMA antibody). In some embodiments, the tag comprises the amino acid glutamine (Q) or the amino acid sequence LQG, LLQGG (SEQ ID NO: 318), LLQG (SEQ ID NO: 454), LSLSQG (SEQ ID NO: 455), GGGLLQGG (SEQ ID NO: 456), GLLQG (SEQ ID NO: 457), LLQ, GSPLAQSHGG (SEQ ID NO: 458), GLLQGGG (SEQ ID NO: 459), GLLQGG (SEQ ID NO: 460), GLLQ (SEQ ID NO: 461), LLQLLQGA (SEQ ID NO: 462), LLQGA (SEQ ID NO: 463), LLQYQGA (SEQ ID NO: 464), LLQGSG (SEQ ID NO: 465), LLQYQG (SEQ ID NO: 466), LLQLLQG (SEQ ID NO: 467), SLLQG (SEQ ID NO: 468). 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).

[0208] In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine engineered at a specific site of the antibody, wherein the tag comprises 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, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine engineered at a specific site of the antibody, wherein the tag comprises the amino acid sequence LLQG (SEQ ID NO: 454) engineered after residue T135 in the heavy chain of the BCMA antibody. In other embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine engineered at a specific site of the antibody, wherein the tag comprises 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 wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted. In some embodiments, a BCMA antibody or conjugate as described herein comprises 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.

[0209] Also provided is an isolated antibody comprising a tag comprising the acyl donor glutamine and an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody, wherein the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. Thus, in some embodiments, a BCMA antibody or conjugate as described herein is provided, comprising a tag containing an acyl donor glutamine (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), LLQGSG (SEQ ID NO: 466), LLQGSG (SEQ ID NO: 467), LLQGSG (SEQ ID NO: 468), LLQGSG (SEQ ID NO: 469), LLQGSG (SEQ ID NO: 470), LLQGSG (SEQ ID NO: 471), LLQGSG (SEQ ID NO: 472), LLQGSG (SEQ ID NO: 473), LLQGSG (SEQ ID NO: 474), LLQGSG (SEQ ID NO: 475), LLQGSG (SEQ ID NO: 476), LLQGSG (SEQ ID NO: 477), LLQGSG (SEQ ID NO: 478), LLQGSG (SEQ ID NO: 479), LLQGSG (SEQ ID NO: 480), LLQGSG (SEQ ID NO: 481), LLQGSG (SEQ ID NO: 482), LLQGSG (SEQ ID NO: NO:465), LLQYQG (SEQ ID NO:466), LLQLLQG (SEQ ID NO:467), SLLQG (SEQ ID NO:468), LLQLQ (SEQ ID NO:469), LLQLLQ (SEQ ID NO:470), LLQGR (SEQ ID NO:471), LLQGPP (SEQ ID NO:472), LLQGPA (SEQ ID NO:473), GGLLQGPP (SEQ ID NO:474), GGLLQGA (SEQ ID NO:475), LLQGPGK (SEQ ID NO:476), LLQGPG (SEQ ID NO:477), LLQGP (SEQ ID NO:478), LLQP (SEQ ID NO:479), LLQPGK (SEQ ID NO:480), LLQAPGK (SEQ ID NO:481), LLQGAPG (SEQ ID NO:482), LLQGAP (SEQ ID NO: 483) and LLQLQG (SEQ ID NO: 484)) and an amino acid modification at position 222, 340 or 370 (EU numbering scheme) of the antibody. In some embodiments, the amino acid modification is a substitution from lysine to arginine (e.g., K222R, K340R or K370R).

[0210] In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine (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, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine (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, a BCMA antibody or conjugate as described herein comprises a tag containing an acyl donor glutamine (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 carboxyl terminus of the heavy chain is deleted. In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing the acyl donor glutamine comprising the sequence LLQG (SEQ ID NO: 454) engineered after residue T135 in the BCMA antibody heavy chain and an amino acid substitution from lysine to arginine at position 222 (EU numbering scheme) of the antibody.

[0211] In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing the acyl donor glutamine (comprising a glutamine engineered at position 297 or an amino acid substitution from asparagine (N) to another amino acid at position 297 in the BCMA antibody) and an amino acid substitution from lysine to arginine at position 222 (EU numbering scheme) of the antibody. For example, in some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing the acyl donor glutamine (comprising the sequence GGLLQGPP (SEQ ID NO: 474) engineered at the C-terminus of the BCMA antibody light chain), an amino acid substitution from asparagine (N) to glutamine (Q) at position 297 of the BCMA antibody, and an amino acid substitution from lysine to arginine at position 222 (EU numbering scheme) of the antibody. In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing the acyl donor glutamine (comprising the sequence LLQG (SEQ ID NO: 454) engineered after residue T135 in the BCMA antibody heavy chain), an amino acid substitution at position 297 of the BCMA antibody from asparagine (N) to alanine (A), and an amino acid substitution at position 222 (EU numbering scheme) of the antibody from lysine to arginine.

[0212] 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.

[0213] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatins, combretastatins, duocarmycin, pyrrole benzodiazepine dimers, indoline-benzodiazepines, dimer, enediyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloid, camptothecin, tubulysin, hemiesterin, spliceostatin, pradienolide, and stereoisomers, isosteres, analogs or derivatives thereof.

[0214] Anthracyclines are derived from the bacterium Strepomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, 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.

[0215] Dolastatins and their peptide analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, US Pat. 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-benzoylvalerate-AE ester (AEVB), and other novel auristatins (such as the auristatins disclosed in U.S. Publication No. 2013 / 0129753). In some embodiments, the auristatin is 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

[0216]

[0217] 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:

[0218]

[0219] 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:

[0220]

[0221] In other embodiments, the auristatin is 0131 (2-methyl-L-prolyl-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:

[0222]

[0223] In other embodiments, the auristatin is 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) having the following structure:

[0224]

[0225] 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.

[0226] Combretastatins are natural phenols that have angiogenic properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

[0227] Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic efficacy. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary Duocarmycin and CC-1065 include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, (+)-CC-1065, and compounds disclosed in international application PCT / IB2015 / 050280, including but not limited to N~2~-acetyl-L-lysyl-L-valyl-N~5~-carbamyl-N-[4-({[(2-{[({(1S) -1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thien-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide:

[0228]

[0229] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pentan-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide having the following structure:

[0230]

[0231] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(4-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}pentacyclo[4.2.0.0-2,5-.0-3,8-.0-4,7-]oct-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide having the following structure:

[0232]

[0233] Enediynes are a class of antitumor bacterial products characterized by the presence of nine-membered and ten-membered ring systems or conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and their derivatives.

[0234] Geldanamycin is a benzoquinone ansa antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an anti-tumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0235] Hemicastelin and its analogs (eg, HTI-286) bind to tubulin, disrupt normal microtubule dynamics, and stoichiometrically depolymerize microtubules.

[0236] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting the polymerization of tubulin and inhibiting microtubule formation during mitosis. See Remillard et al., Science 189:1002-1005, 1975. Exemplary maytansine and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives and ansamitocin.

[0237] Pyrrole-benzodiazepine dimer (PBD) and indoline-benzodiazepine IGNs are antitumor agents containing one or more immine functional groups or their equivalents that bind to double-stranded DNA. PBDs and IGN molecules are based on the natural product atramycin and interact with DNA in a sequence-selective manner, preferring purine-guanine-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

[0238] Spliceostatin and pradienolide are antitumor compounds that inhibit splicing and interact with the spliceosome SF3b. ​​Examples of spliceostatins include, but are not limited to, spliceostatin A, FR901464, and

[0239]

[0240] (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazino-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate of the structure. Examples of pradienolides include, but are not limited to, pradienolide B, pradienolide D, or E7107.

[0241] Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g. ) and docetaxel

[0242] Tubulysin is a natural product isolated from a myxobacterium strain 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.

[0243] Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0244] Thus, in some embodiments, the cytotoxic agent is selected from 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-oxoheptane-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-prolyl-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-oxoheptane-4-yl]-N-methyl L-valinamide), 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}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4-yl]-N-methyl L-valinamide

[0015] The present invention relates to (1) methylpenta-2,4-dien-1-yl, (2) methyltetrahydro-2H-pyran-3-yl, (3) methylpenta-2,4-dien-1-yl, (4) methylpenta-2,4-dien-1-yl, (5) methylpenta-2,4-dien-1-yl, (6) methylpenta-2,4-dien-1-yl, (7) methylpenta-2,4-dien-1-yl, (8) methylpenta-2,4-dien-1-yl, (9) methylpenta-2,4-dien-1-yl, (10) methylpenta-2,4-dien-1-yl, (11) methylpenta-2,4-dien-1-yl, (12) methylpenta-2,4-dien-1-yl, (13) methylpenta-2,4-dien-1-yl, (14) methylpenta-2,4-dien-1-yl, (15) methylpenta-2,4-dien-1-yl, (16) methylpenta-2,4-dien-1-yl, (17) methylpenta-2,4-dien-1-yl, (18) methylpenta-2,4-dien-1-yl, (19) methylpenta-2,4-dien-1-yl, (2 ...

[0245] In some embodiments, the agent is an immunomodulatory agent. Examples of immunomodulators include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, 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 designated "S1 factor," erythropoietin, and thrombopoietin, or a combination thereof.

[0246] In some embodiments, the agent moiety is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, a lanthanide fluorophore, or a derivative thereof, or a radioisotope conjugated to a chelator. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor, fluorescein ... (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5′-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelating agents 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-pentanedioic acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

[0247] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluoresceinamidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor, (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5′-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101).

[0248] In some embodiments, therapeutic or diagnostic radioisotopes or other labels (e.g., PET or SPECT labels) can be incorporated into the agent for conjugation to a BCMA antibody or antigen-binding fragment as 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 Such as 97 Such as 99 Tc, 103 Such as 105 Rh, 105 Such as 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, 166 Dy, 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.

[0249] In some embodiments, the agent is a therapeutic protein, including but not limited to toxins, hormones, enzymes, and growth factors.

[0250] Examples of toxin proteins (or polypeptides) include, but are not limited to, dipththeria (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), α-sarcin, Aleurites fordii protein, dianthin protein, RNase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, mitogellin, restrictocin, phenomycin, enomycin, trichothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxins (e.g., KIIIA or SmIIIa).

[0251] In some embodiments, the pharmaceutical agent is a biocompatible polymer. A BCMA antibody or antigen-binding fragment as described herein can be conjugated to a biocompatible polymer to increase serum half-life and bioactivity, and / or extend in vivo half-life. Examples of biocompatible polymers include water-soluble polymers such as polyethylene glycol (PEG) or its derivatives and biocompatible polymers containing zwitterions (e.g., polymers containing phosphorylcholine).

[0252] In some embodiments, the agent is an oligonucleotide, such as an antisense oligonucleotide.

[0253] In another aspect, the invention provides a conjugate of an antibody or antigen-binding fragment as described herein, wherein the conjugate comprises the formula: antibody-(tag containing acyl donor glutamine)-(linker)-(cytotoxic agent), wherein the tag containing acyl donor glutamine is engineered at a specific site of the antibody or antibody-binding fragment (e.g., at the carboxyl terminus of the heavy or light chain, after residue T135 in the antibody heavy chain, or another site), wherein the tag is conjugated to a linker (e.g., a linker comprising one or more reactive amines (e.g., a primary amine NH2)), and wherein the linker is conjugated to a cytotoxic agent (e.g., MMAD or other auristatins such as 0101, 0131, or 3377).

[0254] 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), yl), amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, or Ac-Lys-putrescine.

[0255] In some embodiments, the conjugate is 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101; 2) antibody-AcLys-VC-PABC-0101 and comprising N297Q; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprising N297Q; 4) antibody-LLQG ( SEQ ID NO: 454) -amino-PEG6-C2-0131 and contains N297A; 5) Antibody - LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377 and contains N297A; 6) Antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101. In some embodiments, a tag containing an acyl donor glutamine, such as GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475), is engineered at the C-terminus of the antibody light chain. In other embodiments, a tag containing an acyl donor glutamine, such as LLQGA (SEQ ID NO: 463) or LLQGPP (SEQ ID NO: 472), is engineered at the C-terminus of the antibody heavy chain, wherein the C-terminal lysine residue is deleted. In some embodiments, a tag containing an acyl donor glutamine, such as LLQG (SEQ ID NO: 454), is engineered after residue T135 in the antibody heavy chain or replaces amino acid residues E294-N297 in the antibody heavy chain. 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 / H 3.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 / P6E 01. 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.<h2 style=";text-align:left;direction:ltr">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.Q L、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 / L 1.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.A E、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_R d4_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_C 18、A02_Rd4_6nM_C10、A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6 nM_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_C 19、COMBO_Rd4_0.6nM_C02、COMBO_Rd4_0.6nM_C23、COMBO_Rd4_0.6nM_C2 9、COMBO_Rd4_0.6nM_C09、COMBO_Rd4_6nM_C12、COMBO_Rd4_0.6nM_C30、CO MBO_Rd4_0.6nM_C14、COMBO_Rd4_6nM_C07、COMBO_Rd4_6nM_C02、COMBO_Rd 4_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.

[0256] In one variation, the conjugate further comprises an amino acid substitution from lysine to arginine at position 222. Thus, for example, the conjugates are 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprising K222R; 2) antibody-AcLys-VC-PABC-0101 comprising N297Q and K222R; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprising N297Q and K222R; 4) antibody-LLQG ( SEQ ID NO: 454) -amino-PEG6-C2-0131 and contains N297A and K222R; 5) Antibody - LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377 and comprising N297A and K222R; and 6) antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101 and comprising K222R. In some embodiments, a tag containing an acyl donor glutamine, such as GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475), is engineered at the C-terminus of the antibody light chain. In other embodiments, a tag containing an acyl donor glutamine, such as LLQGA (SEQ ID NO: 473) or LLQGPP (SEQ ID NO: 472), is engineered at the C-terminus of the antibody heavy chain, wherein the C-terminal lysine residue is deleted. In some embodiments, a tag containing an acyl donor glutamine, such as LLQG (SEQ ID NO: 454), is engineered after residue T135 in the antibody heavy chain or replaces amino acid residues E294-N297 in the antibody heavy chain. 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 / P6E0 1. L3.PY / P6E01, L3.NY / P6E01, L3.PY / L1.PS / P6E01, L3.PY / L1.AH / P6E01, L3.PY / L1.FF / P6E0 1. 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.<h2 style=";text-align:left;direction:ltr">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.D Y, 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 / L 3.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.P Y / 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_R d4_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_C 18、A02_Rd4_6nM_C10、A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6 nM_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_C 19、COMBO_Rd4_0.6nM_C02、COMBO_Rd4_0.6nM_C23、COMBO_Rd4_0.6nM_C2 9、COMBO_Rd4_0.6nM_C09、COMBO_Rd4_6nM_C12、COMBO_Rd4_0.6nM_C30、CO MBO_Rd4_0.6nM_C14、COMBO_Rd4_6nM_C07、COMBO_Rd4_6nM_C02、COMBO_Rd 4_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.

[0257] CD3 antibody and preparation method thereof

[0258] The present invention further provides antibodies that bind to CD3 (eg, human CD3 (SEQ ID NO: 502; or Accession No.: NM_000733.3).

[0259] In one aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CD3, wherein the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the 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 comprises a VH CDR2, a VH CDR3, and a VH CDR3 of the 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; A light chain variable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in 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.

[0260] In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds CD3, wherein 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: NO: 335, 406, 423, 429 or 451; and / or a light chain variable (VL) region comprising (i) a 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.

[0261] In some embodiments, an antibody is provided that has any partial light chain sequence listed in Table 3 and / or any partial heavy chain sequence listed in Table 3.

[0262] Table 3

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] In Table 3, the underlined sequences are CDR sequences according to Kabat, while those in bold are according to Chothia.

[0269] The present invention also provides CDR portions of CD3 antibodies (including Chothia, Kabat CDRs, and CDR contact regions). The determination of CDR regions is within the skill of the art. It should be understood that in some embodiments, the CDRs can be a combination of Kabat and Chothia CDRs (also referred to as "combined CRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments having more than one CDR, the CDRs can be any Kabat, Chothia, combined CDRs, or combinations thereof. Table 4 provides examples of CDR sequences provided herein.

[0270] Table 4

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides.

[0278] In one embodiment, the polynucleotide comprises a polynucleotide encoding the antibody h2B4, h2B4-VH-wt VL_TK, h2B4-VH-hnps VL_TK, h2B4-VH-yaes VL_TK, h2B4-VH-yads VL_TK, h2B4-VH-yaps VL_TK, h2B4-VH-hnps VL_TK-S55Y, h2B4-VH-hnps VL_TK-S105Q, h2B4-vH-hnps The heavy chain of 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 and / or the sequence of variable region of light chain. The sequence of the antibody of interest can be maintained in a carrier in a host cell, which can then be increased and frozen host cells for future use. Carriers (including expression vectors) and host cells are further described herein.

[0279] The present invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody of the present invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 consecutive amino acids of the variable light chain region set forth in SEQ ID NO: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 445, 352, 355, 443, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399, and / or SEQ ID NO: 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, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25 or at least about 30 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 comprises a light chain variable region and / or a heavy chain variable region as set forth in any sequence pair 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 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 comprises one or more antibodies and another amino acid sequence not linked in the native molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags" such as FLAG tags or 6His tags. Tags are well known in the art.

[0280] Fusion polypeptides can be produced by methods known in the art, such as synthesis or recombination. Typically, fusion proteins of the present invention are prepared by utilizing recombinant methods as described herein to prepare expression polynucleotides encoding them, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[0281] Representative material of the CD3 antibody of the present invention was deposited with the American Type Culture Collection (ATCC) on September 11, 2015. The vector with ATCC accession number PTA-122513 is a polynucleotide encoding the heavy chain variable region of the humanized CD3 antibody, while the vector with ATCC accession number PTA-122512 is a polynucleotide encoding the light chain variable region of the humanized CD3 antibody. Deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure and Regulations (Budapest Treaty). This ensures that viable cultures of the deposited material will be maintained for 30 years from the date of deposit. ATCC will make the deposit available under the terms of the Budapest Treaty, and an agreement has been entered into between Pfizer, Inc. and ATCC which ensures the perpetual and non-restricted availability of progeny of cultures of the deposit to the public upon the issuance of a related U.S. patent or disclosure of any U.S. or foreign patent application, whichever occurs first, and to such persons as the Commissioner of the United States Patent and Trademark determines to be authorized pursuant to 35 U.S.C. § 122 and the Commissioner's rules thereunder (including 37 CFR § 1.14, specifically referring to 886 OG 638).

[0282] The assignee of this application has agreed that if a culture of the stored material should die or be lost or destroyed when grown under suitable conditions, the material will be promptly replaced with another of the same material upon notice. The availability of the stored material should not be construed as a violation of any government's right to practice the invention in accordance with rights granted under its patent laws.

[0283] Bispecific antibodies and methods of preparation

[0284] The antibodies disclosed herein can be used to prepare bispecific antibodies, monoclonal antibodies having binding specificity for at least two different antigens. Methods for preparing bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

[0285] According to a method for preparing bispecific antibodies, the antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant region sequences. The fusion preferably has an immunoglobulin heavy chain constant region, comprising at least part of the hinge, CH2 and CH3 regions. Preferably, there is a first heavy chain constant region (CH1), comprising the site necessary for light chain binding, present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and the immunoglobulin light chain (if desired) is inserted into different expression vectors, and co-transfected into a suitable host organism. When the unequal proportions of the 3 polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual ratio of the 3 polypeptide fragments in the embodiment. However, when expressing at least two polypeptide chains of equal proportions and causing high yields or when ratio has no particular significance, the coding sequence of two or all three polypeptide chains can be inserted into an expression vector.

[0286] In one approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with immunoglobulin light chains in only half of the bispecific molecule, facilitates separation of desired bispecific compounds from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94 / 04690.

[0287] In another approach, a bispecific antibody is composed of amino acid modifications in the first hinge region of one arm, and the substituted / replaced amino acid in the first hinge region has a charge opposite to that of the corresponding amino acid in the second hinge region of the other arm. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0288] In another approach, the formation of a desired heteromultimeric or heterodimeric protein (e.g., a bispecific antibody) is enhanced by altering or engineering the interface between a first and a second immunoglobulin-like Fc region (e.g., a hinge region and / or a CH3 region). In this approach, the bispecific antibody can be composed of a CH3 region, wherein the CH3 region comprises a first CH3 polypeptide and a second CH3 polypeptide, which together interact to form a CH3 interface, wherein one or more amino acids within the CH3 interface destabilize and electrostatically disfavor homodimer formation. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0289] In another approach, bispecific antibodies can be generated using a glutamine-containing peptide tag engineered into an antibody against an epitope (e.g., BCMA) in one arm and another peptide tag (e.g., a Lys-containing peptide tag or reactive endogenous Lys) engineered into a second antibody against a second epitope in the other arm in the presence of transglutaminase. This approach is described in International Patent Application No. PCT / IB2011 / 054899 (WO2012 / 059882).

[0290] In another aspect of the present invention, heterodimeric proteins (e.g., bispecific antibodies) as described herein include full-length human antibodies, wherein the first antibody variable domains of the heterodimeric protein can raise human immune effector cell activity by specifically binding to effector antigens located on human immune effector cells, and wherein the second antibody variable domains of the heterodimeric protein can specifically bind to the target antigen. In some embodiments, human antibodies have IgG1, IgG2, IgG3, or IgG4 isotypes. In some embodiments, the heterodimeric protein includes an immunologically inert Fc region.

[0291] Human immune effector cells can be any of various immune effector cells known in the art.For example, immune effector cells can be members of human lymphoid cell lineages, including but not limited to T cells (such as cytotoxic T cells), B cells and natural killer (NK) cells. Immune effector cells can also be, for example but not limited to, members of human myeloid lineages, including but not limited to monocytes, neutrophils and dendritic cells. This type of immune effector cells can have cytotoxic or apoptotic effects or other desired effects on target cells when activated by the combination of effector antigens.

[0292] Effector antigens are antigens (e.g., proteins or polypeptides) expressed on human immune effector cells. Examples of effector antigens that heterodimeric proteins (e.g., heterodimeric antibodies or bispecific antibodies) can bind to include, but are not limited to, human CD3 (or CD3 (cluster of differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

[0293] Target cells can be natural or exogenous to a person. In natural target cells, the cells may have been transformed into malignant cells or pathologically modified (e.g., natural target cells infected with viruses, malarial parasites, or bacteria). In exogenous target cells, the cells are invading pathogens, such as bacteria, malarial parasites, or viruses.

[0294] The target antigen is expressed on the target cell under disease conditions (e.g., inflammatory diseases, proliferative diseases (e.g., cancer), immune disorders, neurological diseases, neurodegenerative diseases, autoimmune diseases, infectious diseases (e.g., viral infections or parasitic infections), allergic reactions, graft-versus-host diseases, or host-versus-graft diseases). 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, Poly SA, GD2, Carboanhydrase IX (MN / CAIX), CD44v6, Shh (Sonic Hedgehog), Wue-1, plasma cell antigen, (membrane-bound) IgE / MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF-α precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; desmoglein 4, E-cadherin neoepitope, embryonic acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and MIS (Muellerian inhibitory substance) receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

[0295] In some embodiments, the heterodimeric protein (e.g., a bispecific antibody) as described herein comprises a full-length human antibody, wherein the first antibody variable domain of the heterodimeric protein is capable of recruiting human immune effector cell activity by specifically binding to an effector antigen (e.g., a CD3 antigen) located on human immune effector cells, wherein the second antibody variable domain of the heterodimeric protein is capable of specifically binding to a target antigen (e.g., a CD20 antigen or EpCAM), wherein the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 223, 225, and 228 in the hinge region (e.g., (C223E or C223R), (E225R), and (P228E or P228R)) and at position 409 or 368 in the CH3 region of human IgG2 (SEQ ID NO: 493) (e.g., K409R or L368E (EU numbering scheme)).

[0296] In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 221 and 228 (e.g., (D221R or D221E) and (P228R or P228E)) in the hinge region and position 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in the CH3 region of human IgG1 (SEQ ID NO: 494).

[0297] In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at position 228 (e.g., (P228E or P228R)) in the hinge region and position 409 or 368 (e.g., R409 or L368E (EU numbering scheme)) in the CH3 region of human IgG4 (SEQ ID NO: 495).

[0298] In another embodiment, the first antibody variable domain of the heterodimeric protein comprises a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384386, 388, 390, 392, 394, 396, 398, or 400; and / or a light chain variable (VL) region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382, ​​384386, 388, 390, 392, 394, 396, 398, or 400; 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, and the second antibody variable domain of the heterodimeric protein comprises a VH region comprising 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 SEQ IDNO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38 ,40,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,31 7, 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, a VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in FIG.

[0299] In another embodiment, the first antibody variable domain comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 324 or 388; and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 323 or 387; and the second antibody variable domain comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 112; and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 38.

[0300] The antibodies that can be used in the present invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heterologous conjugate antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired 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).

[0301] In some embodiments, the BCMA or CD3 antibody as described herein is a monoclonal antibody, for example a BCMA or CD3 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

[0302] In some embodiments, the antibodies comprise a modified constant region, for example, but not limited to, a constant region with increased potential to stimulate an immune response. For example, the constant region can be modified to have increased affinity for an Fcγ receptor such as FcγRI, FcγRIIA, or FcγIII.

[0303] In some embodiments, the antibody comprises a modified constant region, such as an immunologically inert constant region, i.e., one with reduced potential to stimulate an immune response. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 29:2613-2624, 1999; PCT Publication 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) comprising the mutations A330P331 to S330S331, wherein 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 an IgG4 constant region comprising the following mutations (Armour et al., Molecular Immunology 40, 585-593, 2003): E233F234L235 to P233V234A235 (IgG4Δc), wherein the numbering is with reference to wild-type IgG4. In another embodiment, the Fc is human IgG4 E233F234L235 to P233V234A235, with deletion G236 (IgG4Δb). In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb, or IgG4Δc) comprising the hinge stabilizing mutation S228 to P228 (Aalberse et al., Immunology 105, 9-19, 2002). In another embodiment, the Fc may be a non-glycosylated Fc.

[0304] In some embodiments, the constant region is rendered aglycosylated by mutating the oligosaccharide attachment residue (e.g., Asn297) and / or flanking residues that are part of the glycosylation recognition sequence in the constant region. In some embodiments, the constant region is enzymatically rendered aglycosylated for N-linked glycosylation. The constant region can be rendered aglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

[0305] In some embodiments, the constant region has a modified constant region that removes or reduces Fcγ receptor binding.For example, the Fc can be a human IgG2 comprising the mutation D265, wherein 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 a modified constant region having the sequence set forth in SEQ ID NO: 496: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0306] In some embodiments, the constant region has a modified constant region having the sequence set forth in SEQ ID NO: 497:

[0307] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0308] One method of determining the binding affinity of an antibody for BCMA or CD3 is to measure the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (e.g., IgG) can be cleaved with papain or recombinantly expressed. The affinity of the BCMA Fab fragment of the antibody can be measured by surface plasmon resonance (Biacore) on a pre-immobilized streptavidin sensor chip (SA). TM 3000 TM Surface plasmon resonance (SPR) system, Biacore TM , INC, Piscataway NJ) or anti-mouse Fc or anti-human Fc determined 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 can be diluted to a concentration of less than 0.5 μg / mL in HBS-EP buffer and injected through separate chip channels using variable contact times to obtain two ranges of antigen density, 50-200 response units (RU) for detailed kinetic studies or 800-1,000 RU for screening assays. Regeneration studies showed that 25 mM NaOH in 25% v / v ethanol effectively removed bound Fab while maintaining activity of BCMA on the chip for up to 200 injections. Typically, serial dilutions of purified Fab samples (spanning 0.1-10x estimated K D The concentration of the Fab protein was determined by ELISA and / or SDS-PAGE electrophoresis using a known concentration of Fab (as determined by amino acid analysis) as a standard. The kinetic binding rate (k) was obtained by 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 a BIA evaluation program. on ) and dissociation rate (k off ). 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 for any BCMA, including human BCMA, BCMA from another mammal (such as mouse BCMA, rat BCMA, or primate BCMA), and different forms of BCMA (such as glycosylated BCMA). Antibody binding affinity is typically measured at 25°C, but can also be measured at 37°C.

[0309] Antibodies as described herein can be prepared by any method known in the art. As further described herein, for the preparation of hybridoma cell lines, the route and schedule of host animal immunization generally follow established and conventional techniques for antibody stimulation and production. General techniques for preparing human and mouse antibodies are known in the art and / or described herein.

[0310] It is contemplated that any mammalian individual, including humans, or its antibody-producing cells, including human and hybridoma cell lines, can be manipulated to serve as the basis for mammalian production. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intrapedicularly, and / or intradermally with an amount of the immunogen, including as described herein.

[0311] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256:495-497, 1975 or as modified by Buck, DW, et al., In Vitro, 18:377-381, 1982. Available myeloma lines can be used in hybridization, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA. Typically, the technique comprises fusing myeloma cells and lymphoid cells using a fusing agent such as polyethylene glycol or by electrical methods well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown on a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) to eliminate unhybridized parental cells. Any culture medium described herein, supplemented or not supplemented with serum, can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion technology, EBV immortalized B cells can be used to produce the monoclonal antibodies of the present invention. The hybridomas are expanded and subcloned, if desired, and the supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0312] Hybridomas that can be used as a source of antibodies encompass all derivatives and progeny cells of the parent hybridoma that produce monoclonal antibodies specific for BCMA, CD3, or portions thereof.

[0313] Known methods can be utilized to make the hybridoma producing this type of antibody grow in vitro or in vivo. If desired, monoclonal antibodies can be separated from culture medium or body fluids by conventional immunoglobulin purification methods such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration. Undesirable activity (if present) can be removed by, for example, running products on an adsorbent made from the immunogen being connected to a solid phase and eluting or releasing the desired antibody from the immunogen. Utilize bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated by cysteine ​​residues), N-hydroxysuccinimide (by lysine residues), glutaraldehyde, succinic anhydride, SOCl or R 1 N=C=NR (where R and R 1 A population of antibodies (e.g., monoclonal antibodies) can be generated by immunizing human BCMA or CD3 or a fragment comprising the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or trypsin inhibitor).

[0314] If desired, the antibody of interest (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be expanded and frozen for future use. Recombinant monoclonal antibodies can be produced in cell culture by cloning antibody genes from B cells in a manner known in the art. See, for example, Tiller et al., J. Immunol. Methods 329, 112, 2008; US Pat. No. 7,314,622.

[0315] In one option, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or improve affinity, or other characteristics of the antibody. For example, the constant region can be engineered to be closer to that of a human constant region to avoid an immune response if the antibody is used in clinical trials and therapeutics in humans. It is desirable to genetically manipulate the antibody sequence to obtain greater affinity for BCMA or CD3 and greater potency in inhibiting BCMA.

[0316] There are four general steps to humanizing a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains, (2) designing the humanized antibody, i.e., deciding which antibody framework regions to use in the humanization process, (3) the actual humanization method / technique, and (4) transfection and expression of the humanized antibody. See, for example, US Pat. 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.

[0317] Many "humanized" antibody molecules comprising antigen-binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies having rodent or modified rodent V regions fused to human constant regions and their associated CDRs. See, for example, Winter et al. Nature 349: 293-299, 1991, Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220-4224, 1989, Shaw et al. J Immunol. 138: 4534-4538, 1987, and Brown et al. Cancer Res. 47: 3577-3583, 1987. Other references describe rodent CDRs that are grafted into human supporting framework regions (FRs) prior to fusion with appropriate human antibody constant regions. See, for example, 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 recombinantly engineered rodent framework regions. See, for example, European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize undesirable immune responses to rodent anti-human antibody molecules, which limit the duration and effectiveness of therapeutic applications of those parts in human recipients. For example, antibody constant regions can be engineered so that they are immunologically inert (e.g., do not trigger complement lysis). See, for example, PCT Publication No. PCT / GB99 / 01441; UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that may also be used are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991 and in US Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01 / 27160.

[0318] The general principles associated with humanized antibodies discussed above can also be applied to custom antibodies for, for example, dogs, cats, primates, horses, and cattle. In addition, one or more aspects of humanizing the antibodies described herein can combine, for example, CDR grafting, framework mutations, and CDR mutations.

[0319] In one variation, fully human antibodies can be obtained by utilizing commercially available mice that have been engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to produce humanized or human antibodies. An example of such technology is the Xenomouse® from Abgenix, Inc. (Fremont, CA). TM and from Medarex, Inc. (Princeton, NJ) and TC Mouse TM .

[0320] In one selection, antibodies can be recombinantly prepared and expressed using any method known in the art. In another selection, antibodies can be recombinantly prepared by phage display technology. See, for example, US Pat. No. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12: 433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553, 1990) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technology, the antibody V domain gene is cloned in frame into the major or minor capsid protein gene of a filamentous phage such as M13 or fd and displayed on the surface of the phage particle as a functional antibody fragment. Because the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies that exhibit these properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in various formats; for review, see, for example, 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 set of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene libraries from unimmunized human donors can be constructed, and antibodies to diverse antigens (including self-antigens) can be separated essentially according to the techniques described in 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 changes introduced confer high affinity, and B cells that exhibit high-affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigenic attack. This natural process can be mimicked by employing a technique known as "chain shuffling." (Marks et al., Bio / Technol. 10: 779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by replacing the heavy and light chain V regions with the library order of naturally occurring variants (libraries) of the V domains obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range.Waterhouse et al., Nucl. Acids Res. 21: 2265-2266, 1993 have described a strategy for preparing very large phage antibody libraries (also referred to as "root libraries"). Gene shuffling can also be used to derive human antibodies from rodent antibodies, wherein the human antibodies have similar affinity and specificity to the starting rodent antibodies. According to this method, which is also referred to as "epitope imprinting", the heavy chain or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a library of human V domain genes to produce rodent-human chimeras. Antigen selection results in the separation of human variable regions that can restore functional antigen binding sites, i.e., the selection of epitope-dominated (imprinted) partners. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication No. WO 93 / 06213). Unlike traditional humanization of rodent antibodies by CDR transplantation, this technology provides fully human antibodies that do not have framework or CDR residues of rodent origin.

[0321] Antibodies can be recombinantly produced by first isolating the antibody or antibody-producing cells from the 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 transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for preparing antibody derivatives such as humanized, single-chain, etc. are known in the art.

[0322] Immunoassays and flow cytometric sorting techniques such as fluorescence activated cell sorting (FACS) can also be used to isolate antibodies specific for BCMA, CD3, or the tumor antigen of interest.

[0323] Antibodies as described herein can be bound to many different carriers. The carrier can be active and / or inert. Examples of known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier can be soluble or insoluble. Those skilled in the art will know other suitable carriers for binding antibodies or will be able to determine such carriers using routine experimentation. In some embodiments, the carrier comprises a portion that targets the myocardium.

[0324] DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional methods (e.g., by utilizing oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells are used as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector (e.g., an expression vector disclosed in PCT Publication No. WO 87 / 04462), which is then transfected into a host cell that does not produce immunoglobulins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, in order to obtain the synthesis of the monoclonal antibody in the recombinant host cell. See, for example, PCT Publication No. WO 87 / 04462. The DNA can also be modified, for example, by replacing the coding sequences of the human heavy and light chain constant regions with the homologous mouse sequences, Morrison et al., Proc. Nat. Acad. Sci. 81: 6851, 1984, or by covalently linking to the immunoglobulin coding sequence, all or part of the coding sequence of a non-immunoglobulin polypeptide. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the monoclonal antibodies herein.

[0325] BCMA or the infused tumor antigen antibodies as described herein can be identified or characterized using methods known in the art to detect and / or measure a reduction in the expression level of BCMA or other tumor antigens. In some embodiments, BCMA antibodies are identified by incubating a candidate substance with BCMA and monitoring binding and / or the accompanying reduction in BCMA expression levels. Binding assays can be performed using purified BCMA polypeptides, or using cells that naturally express or transfected to express BCMA polypeptides. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete with a known BCMA antibody for BCMA binding is evaluated. The assay can be performed in various formats, including an ELISA format.

[0326] After initial identification, the activity of candidate BCMA, CD3, or other tumor antigen antibodies can be further confirmed and refined using bioassays known to detect target biological activity. Alternatively, bioassays can be used to directly screen candidates. Some methods for identifying and characterizing antibodies are described in detail in the Examples.

[0327] BCMA, CD3 or other tumor antigen antibodies can be characterized using methods known in the art. For example, one method is to identify the epitope to which it binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including crystal structure analysis of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, such as those described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In additional examples, epitope mapping can be used to determine the sequence to which the antibody binds. Epitope mapping is commercially available from various sources, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). An epitope can be a linear epitope, i.e., contained in a single amino acid stretch, or a conformational epitope formed by the three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch. Peptides of different lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with BCMA, CD3 or other tumor antigen antibodies. In another example, the epitope bound by BCMA, CD3 or other tumor antigen antibodies can be determined in a systematic screening by utilizing overlapping peptides derived from BCMA, CD3 or other tumor antigen sequences and determining binding by BCMA, CD3 or other tumor antigen antibodies. According to gene fragment expression assays, the open reading frame encoding BCMA, CD3 or other tumor antigens is fragmented randomly or by a specific genetic construct, and the reactivity of the expressed BCMA, CD3 or other tumor antigen fragments with the antibody to be tested is determined. Gene fragments can be generated, for example, by PCR and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled BCMA, CD3 or other tumor antigen fragment is then determined by immunoprecipitation or gel electrophoresis. Certain epitopes can also be identified by utilizing a large library of random peptide sequences displayed on the surface of phage particles (phage library). 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 swapping experiments, and alanine scanning mutagenesis can be performed to identify residues that are required, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutant BCMA, CD3, or other tumor antigens, in which various fragments of the BCMA, CD3, or other tumor antigen protein have been replaced (exchanged) with sequences from BCMA from another species (e.g., mouse) or a closely related but antigenically distinct protein (e.g., Trop-1).By evaluating antibody binding to mutant BCMA, CD3, or other tumor antigens, one can assess the importance of specific BCMA, CD3, or other tumor antigen fragments for antibody binding.

[0328] 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 the same antigen (i.e., various fragments on BCMA, CD3 or other tumor antigens) to determine whether the BCMA, CD3 or other tumor antigen antibody binds to the same epitope as the other antibody. Competition assays are well known to those skilled in the art.

[0329] Expression vectors can be used to directly express BCMA, CD3 or other tumor antigen antibodies. Those skilled in the art are familiar with the administration of expression vectors to obtain expression of exogenous proteins in vivo. See, for example, US Pat. No. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or intubation administration, and topical administration. In another embodiment, the expression vector is administered directly to a sympathetic nerve trunk or ganglion, or to a coronary artery, atrium, ventricle, or pericardium.

[0330] Targeted delivery of therapeutic compositions comprising 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, JA Wolff, ed., 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. For local administration in gene therapy protocols, therapeutic compositions comprising polynucleotides are administered in the range of about 100 ng to about 200 mg of DNA. 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 in gene therapy protocols. Gene delivery vehicles can be used to deliver therapeutic polynucleotides and polypeptides. Gene delivery vehicles can be viral or non-viral in origin (see generally, Jolly, Cancer Gene Therapy, 1:51, 1994; Kimura, HμMan Gene Therapy, 5:845, 1994; Connelly, HμMan Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 6:148, 1994). Endogenous mammalian or heterologous promoters can be used to induce the expression of such coding sequences. The expression of coding sequences can be constitutive or regulated.

[0331] Viral-based vectors for delivery of desired polynucleotides and expression in desired cells are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Pat. No. 2,200,651; and EP Pat. No. 0 345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-1250). VR-532)) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to killed adenovirus as described in Curiel, HμM. Gene Ther., 1992, 3:147 can also be used.

[0332] Non-viral delivery vehicles and methods can also be used, including but not limited to polyoxygen ion condensed DNA linked or unlinked to killed adenovirus (see, e.g., Curiel, H. μM. Gene Ther., 3: 147, 1992); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264: 16985, 1989); eukaryotic cell delivery vehicles (see, e.g., US Pat. No. 5,814,482; PCT Publication Nos. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90 / 11092 and US Pat. No. 5,580,859. US Pat. No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP 0524968 describe liposomes that can serve as gene delivery vehicles. Additional methods are described in Philip, Mol. Cell Biol., 14:2411, 1994 and Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

[0333] In some embodiments, the present invention encompasses compositions, including pharmaceutical compositions, comprising antibodies described herein or prepared by the methods described herein and having the characteristics described herein. As used herein, compositions comprise one or more antibodies that bind to CD3 or a tumor antigen (e.g., BCMA), and / or one or more polynucleotides comprising sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

[0334] The present invention also provides methods for preparing any of these antibodies. The antibodies of the present invention can be prepared by methods known in the art. The polypeptides can be prepared by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Polypeptides of the antibody, particularly shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, the antibodies can be prepared by an automated polypeptide synthesizer using a solid phase method. See also, US Pat. No. 5,807,715; 4,816,567; and 6,331,415.

[0335] Heteroconjugate antibodies comprising two covalently linked antibodies are also within the scope of the present invention. Such antibodies have been used to target immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (PCT Publication Nos. WO 91 / 00360 and WO 92 / 200373; EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are described in US Pat. No. 4,676,980.

[0336] 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 suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0337] In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction with Fcγ receptors, complement, and the immune system. WO 99 / 58572 describes techniques for preparing such antibodies. For example, the constant region can be engineered to be more human-like so as to avoid immune responses if the antibody is used in clinical trials and therapeutics in humans. See, for example, US Pat. Nos. 5,997,867 and 5,866,692.

[0338] The present invention encompasses modifications to the antibodies and polypeptides of the invention as described herein, including functionally equivalent antibodies that do not significantly affect their properties and variants with enhanced or reduced activity and / or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with a desired binding affinity to BCMA and / or CD3. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly deleteriously alter functional activity, or the affinity of the mature (enhanced) polypeptide for its ligand, or the use of chemical analogs.

[0339] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides comprising one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to epitope tags. Other insertion variants of the antibody molecule include fusions of enzymes or polypeptides that increase the half-life of the antibody in the blood circulation to the N- or C-terminus of the antibody.

[0340] The substitution variant has at least one amino acid residue removed and a different residue inserted in its position in the antibody molecule. The sites of greatest concern for substitution mutagenesis include hypervariable regions, but FR changes are also considered. Conservative substitutions are shown in Table 5 under the heading "conservative substitutions." If this type of substitution results in a change in biological activity, more substantial variations can be introduced, designated as "exemplary substitutions" in Table 5, or as further described below with reference to amino acid classes, and the product screened.

[0341] Table 5: Amino acid substitutions

[0342]

[0343]

[0344] Substantial changes in the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring amino acid residues are grouped based on common side chain properties:

[0345] (1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0346] (2) Polarity has no charge: Cys, Ser, Thr, Asn, Gln;

[0347] (3) Acidic (negatively charged): Asp, Glu;

[0348] (4) Basic (positively charged): Lys, Arg;

[0349] (5) Residues that affect chain direction: Gly, Pro; and

[0350] (6) Aromatic: Trp, Tyr, Phe, His.

[0351] Exchanging a member of one of these classes for another class results in non-conservative substitutions.

[0352] Any cysteine ​​residues not involved in maintaining the correct conformation of the antibody can also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

[0353] Amino acid modifications can range from changing or modifying one or more amino acids to completely redesigning a region, such as a variable region. Changes in the variable region can alter binding affinity and / or specificity. In some embodiments, no more than 1-5 conservative amino acid substitutions are made within the CDR domain. In other embodiments, no more than 1-3 conservative amino acid substitutions are made within the CDR domain. In other embodiments, the CDR domain is CDR H3 and / or CDR L3.

[0354] Modifications also include glycosylated or non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 65: 111-128, 1997; Wright and Morrison, TibTECH 15: 26-32, 1997). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., Mol. Immunol. 32: 1311-1318, 1996; Wittwe and Howard, Biochem. 29: 4175-4180, 1990), and the intramolecular interactions between glycoprotein moieties can affect the conformation and present the three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7: 409-416, 1996). Based on specific recognition structures, oligosaccharides can also be used to target a given glycoprotein to certain molecules. It has also been reported that the glycosylation of antibodies affects antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the bisecting formation of GlcNAc, were reported to have enhanced ADCC activity (Mana et al., Mature Biotech. 17:176-180, 1999).

[0355] Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine ​​(wherein X is any amino acid except proline) are recognition sequences for the enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to an amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0356] Glycosylation sites are conveniently added to antibodies by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0357] The glycosylation pattern of an antibody can also be altered without changing the underlying nucleotide sequence. Glycosylation is primarily dependent on the host cell used to express the antibody. Because the cell types used to express recombinant glycoproteins such as antibodies as potential therapeutic agents are rarely native cells, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al., J. Biol. Chem. 272:9062-9070, 1997).

[0358] In addition to the choice of host cells, factors that affect glycosylation during the recombinant production of antibodies include growth mode, medium formulation, culture density, oxidation, pH, purification schemes, etc. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (US Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation or certain types of glycosylation can be enzymatically removed from glycosyltransferases, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, recombinant host cells can be genetically engineered to become defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

[0359] Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic methods, oxidative substitution, and chelation. For example, the modification can be used to attach a label for immunoassays. Modified polypeptides can be prepared using methods 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.

[0360] In some embodiments of the invention, the antibody comprises a modified constant region, such as a constant region with increased affinity for human Fcγ receptors, is immunologically inert or partially inert, for example, does not trigger 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: triggering complement-mediated lysis, stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or activating microglia. Different modifications of the constant region can be used to obtain optimal levels and / or combinations of effector functions. See, e.g., Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-4969, 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 Publication No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence). Eur. J. Immunol., 1999, 29:2613-2624. In other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the glycosylated amino acid residues in the constant region or the flanking residues of the N-glycosylation recognition sequence. 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 aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation either enzymatically (eg, by removal of sugars by the enzyme PNGase) or by expression in a glycosylation-deficient host cell.

[0361] Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99 / 58572. In addition to the binding domain for the target molecule, these antibodies contain an effector domain having an amino acid sequence substantially homologous to all or part of the constant region of a human immunoglobulin heavy chain. These antibodies are capable of binding to the target molecule without triggering significant complement-dependent lysis or cell-mediated target destruction. In some embodiments, the effector domain is capable of specifically binding to FcRn and / or FcγRIIb. These are typically based on proteins derived from two or more human immunoglobulin heavy chain C H Antibodies modified in this way are particularly suitable for chronic antibody therapy in order to avoid the inflammation and other adverse reactions of conventional antibody therapy.

[0362] The present invention includes affinity matured embodiments. For example, affinity matured antibodies can be prepared by methods 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).

[0363] The following methods can be used to adjust the affinity of antibodies and characterize CDRs. A method for characterizing the CDRs of antibodies and / or changing (such as improving) the binding affinity of polypeptides such as antibodies is called "library scanning mutagenesis." Typically, library scanning mutagenesis works as follows. Utilize methods well known in the art to replace one or more amino acid positions in the CDR 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) amino acids. This produces a small library of clones (in some embodiments, one for each amino acid position analyzed), each with a complexity of two or more members (if two or more amino acids are replaced at each position). Typically, the library also includes clones containing natural (unsubstituted) amino acids. A small amount of clones from each library, such as about 20-80 clones (depending on the complexity of the library), are screened for binding affinity to the target polypeptide (or other binding targets), and candidates with increased, identical, reduced or no binding are identified. Methods for determining binding affinity are well known in the art. Biacore TM Binding affinity is determined by surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater. D When combined, Biacore TM The use of Biacore is described in the examples herein. TM Screening by surface plasmon resonance.

[0364] Binding affinity can be established using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast display. Binding affinity can also be screened using a suitable bioassay.

[0365] In some embodiments, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids using mutagenesis methods known in the art (some of which are described herein). This generates a small library of clones (in some embodiments, one for each amino acid position analyzed), each with a complexity of 20 members (if all 20 amino acids are substituted at each position).

[0366] In some embodiments, the library to be screened comprises substitutions at two or more positions, which can be in the same CDR or in two or more CDRs. Thus, the library can comprise substitutions at two or more positions in one CDR. The library can comprise substitutions at two or more positions in two or more CDRs. The library can comprise substitutions at 3, 4, 5 or more positions, which are located in 2, 3, 4, 5 or 6 CDRs. Substitutions can be made using low redundancy codons. See, for example, Table 2 of Balint et al., Gene 137(1):109-18, 1993.

[0367] The CDR may be CDRH3 and / or CDRL3. The CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and / or CDRH3. The CDR may be a Kabat CDR, a Chothia CDR, or an extended CDR.

[0368] Candidates with improved binding can be sequenced to identify CDR substitution mutations that result in improved affinity (also referred to as "improving" substitutions). Candidates that bind can also be sequenced to identify CDR substitutions that maintain binding.

[0369] Multiple rounds of screening can be performed. For example, candidates with improved binding (each comprising an amino acid substitution at one or more positions in one or more CDRs) can also be used to design a second library comprising at least the original and substituted amino acids at each improved CDR position (i.e., the amino acid position in the CDR where the substitution mutant exhibits improved binding). Preparation of such libraries and screening or selection are discussed further below.

[0370] Library screening mutagenesis also provides the method for characterizing CDR, because the frequency of the clone with the combination of raising, identical combination, minimization or no combination also provides the information about the importance of each amino acid to antibody-antigen complex stability.For example, if the position of CDR keeps combining when being changed to whole 20 kinds of amino acid, then this position is accredited as the position that antigen combines and is unlikely to need.On the contrary, if the position of CDR only keeps combining in a small portion replacement, then this position is accredited as the position important to CDR function.Therefore, library screening mutagenesis method produces about the position in the CDR that can be changed to many different amino acids (comprising whole 20 kinds of amino acid) and can not change or only can change to the information of the position in several amino acid whose CDR.

[0371] The candidate with the affinity of raising can be merged in the second library, and it comprises the amino acid that raises, the original amino acid at this position, and can further comprise extra replacement at this position, depend on expectation or utilize the library complexity that the screening or selection method of expectation allows.In addition, if expectation, adjacent amino acid position can be randomized to at least two or more amino acid.The randomization of adjacent amino acid can allow extra conformational flexibility in the sudden change CDR, and conversely it can allow or promote to introduce a large amount of raising sudden changes.The library can also comprise replacement at the position that does not show the affinity of raising in the first round screening.

[0372] The second library is screened or selected for library members having improved and / or altered binding affinity using any method known in the art, including using Biacore TM Screening can be performed by surface plasmon resonance analysis, and selection can be performed using any method known in the art for selection, including phage display, yeast display, and ribosome display.

[0373] The present invention also provides compositions comprising an antibody conjugated to an agent that facilitates coupling to a solid support, such as biotin or avidin. For simplicity, reference will generally be made to antibodies, with the understanding that these methods apply to any BCMA antibody embodiment described herein. Conjugation generally refers to linking these components as described herein. Linking (which generally fixes these components in a directly associated manner, at least for administration) can be accomplished in any manner. For example, direct reaction between the agent and the antibody is possible when each has substituents that are capable of reacting with each other. For example, a nucleophilic group on one, such as an amino group or a thiol group, may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group on the other containing a good leaving group (e.g., a halide).

[0374] In another aspect, the invention provides a method of making any of the polynucleotides described herein.

[0375] The present invention also encompasses polynucleotide complementarity of any such sequences. 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, which contain introns and correspond to DNA molecules in a one-to-one manner; and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present invention, and polynucleotides may, but need not, be linked to other molecules and / or support materials.

[0376] 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. The polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be evaluated as described herein. The variant preferably exhibits at least about 70% identity to the polynucleotide sequence encoding the native antibody or a portion thereof, more preferably, at least about 80% identity, more preferably, at least about 90% identity, and most preferably, at least about 95% identity.

[0377] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically 30 to about 75, or 40 to about 50, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned.

[0378] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program includes several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0379] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less additions or deletions (i.e., gaps), typically 5-15%, or 10-12%, when compared to a reference sequence (which does not contain 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 occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence and multiplying the result by 100 to produce the percentage of sequence identity.

[0380] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0381] Suitable "moderately stringent conditions" include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C-65°C, 5X SSC; and then washing twice at 65°C for 20 minutes, each time with 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[0382] As used herein, "high stringency conditions" or "high stringency conditions" are: (1) using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, during hybridization, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 at 42°C with 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate ... Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate, washes in 0.2× SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1× SSC containing EDTA at 55° C. The skilled artisan will recognize how to adjust temperature, ionic strength, etc., and, if necessary, factors such as probe length.

[0383] Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that differ due to differences in codon usage. Moreover, alleles of genes comprising 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 deletions, additions, and / or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0384] Polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. The method for chemical polynucleotide synthesis is well known in the art and need not be described in detail herein. Those skilled in the art can use the sequence provided by this paper and commercial DNA synthesizer to prepare the desired DNA sequence.

[0385] In order to utilize recombinant methods to prepare polynucleotides, as further discussed herein, the polynucleotide comprising the desired sequence can be inserted into a suitable vector, and then the vector is introduced into a suitable host cell for replication and amplification. Polynucleotides can be inserted into host cells by any method known in the art. Exogenous polynucleotides are introduced into cells by direct absorption, endocytosis, transfection, F-conjugation or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as a non-integrated vector (e.g., plasmid) or integrated into the host cell genome. The polynucleotides amplified in this way can be separated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0386] Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in the art and are described in, for example, US Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

[0387] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, such as shown in Sambrook et al., 1989, supra.

[0388] Suitable cloning vector can be made up according to standard techniques, or can be selected from a large amount of cloning vectors available in this area. Although selected cloning vector can be used according to the host cell change of expectation, available cloning vector generally has the ability of self-replication, can have the single target of specific restriction endonuclease, and / or can carry the gene of the marker that can be used for selecting the clone containing described vector.Suitable example comprises plasmid and bacterial virus, for example pUC18, pUC19, Bluescript (for example pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vector, as pSA3 and pAT28.These and many other cloning vectors can be obtained from commercial suppliers, for example BioRad, Strategene and Invitrogen.

[0389] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide of the present invention. This implies that the expression vector must be replicable in the host cell as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components generally may include, but are not limited to, one or more of the following components: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are generally also required.

[0390] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, when the vector is an infectious agent, such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0391] 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 genes encoding antibodies, polypeptides, or proteins 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. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe; or Kluyveromyces lactis). Preferably, the host cell expresses the cDNA at a level that is about 5 times higher, more preferably 10 times higher, and even more preferably 20 times higher than the corresponding endogenous antibody or protein of interest (if present) in the host cell. Screening for host cells that specifically bind to BCMA or BCMA domains (e.g., domains 1-4) is performed by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

[0392] Methods of using bispecific antibodies [Therapeutic applications]

[0393] The antibodies (e.g., BCMA, CD3, or bispecific) and antibody conjugates (e.g., BCMA antibody-drug conjugates) of the invention can be used in a variety of applications, including, but not limited to, therapeutic treatment methods and diagnostic treatment methods.

[0394] In one aspect, the present invention provides a method for treating a disease condition associated with BCMA expression in an individual. In some embodiments, the method for treating a disease condition associated with BCMA expression in an individual comprises administering to an individual in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising a BCMA antibody or BCMA antibody conjugate as described herein. Disease conditions associated with BCMA expression include, but are not limited to, abnormal BCMA expression, altered or aberrant BCMA expression, malignant cells expressing BCMA, and proliferative disorders (e.g., cancer) or autoimmune disorders.

[0395] In another aspect, the present invention provides a method for treating a B-cell-associated cancer or a malignant cell expressing a tumor antigen. In some embodiments, a method for treating a B-cell-associated cancer in an individual in need thereof is provided, comprising a) providing a bispecific antibody as described herein, and b) administering the bispecific antibody to the patient. In some embodiments, a method for treating a disease condition associated with a malignant cell expressing a tumor antigen in an individual is provided, comprising administering to the individual in need thereof an effective amount of a pharmaceutical composition comprising a bispecific antibody as described herein.

[0396] Thus, in some embodiments, a method of treating cancer in an individual is provided, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody (e.g., BCMA or CD3-BCMA bispecific antibody) or BCMA antibody conjugate as described herein. As used herein, cancer can be a B-cell related cancer, including but not limited to multiple myeloma, malignant plasmacytoma, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Kahler's disease and myeloid leukemia, 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 (ALCL), and leukemia. ALL, chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphocytic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, inflammation-associated diffuse large B-cell lymphoma B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-related lymphomas.

[0397] In some embodiments, a method of inhibiting tumor growth or development in an individual with malignant cells expressing BCMA is provided, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate as described herein. In other embodiments, a method of inhibiting metastasis of cells expressing BCMA in an individual is provided, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate as described herein. In other embodiments, a method of inducing tumor regression of malignant cells in an individual is provided, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate as described herein.

[0398] In some embodiments, a method of treating an autoimmune disorder in an individual is provided, comprising administering to the individual in need thereof an effective amount of a composition comprising a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate as described herein.

[0399] As used herein, autoimmune disorders include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus (type I), multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, Hashimoto's encephalopathy, myasthenia gravis, pernicious anemia, reactive arthritis, Sjögren's syndrome, acute disseminated encephalomyelitis, agammaglobulinemia, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune bowel disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune Autoimmune pancreatitis, autoimmune endocrine polyglandular syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Behçet's disease, Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, 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.

[0400] In another aspect, the present invention provides an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein for use in treating a disease state (e.g., cancer or an autoimmune disorder) associated with BCMA expression in an individual in need thereof. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein is provided for inhibiting tumor growth or progression in an individual with malignant cells expressing BCMA. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein is provided for inhibiting metastasis of malignant cells expressing BCMA in an individual in need thereof. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein is provided for inducing tumor regression in an individual with malignant cells expressing BCMA.

[0401] In another aspect, the present invention provides antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates as described herein for use in treating a disease state associated with BCMA expression (e.g., cancer or an autoimmune disorder) in an individual in need thereof. In some embodiments, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates as described herein are provided for use in inhibiting tumor growth or development in an individual with malignant cells expressing BCMA. In some embodiments, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates as described herein are provided for use in inhibiting metastasis of malignant cells expressing BCMA in an individual in need thereof. In some embodiments, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates as described herein are provided for inducing tumor regression in an individual with malignant cells expressing BCMA.

[0402] In another aspect, the present invention provides the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein in the manufacture of a medicament for treating a disease state associated with BCMA expression (e.g., cancer or an autoimmune disorder). In some embodiments, the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein in the manufacture of a medicament for inhibiting tumor growth or development is provided. In some embodiments, the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing BCMA is provided. In some embodiments, the use of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein in the manufacture of a medicament for inducing tumor regression is provided.

[0403] In another aspect, a method for detecting, diagnosing, and / or monitoring a disease condition associated with BCMA expression is provided. For example, an antibody as described herein (e.g., BCMA or CD3-BCMA bispecific) can be labeled with a detectable moiety such as an imaging agent and an enzyme-substrate label. The antibodies as described herein can also be used in in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or staining reagents.

[0404] In some embodiments, the methods described herein further comprise the step of treating the individual 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.

[0405] In some embodiments, the additional form of therapy comprises administering one or more therapeutic agents in addition to an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein. The one or more therapeutic agents may be chemotherapeutic agents, including but not limited to secondary antibodies (e.g., anti-VEGF (vascular endothelial growth factor) antibodies (e.g., ), anti-HER2 antibodies (e.g. ), anti-CD25 antibodies, anti-CD33 antibodies, anti-CD20 antibodies (e.g. ), anti-mucin-like glycoprotein antibodies, anti-TNF antibodies, and / or epidermal growth factor receptor (EGFR) antibodies (e.g. )), angiogenesis inhibitors, cytotoxic agents (e.g., anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone), taxanes (e.g., paclitaxel and docetaxel), dolastatins, duocarmycins, enediynes, geldanamycins, maytansines, puromycins, 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 hemicycline), immunomodulators (e.g., prednisone and lenalidomide) ), anti-inflammatory agents (e.g., dexamethasone), aromatase inhibitors (e.g., anastrozole, exemestane, letrozole, vorozole, formestane, or testolactone), proteasome inhibitors (e.g., bortezomib, ([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propy-1]amino]butyl]boronic acid or carfilzomib) and other agents such as tamoxifen.

[0406] For example, in some embodiments, a method of treating multiple myeloma is provided, comprising administering to a patient in need thereof an effective amount of a composition comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody as described herein and one or more therapeutic agents such as a chemotherapeutic agent (e.g., doxorubicin or carfilzomib) or thalidomide or a derivative thereof (e.g., lenalidomide). In some embodiments, the one or more additional therapeutic agents are selected from bortezomib (e.g. ), melphalan, prednisone, doxorubicin, lenalidomide, thalidomide, prednisone, carmustine, etoposide, cisplatin, cyclophosphamide, carfilzomib, and vincristine. In some embodiments, the additional therapeutic agent is bortezomib (e.g., ), melphalan, lenalidomide Carfilzomib, doxorubicin or prednisone. Therefore, a method of treating multiple myeloma is provided, comprising administering to a patient in need thereof an effective amount of a composition comprising an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate as described herein and one or more other therapeutic agents selected from bortezomib, lenalidomide, carfilzomib and doxorubicin. In some embodiments, the patient has relapsed or is refractory to previous multiple myeloma therapy.

[0407] The antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be administered to an individual by any suitable route. It will be understood by those skilled in the art that the examples described herein are not intended to limit but to illustrate available technology. Therefore, in some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate is administered to an individual according to known methods, such as intravenous administration, for example as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, by insufflation, intrathecal, oral, inhalation or body surface routes. Administration can be systemic, such as intravenous administration, or local. Commercially available liquid formulation nebulizers, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid formulations can be directly atomized, while lyophilized powders can be atomized after reconstitution. Alternatively, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be nebulized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and ground powder.

[0408] In one embodiment, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate is administered by site-specific or targeted local delivery technology. Examples of site-specific or targeted local delivery technologies include various implantable storage sources or local delivery catheters of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate, such as perfusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00 / 53211 and US Pat. No. 5,981,568.

[0409] Various formulations of the antibody (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 conjugates can be administered neat. In some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates and pharmaceutically acceptable excipients can be in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that promote the administration of pharmacologically effective substances. For example, excipients can be given form or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts that change osmotic pressure, encapsulation agents, and skin penetration enhancers. Excipients and formulations for parenteral and non-parenteral drug delivery are shown in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005.

[0410] 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., dose, timing, and repetition, will depend on the particular individual and that individual's medical history.

[0411] As described herein, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can be administered using any suitable method, including by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). As described herein, the antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can also be administered by inhalation. Typically, for the administration of antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates, the initial candidate dose can be about 2 mg / kg. For the purposes of the present invention, a typical daily dose can range from about any 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 mentioned 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 longer, depending on the disease state, treatment is continued until the desired symptom suppression occurs or until sufficient therapeutic levels are reached, such as suppressing or delaying tumor growth / development or metastasis of cancer cells. An exemplary dosage administration regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate, or then a maintenance dose of about 1 mg / kg every other week. Other exemplary dosage administration regimens include administering increasing doses (e.g., an initial dose of 1 mg / kg and gradually increasing to one or more higher doses per week or longer). Other dosage regimens are also available, depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, in some embodiments, 1-4 doses are considered per week. In other embodiments, dose administration is considered once a month, once every other month, or once every three months. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage administration regimen (including the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate used) can vary over time.

[0412] For the purposes of the present invention, the appropriate dosage of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate depends on the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate employed, the type and severity of the symptom to be treated, whether the agent is administered for therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, the patient's clearance of the administered agent, and the discretion of the attending physician. Typically, the physician administers the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate until a dose that achieves the desired result is reached. The dosage and / or frequency may vary over the course of treatment. Empirical considerations such as half-life generally aid in determining dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, based on the treatment and / or inhibition and / or relief and / or delay of symptoms, such as tumor growth inhibition or delay. Alternatively, sustained-release formulations of antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA-antibody conjugates may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0413] In one embodiment, the dosage of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be determined empirically in an individual who has been given one or more administrations of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. Increasing doses of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate are administered to the individual. Indicators of disease can be tracked to assess efficacy.

[0414] Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate according to the methods of the present invention can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be substantially continuous over a preselected time period or can be a series of spaced doses.

[0415] In some embodiments, more than one antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate may be present. There may be at least 1, at least 2, at least 3, at least 4, at least 5 different or more antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates. Typically, these antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates may have complementary activities that do not adversely affect each other. For example, one or more of the following antibodies may be used: a first BCMA or CD3 antibody directed against one epitope on BCMA or CD3 and a second BCMA or CD3 antibody directed against a different epitope on BCMA or CD3.

[0416] Therapeutic formulations of antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates used in accordance with the present invention are prepared for storage by mixing the antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005) in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the doses and concentrations employed and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0417] Liposomes containing antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates are prepared by methods known in the art, such as Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688, 1985; Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030, 1980; and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to produce liposomes with a desired diameter.

[0418] The active ingredient can also be embedded in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005.

[0419] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-propylene acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®, and the like. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0420] Formulations for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

[0421] The compositions of the present invention may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0422] To prepare solid compositions such as tablets, the primary active ingredient is mixed 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 preformulated composition comprising a homogeneous mixture of the compound of the invention or a nontoxic, pharmaceutically acceptable salt thereof. When these preformulated compositions are referred to as homogeneous, it is meant that the active ingredient is evenly dispersed throughout the composition, allowing the composition to be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulated composition is then subdivided into unit dosage forms of the above type containing 0.1 to about 500 mg of the active ingredient of the invention. The tablets or pills of the novel composition can be coated or compounded to provide a dosage form having the advantage of a long-lasting effect. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of an envelope covering the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0423] In particular, suitable surfactants include nonionic agents such as polyoxyethylene sorbitan (e.g. Tween TM 20, 40, 60, 80 or 85) and other sorbitan (such as Span TM 20, 40, 60, 80 or 85). Compositions with surfactant will conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added if necessary.

[0424] Commercially available fat emulsions such as Intralipid TM , Liposyn TM Infonutrol TM 、Lipofundin TM and Lipiphysan TMPrepare a suitable emulsion. The active ingredient can be dissolved in a premixed emulsion composition, or it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and formed into an emulsion when mixed with a phospholipid (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tension of the emulsion. Suitable emulsions typically contain up to 20% oil, e.g., 5-20%. Fat emulsions can contain fat droplets of 0.1-1.0 μm, particularly 0.1-0.5 μm, and have a pH in the range of 5.5-8.0.

[0425] The emulsion composition can be prepared by mixing an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate with an intralipid. TM or those compositions prepared from its components (soybean oil, egg yolk phospholipids, glycerol and water).

[0426] Compositions for inhalation or insufflation include pharmaceutically acceptable, solutions or suspensions in aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as indicated above. In some embodiments, for local or systemic effect, the compositions are administered by oral or nasal respiratory route. Compositions in preferably sterile pharmaceutically acceptable solvents can be atomized using a gas. Atomized solutions can be breathed directly from the atomizing device, or the atomizing device can be connected to a mask, tent, or intermittent positive pressure respirator. Solutions, suspensions, or powder compositions can be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

[0427] Composition

[0428] The composition used in the method of the present invention comprises an effective amount of an antibody as described herein (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate. Examples of such compositions and how to prepare them are also described in the previous sections and below. In some embodiments, the composition comprises one or more antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates. For example, BCMA antibodies or CD3-BCMA bispecific antibodies recognize human BCMA or CD3-BCMA. In some embodiments, BCMA or CD3-BCMA antibodies are human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, BCMA antibodies or CD3-BCMA antibodies comprise a constant region that can trigger a desired immune response such as antibody-mediated lysis or ADCC. In other embodiments, BCMA antibodies or CD3-BCMA antibodies comprise a constant region that does not trigger an unwanted or undesirable immune response such as antibody-mediated lysis or ADCC.

[0429] It should be understood that the composition can include more than one antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate (e.g., a mixture of BCMA antibodies or CD3-BCMA bispecific antibodies that recognize different epitopes of BCMA or CD3 and BCMA). Other exemplary compositions include more than one BCMA antibody, CD3-BCMA antibody, or BCMA antibody conjugate that recognizes the same epitope, or BCMA antibodies, CD3-BCMA bispecific antibodies, or BCMA antibody conjugates from different species that bind to different epitopes of BCMA (e.g., human BCMA) or CD3 and BCMA (human CD3 and BCMA).

[0430] The composition used in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: The Science and practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. KE Hoover), in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosage and concentration, and may comprise buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 1% hydroxybenzoic acid); 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 nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0431] Reagent test kit

[0432] The present invention also provides kits for use in the present methods. The kits of the present invention include one or more containers containing a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody as described herein, and instructions for use according to any of the methods of the invention described herein. Typically, these instructions include a description of administering the BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate for the above-described therapeutic treatments.

[0433] Instructions for use of a BCMA antibody, CD3-BCMA bispecific antibody, or BCMA antibody conjugate as described herein generally include information about the dose, dosage schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions on a magnetic or optical storage disk) are also acceptable.

[0434] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Also contemplated are packaging used in conjunction with specific devices, such as inhalers, nasal administration devices (e.g., sprayers), or infusion devices such as miniature pumps. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper 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 may further comprise a second pharmaceutically active agent.

[0435] The kit may optionally provide additional components such as buffers and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0436] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Example

[0437] Example 1: Determination of the kinetics and affinity of hBCMA / human IgG interactions 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.

[0438] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). Anti-BCMA antibody arrays were prepared on Bio-Rad GLC sensor chips using an amine-coupling method similar to that described in Abdiche, et al., Anal. Biochem. 411, 139-151 (2011). The assay temperature was fixed at 25°C, and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) direction by injecting a mixture of 1 mM ECD and 0.25 mM NHS at a flow rate of 30 μL / min for 3 minutes. IgG was immobilized on the activated spots in the ligand (vertical) direction by injecting 20 μg / mL in 10 mM acetate pH 4.5 buffer for 1.5 minutes (30 μg / mL). The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 minutes in the direction of the analyte.

[0439] The hBCMA binding assay was performed at either 37°C or 25°C in HBS-T+ running buffer supplemented with 1 mg / mL BSA. A kinetic titration method was used for interaction analysis as described by Abdiche et al. The hBCMA (human BCMA) analyte was injected in the analyte direction using a series of injections from low to high concentrations. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM (a 5-way series with 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 following the 50 nM hBCMA injection. Prior to hBCMA analyte injection, buffer was injected five times during the hBCMA analyte cycle using the same association and dissociation times to prepare a buffer blank sensorgram for double referencing purposes (as described in Myszka, J. Mol. Recognit. 12, 279-284 (1999)).

[0440] The sensorgrams were double-referenced and fitted to a 1:1 Langmuir and mass transport kinetic titration model in BIA Evaluation Software Version 4.1.1 (GE Lifesciences, Piscataway, NJ). The sensorgrams and fits are shown in FIG1 , and the kinetic and affinity parameters of various anti-BCMA antibodies of the invention are shown in Tables 6A-6C.

[0441] Table 6A

[0442] sample <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[t 1 / 2 (min)]]> <![CDATA[K D (pM)]]> A02_Rd4_6nM_C01 1.2E+06 2.8E-05 411 24 A02_Rd4_6nM_C16 1.1E+06 6.2E-05 187 59 Combo_Rd4_0.6nM_C29 6.6E+06 1.4E-04 83 21 L3PY / H3TAQ 2.6E+06 1.4E-04 84 53

[0443] Table 6B

[0444]

[0445]

[0446]

[0447]

[0448]

[0449] Table 6C*

[0450]

[0451]

[0452]

[0453] *Binding assays were performed at 37°C.

[0454] Example 2: Flow cytometry of human anti-BCMA antibodies on BCMA-positive tumor cells

[0455] This example demonstrates that various BCMA antibodies of the present invention bind to BCMA-positive tumor cells.

[0456] Binding of human anti-hBCMA expressed in mouse IgG2a was evaluated by flow cytometry on BCMA-expressing cells (KMS12BM, L363, MM1S, and KMS12PE). 250,000 cells were incubated with 0.5 ug of antibody in 100 uL of binding buffer (PBS (phosphate-buffered saline) + 0.2% BSA (bovine serum albumin)) and then incubated with Alex Fluor 647-conjugated anti-mouse IgG (Biolegend). Table 7 shows the MFI (mean fluorescence intensity) on BCMA-positive tumor cells by various BCMA antibodies (e.g., Combo_Rd4_0.6nM_C29, A02_Rd4_6nM_C01, A02_Rd4_6nM_C16, and P6E01 / H3TAQ).

[0457] Table 7

[0458]

[0459] Example 3: Cytotoxicity of anti-BCMA ADC in BCMA-positive cells

[0460] This example demonstrates the efficacy of anti-BCMA ADC in BCMA-positive cells.

[0461] Human anti-BCMA (L3.PY / P6E01, L3.PY / H3.TAQ, Combo_Rd4_0.6nM_C29, A02_Rd4_6nM_C01, and A02_Rd4_6nM_C16) were expressed as human IgG1 subtype engineered with a glutamine-containing transglutaminase ("Q") tag (e.g., LCQ05, H7c, N297A, N297Q, N297A / H7c, N297Q / LCQ05) for drug-antibody ratios (DARs) of 2, 4, and 6. TG17 corresponds to SEQ ID NO: 472 (LLQGPP); LCQ05 corresponds to SEQ ID NO: 474 (GGLLQGPP); and H7c corresponds to SEQ ID NO: 454 (LLQG), respectively, and is conjugated to 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, a transglutaminase tag can be engineered into the light chain, the heavy chain, or a combination of the light and heavy chains. In other examples, a transglutaminase tag (e.g., Q) is engineered into a site of the antibody, such as position 297 (EU numbering scheme) of human IgG. 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). The anti-BCMA antibody is then conjugated to Aur0101, Aur3377, and Aur0131 by a transamidation reaction catalyzed by a microbial transglutaminase between an anti-BCMA antibody carrying a targeting glutamine or glutamine tag at a specific site (e.g., the carboxyl or amino terminus of the heavy or light chain, position 297, or at another site of the antibody) and an amine-containing derivative of the payload (e.g., MMAD, Aur0101, Aur3377, or Aur0131). In some cases, the wild-type amino acid lysine at positions 222, 340, or 370 (according to the EU numbering scheme) is replaced with the amino acid arginine ("K222R," "K340R," or "K370R"). For example, it was found that the K222R substitution had a surprising effect, resulting in more homologous antibody and payload conjugates, better intermolecular cross-linking between the antibody and the payload, and / or a significant reduction in interchain cross-linking with the glutamine tag on the C-terminus of the antibody light chain.

[0462] In the transamidation reaction, glutamine on the antibody acts as an acyl donor and an amine-containing compound acts as an acyl acceptor (amine donor). Purified anti-BCMA antibodies at a concentration of 1-150 μM are treated with 5-100 molar excess of an acyl acceptor in the range of 5 μM-15 mM in 10-1000 mM NaCl, and 25 mM MES, HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] or 0.23-0.55% (w / v) Streptoverticilli μM mobaraense transglutaminase (ACTIVA) in Tris HCl buffer at a pH range of 6.2-8.8. TM , Ajinomoto, Japan). Reaction conditions were adjusted for individual acyl acceptor derivatives, and optimal efficiency and specificity were generally observed for 33 μM antibody, 0.67 mM derivative, and 0.378% (w / v) transglutaminase in 75 mM NaCl, 25 mM Tris HCl, pH 8.5. After incubation for 1-24 hours at 20-37 degrees Celsius, the antibodies were purified on butyl sepharose high performance (butyl HP) resin (GE Healthcare, Waukesha, WI) using standard chromatography methods known to those skilled in the art, such as commercial hydrophobic interaction chromatography from GE Healthcare.

[0463] Target expressing (MM1.S, KMS12BM and L363) cells were then seeded at 3000 cells / well on clear bottom plates. Cells were treated with 4-fold serial dilutions of the antibody-drug conjugate in triplicate. 96 hours after treatment, the cells were Cell viability was determined using the Luminescent Cell Viability Assay 96 (Promega, Madison, WI). Relative cell viability was determined as a percentage of the untreated control. EC50 was calculated using Prism software. Table 8 shows that all human anti-BCMA antibodies of the present invention conjugated to the cytotoxic agents 0101, 3377, and 0131 via a transglutaminase tag and a linker exerted potent cell-killing activity in BCMA-expressing cells.

[0464] Table 8

[0465]

[0466]

[0467] Example 4: Anti-BCMA ADC induces tumor regression in an orthotopic multiple myeloma model

[0468] This example demonstrates the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model.

[0469] In vivo efficacy studies of BCMA ADC were conducted using the multiple myeloma cell line MM1.S expressing luciferase and GFP (green fluorescent protein) in an orthotopic model. 7 MM1.S LucGFP cells were injected intravenously into 6-8 week old female CB17 / SCID animals via the tail vein. D-luciferin (Regis Technologies, Morton Grove, IL) (15 mg / mL, 200 uL / animal) was injected intraperitoneally, followed by isoflurane anesthesia and whole-body bioluminescence imaging (BLI) to monitor tumor burden. The bioluminescent signal, generated by the interaction between luciferase expressed by tumor cells and luciferin, was captured using IVIS SpectrμM CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image 4.4 (Caliper LifeSciences, Alameda, CA). When the total flux reached an average of 1-3E6 for all animals, animals were randomized and administered a single dose of a human anti-BCMA antibody conjugated to 1) LCQ05 / K222R-vc0101 at the C-terminus of the antibody light chain and a control conjugate via bolus tail vein injection. Animals were terminated when they exhibited hind limb paralysis, which is the endpoint of the MM1.S orthotopic model. Figure 2 A single dose of 3 mg / kg of various human anti-BCMA ADCs, 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, were shown to inhibit tumor development compared to negative control (NNC).

[0470] This study demonstrated that treatment with BCMA-ADC inhibited the progression of multiple myeloma.

[0471] Example 5: Anti-BCMA ADC induces tumor regression and inhibition in an orthotopic multiple myeloma model

[0472] This example also demonstrates the in vivo efficacy of anti-BCMA ADC in the MM1.S orthotopic multiple myeloma model.

[0473] The in vivo efficacy of BCMA ADC was studied using the multiple myeloma cell line MM1.S expressing luciferase and GFP in an orthotopic model. 7Individual MM1.S LucGFP cells were injected intravenously into 6-8 week old female CB17 / SCID animals via the tail vein. D-luciferin (Regis Technologies, Morton Grove, IL) (15 mg / mL, 200 uL / animal) was injected intraperitoneally and then anesthetized with isoflurane followed by whole-body bioluminescence imaging (BLI) to monitor tumor burden. Bioluminescent signals emitted by the interaction between luciferase and luciferin expressed by tumor cells were captured by imaging with IVIS Spectr μM 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, 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) the control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugate was administered by bolus tail vein injection. Animals were terminated when they exhibited hind limb paralysis, the endpoint of the MM1.S orthotopic model. Figure 3 A single dose of human anti-BCMAL3.PY / P6E01 antibodies conjugated to 1) H7c / N297A / K222R-amino-PEG6-C2-0131 and 2) H7c / N297A / K222R-amino-PEG6-C2-3377 resulted in tumor regression. A single dose of human anti-BCMAL3.PY / P6E01 antibodies conjugated to 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.

[0474] Thus, this study demonstrates that treatment with BCMA-ADC induces regression and inhibits the progression of multiple myeloma.

[0475] Example 6: Anti-BCMA ADC induces tumor suppression in an orthotopic multiple myeloma model

[0476] This example also demonstrates the in vivo efficacy of anti-BCMA ADC in the KMS12BM orthotopic multiple myeloma model.

[0477] In vivo efficacy studies of BCMA ADCs were performed using the multiple myeloma cell line KMS12BM expressing luciferase and GFP in an orthotopic model. Female NSG animals aged 6-8 weeks were irradiated with 100 cGy and injected intravenously via the tail vein with 1 × 10 7 KMS12BM LucGFP cells were injected intraperitoneally with D-luciferin (Regis Technologies, Morton Grove, IL) (15 mg / mL, 200 uL / animal) and then anesthetized with isoflurane and whole-body bioluminescence imaging (BLI) was used to monitor tumor burden. The bioluminescent signal emitted by the interaction between luciferase and luciferin expressed by tumor cells was captured by imaging with IVIS Spectr μM CT (Perkin Elmer, MA) and was quantified as total flux (photons / sec) using LivingImage 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 5E6 for all animals, the animals were randomized into the following groups: 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur010, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugate was administered by bolus tail vein injection. Animals were terminated when they lost more than 15% of total body weight, which is the endpoint of the KMS12BM orthotopic model. Figure 4It was shown that a single dose of human anti-BCMA L3.PY / P6E01 antibody conjugated to 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 resulted in tumor inhibition.

[0478] Thus, this study further demonstrates that treatment with BCMA-ADC induces regression and inhibits the progression of multiple myeloma.

[0479] Example 7: Dose-response curve of anti-BCMA ADC in the MM1S orthotopic model

[0480] This example further demonstrates the in vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model.

[0481] The in vivo efficacy of BCMA ADC was studied using the multiple myeloma cell line MM1.S expressing luciferase and GFP in an orthotopic model. 7Individual MM1.S LucGFP cells were injected intravenously into 6-8 week old female CB17 / SCID animals via the tail vein. D-luciferin (Regis Technologies, Morton Grove, IL) (15 mg / mL, 200 uL / animal) was injected intraperitoneally and then anesthetized with isoflurane followed by whole-body bioluminescence imaging (BLI) to monitor tumor burden. Bioluminescent signals emitted by the interaction between luciferase and luciferin expressed by tumor cells were captured by imaging with IVIS Spectr μM 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 the following groups: 1) 0.1 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 2) 0.38 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 3) 0.75 mg / kg H7c / N297A / K222-amino-PEG6-C2-0131, 4) 1.5 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) 3 mg / kg control conjugate N297Q / K222R-AcLys-VC-0101. A single dose of human anti-BCMA ADC and control conjugate was administered by bolus tail vein injection. Animals were terminated when they showed hind limb paralysis, which is the endpoint of the MM1.S orthotopic model. Figure 5 It was shown that a single dose of human anti-BCMA COMBO_Rd4_0.6nM_C29 antibody conjugated to groups 1)-4) above resulted in tumor regression starting at 0.1 mg / kg and tumor inhibition for up to 100 days starting at 0.75 mg / kg.

[0482] Thus, this study demonstrates that treatment with BCMA-ADC induces tumor regression and tumor suppression in multiple myeloma.

[0483] Example 8: Production and purification of heterodimeric antibodies

[0484] This example describes the production and purification of heterodimeric antibodies of the invention.

[0485] The variable regions of the human-specific anti-CD3 antibodies were cloned into human IgG1 or IgG2ΔA comprising the following mutations 221R, 228R and K409R; or 223R, 225R, 228R and K409R, respectively, and referred to as hIgG1 RRR or IgG2ΔA-RRRR.

[0486] The variable regions of the anti-target antibodies were cloned into human IgG1 or IgG2ΔA containing the following mutations 221E, 228E, L368E or 223E, 225E, 228E and L368E, respectively, and designated hIgG1EEE or hIgG2ΔA-EEEE.

[0487] Heterodimers were prepared by incubating anti-CD3 IgG1 or IgG2ΔA with hIgG1RRR or IgG2ΔA-RRRR mutations with anti-target antibodies with hIgG1EEE or hIgG2ΔA-EEEE mutations in PBS with 1 mM or 2 mM GSH for 24 hours at 37° C. as described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545). Heterodimers were purified by ion exchange chromatography as described below.

[0488] All heterodimers were purified by ion exchange chromatography. Briefly, analytical ion exchange separations of Fc-heterodimers and Fc-homodimers were 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 (4x50mm) column. The protein was injected in 5% buffer A (20mM MES pH 5.4) and eluted in a 25%-75% buffer B (20mM MES pH 5.4 and 500mM NaCl) gradient at a flow rate of 1 ml / min over 20 minutes. Large-scale Fc-heterodimer purification was performed on an Akta Explorer (GE) equipped with a weak cation exchange DIONEX Propac WCX-10G (4x250mm) column. Proteins were injected in 5% buffer A (20 mM MES pH 5.4) and eluted in a 15%-75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) gradient at a flow rate of 1 ml / min over a period of 20 minutes.

[0489] Example 9: Determination of the kinetics and affinity of hCD3 / human IgG interactions at 25°C and / or 37°C

[0490] This example determines the kinetics and affinity of various anti-CD3 antibodies at 25°C and 37°C.

[0491] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). Anti-CD3 antibody arrays were prepared on a Bio-Rad GLC sensor chip using an amine-coupling method similar to that described in Abdiche et al., Anal. Biochem. 411, 139-151 (2011). The assay temperature was fixed at 25°C, and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) orientation 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 in the ligand (vertical) orientation by injecting 20 μg / mL in 10 mM acetate pH 4.5 buffer for 1.5 minutes (30 μg / mL). The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 minutes in the direction of the analyte.

[0492] The hCD3 binding assay was performed at either 37°C or 25°C in HBS-T+ running buffer supplemented with 1 mg / mL BSA. A kinetic titration method 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 injections from low to high concentrations. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM (a 5-way series with 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 following the 50 nM hCD3 injection. Prior to hCD3 analyte injection, buffer was injected five times during the hCD3 analyte cycle using the same association and dissociation times to prepare a buffer blank sensorgram for double referencing purposes (as described in Myszka, J. Mol. Recognit. 12, 279-284 (1999)).

[0493] Sensorgrams were double referenced and fitted to a 1:1 Langmuir and mass transport kinetic titration model in BIA Evaluation software version 4.1.1 (GE Lifesciences, Piscataway, NJ).The kinetic and affinity parameters of various anti-CD3 antibodies of the invention are shown in Table 9.

[0494] Table 9

[0495]

[0496] Example 10: Flow cytometry of human anti-CD3 bispecific antibodies on B cells and CD8+ T cells

[0497] This example demonstrates the efficacy of an anti-CD3-anti-CD20 bispecific antibody in CD20+ cells.

[0498] 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 bispecific anti-CD20 / h2B4 antibodies at doses of 500 μg / kg, 100 μg / kg, 20 μg / kg, 2 μg / kg, 0.2 μg / kg, or 0.02 μg / kg 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 from a peripheral blood vessel not used for IV dosing into K2EDTA tubes.

[0499] The B cells and T cells in peripheral blood were measured by flow cytometry to determine efficacy. Whole blood was collected at the indicated time points and maintained at 4°C until analysis. Red blood cells were lysed with ACK buffer (Gibco) for 5 minutes at room temperature, and leukocytes were precipitated by centrifugation. Cells were stained for 1 hour at 4°C with a mixture of antibodies containing fluorescently labeled recognition cyno CD19 (Beckman Coulter), CD45, CD4, CD8, Ki67 (BD Biosciences) in PBS+2% FBS. For Ki67 analysis, cells were first stained with CD4 and CD8, then fixed / permeabilized with BD cyotfix / cytoperm kit (BD Biosciences) according to the manufacturer's instructions, and subsequently to Ki67 intracellular staining. Immediately after staining, cells were obtained on a BD LSRII flow cytometer.

[0500] exist Figures 6A-6F The resulting B cell counts were plotted as a percentage of the pre-study B cell count. Prolonged B cell depletion after a single dose was achieved with doses as low as 2 μg / kg. B cell depletion was seen at all doses. The duration of the depletion effect was dose-dependent.

[0501] exist Figures 7A-7F The resulting CD8+ T cell counts were plotted as a percentage of the pre-study CD8+ T cell counts in After the initial repositioning, T cell levels returned to or above baseline levels during the study.

[0502] Example 11: Flow cytometry of human anti-CD3 bispecific antibodies on CD8+ T cells

[0503] This example demonstrates the efficacy of monovalent anti-CD3 antibodies on T cell kinetics and activation.

[0504] Cynomolgus monkey studies were conducted as described in Example 3 and efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Cynomolgus monkeys (n=2) were administered anti-CD20 / h2B4 or NNC (non-specific antibody) / h2B4 at a weekly iv dose of 0.2 ug / kg. In contrast to the CD20-targeting bispecific antibody, NNC / h2B4 had little to no effect on CD8+ T cell dynamics in the blood as measured by flow cytometry. Ki67 was used as a marker of T cell activation.

[0505] exist Figure 8A and 8B The resulting T cell counts were plotted as a percentage of pre-study T cell counts. In cynomolgus monkeys dosed with the CD20 / h2B4 bispecific antibody, Ki67+ T cells increased and peaked between days 3 and 7 post-dose, indicating T cell activation. However, in cynomolgus monkeys dosed with the NNC / h2B4 antibody, Ki67+ T cells did not increase.

[0506] Example 12: Flow cytometry of human anti-CD3 bispecific antibodies on B cells

[0507] This example demonstrates the effect of anti-CD3 arm affinity on B cell depletion.

[0508] Cynomolgus monkey studies were conducted as described in Example 10 and efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Bispecific antibodies were prepared using an anti-CD20 arm paired with four anti-CD3 antibody arms of varying affinity. Efficacy was determined by measuring B cells in peripheral blood by flow cytometry after a single iv dose of 0.2 μg / kg.

[0509] exist Figures 9A-9D The resulting B cell counts were plotted as a percentage of the pre-study B cell count. The efficacy of B cell depletion correlated with the affinity of the anti-CD3 arm.

[0510] Example 13: In vitro study of bispecific antibodies on T-cell-mediated killing of BCMA-positive cells

[0511] This example demonstrates the in vitro cytotoxicity of the anti-BCMA / CD3 hIgG2ΔA bispecific in BCMA-positive cells.

[0512] 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) antibodies were expressed as human IgG2dAs engineered with EEEE for bispecific exchange as described in Example 8.

[0513] CD3+ T cells from PBMC were negatively selected using the Pan T cell isolation kit (Miltenyi, San Diego CA). Target expression (KMS12PE, L363, and Molp8) cells and CD3+ T- cells were seeded at 20,000 and 100,000 cells / well, respectively, on clear U-bottom plates. Cells were treated with 10-fold serial dilutions of the bispecific antibody in triplicate. CytoTox was used 20 hours after treatment. Cell death was determined using a non-radioactive cytotoxicity assay (Promega, Madison, WI). Cytotoxicity was determined as the percentage of untreated effector plus target control wells. EC50 was calculated using Prism software. Table 10 shows that all human anti-BCMA-H2B4 bispecific antibodies exerted cell-killing activity in BCMA-expressing cells.

[0514] Table 10

[0515]

[0516] UND means undetermined; N / A means EC50 could not be determined.

[0517] Example 14: In vitro characterization of anti-CD3 antibodies cloned from mouse hybridomas

[0518] This example demonstrates in vitro T cell activation / expansion of anti-CD3 cloned from mouse hybridoma in human / cynomolgus monkey PBMC cells for antibody screening.

[0519] Human anti-CD3 antibodies were cloned from immunized mice, expressed as mouse IgG1, and purified by protein A affinity beads. Human / cynomolgus monkey peripheral blood mononuclear cells (hu / cyPBMC) were prepared from blood filters obtained from local blood banks by Ficoll (density: 1.083 g / mL, GE) density gradient centrifugation. Red blood cells were removed by incubation for 3 minutes in LCK buffer (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA; Gibco) at room temperature. The cells were centrifuged at 600 g for 5 min. The supernatant containing the lysed red blood cells was discarded, and PBMC was washed twice in 50 ml 1xPBS / 1% BSA / 1 mM EDTA. The precipitated cells were adjusted to 10 7 cells / ml, and PBMC were cultured at 10 6(100ul) / well was seeded into a round-bottom 96-well tissue culture plate. The selected Ab was serially diluted from 1000ng to 1ng / mL 10x for mixing with human PBMCs, and serially diluted from 5000ng to 200ng / mL 5x for mixing with cynomolgus monkey PBMCs. 3 PBMC T cell proliferation was analyzed by H-thymidine incorporation in triplicate for 2 days. 3 H-thymidine (0.5 mCi / well) was added and incorporation was measured. Cells were harvested and lysed, and DNA was captured on glass-fiber filters. Radioactivity (cpm) was used as a measure of proliferation by counting in a scintillation β-counter.

[0520] Figure 10A and 10B Selected anti-CD3 1A4, 1C10, 2B4, and 7A3 antibodies are shown to have thymidine incorporation readouts on human and cynomolgus monkey PBMCs (peripheral blood mononuclear cells). Table 11 shows their KD measured by Biacore. In vitro characterization showed that the anti-CD3 1C10 and 2B4 antibodies were similar to the positive control SP34 anti-CD3 antibody (BD Biosciences).

[0521] Table 11 Anti-xCD3e ab / bsc_hCD3ed kinetic results from data fitted from 80 nM to 0.64 nM

[0522] ligand ka kd <![CDATA[t 1 / 2 (min)]]> KD (nM) UCHT1(+) 1.80E+05 <8.55E-04 >13.5 <4.74 2B4 3.74E+05 2.74E-03 4.21 7.33 1C10 2.96E+05 2.37E-03 4.88 8.00 SP34(+) 2.84E+05 3.04E-03 3.80 10.73 7A3* 82.70 1A4* 99.97

[0523] Notice:

[0524] Data are reported only for satisfactory kinetic fits. (+) = positive control

[0525] *Kinetic determinations are rough estimates because the antibody is heterologous. Only steady-state affinity was measured.

[0526] Example 15: In vitro study of T cell-mediated killing by bispecific antibodies

[0527] This example demonstrates the in vitro cytotoxicity of an anti-EpCam / CD3 bispecific in SW480 cells mixed with Pan T cells isolated from healthy donors.

[0528] A: Anti-CD antibodies h2B4-1d, TK, hnpsTK, and yaesTK

[0529] Human anti-CD3 (h2B4-1d (or h2B4), h2B4-TK (or h2B4-VH-wt VL_TK), h2B4-hnpsTK (or h2B4-VH-hnps VL_TK), and h2B4-yaesTK (or h2B4-VH-yaes VL_TK)) antibodies and human anti-EpCam antibodies were expressed as human IgG2dA engineered with RRRR or EEEE for bispecific exchange as described in Example 8.

[0530] SW480 was selected as the target cell line for cell killing assays, and effector cells and human T cells were purified from human peripheral blood mononuclear cells (huPBMC). Target and effector cells were seeded in 96-well round-bottom plates in cell culture medium containing 5% fetal bovine serum (FBS). The number of target cells was kept constant at 2×10 4 10-fold serial dilutions of the bispecific antibody at 3 μg to 3 pg / mL were added to the cells in triplicate. The total reaction volume was 200 μL. The reactions were incubated for 48 and 72 hours. For cytotoxicity analysis, the cells were analyzed by CytoTox. A non-radioactive cytotoxicity assay kit (Promega, G1780) quantitatively measures the stable cytosolic enzyme lactate dehydrogenase (LDH) released upon cell lysis. Plates were read at 490 nM on a Vmax kinetic microplate reader (Molecular Devices). Optical density values ​​were corrected for culture medium background and spontaneous lysis of target and effector cells. Specific cytotoxicity was calculated according to the following formula:

[0531] [% specific lysis = 490 nM reading of sample - 490 nM reading of E+T mixed control) / (490 nM reading of total T lysis - 490 nM reading of medium control) x 100%]

[0532] Figure 11A and 11B All human anti-EpCam-h2B4 bispecific antibodies were shown to have cell killing activity in vitro, and antibody-mediated T cell activation was monitored by T cell activation markers. Table 12A shows their EC50s. Table 12B shows the Biacore KD of the bispecific antibody formats.

[0533] Table 12A

[0534] Day 2: EC50 (nM) h2B4-1d_Ep 537.1 h2B4-TK_Ep 405.3 h2B4-hnpsTK_Ep 424.7 h2B4-yaesTK_Ep 1126

[0535] Table 12B Summary of anti-CD3 hIgG2dA bispecific kinetics at 37°C

[0536] Sample ID-bschIgG2dA ka(1 / Ms) kd(1 / s) t1 / 2(min) KD (nM) h2B4-1d_Ep 5.86E+05 2.37E-02 0.49 40.4 h2B4-TK_Ep 6.87E+05 2.13E-02 0.54 31.0 h2B4-hnpsTK_Ep 7.54E+05 2.35E-02 0.49 31.2 h2B4-yaesTK_Ep 4.74E+05 2.58E-02 0.45 54.4

[0537] B: anti-CD antibodies m25A8, h25A8-B12, and h25A8-B13

[0538] Human anti-CD3 h2B4 (h2B4_ld) and h25A8 (m25A8, h25A8-B12, and h25A8-B13) and human anti-EpCam antibodies were expressed as human IgG2dAs engineered with RRRR or EEEE for bispecific exchange as described in Example 8.

[0539] Figure 11C and 11D All human anti-EpCam-anti-CD3 bispecific antibodies were shown to have cell killing activity in vitro, and antibody-mediated T cell activation was monitored by T cell activation markers. Table 12C shows the EC50 for in vitro cell killing. Table 12D shows the Biacore kinetics of the bispecific antibody formats at 37°C. Table 12E shows in vitro characterization using SEC-MALS (size exclusion chromatography with multi-angle light scattering) and DSC (differential scanning calorimetry).

[0540] Table 12C: EC50 of in vitro cell killing

[0541] Day 1: EC50 (nM) h2B4-1d_Ep 52.9 m25A8_Ep 127.6 h25A8-B12_Ep 99.36 h25A8-B13_Ep 57.11

[0542] Table 12D: Summary of anti-CD3 hIgG2dA bispecific kinetics at 37°C

[0543]

[0544] Table 12E: In vitro characterization

[0545]

[0546] Example 16: In vitro T-cell mediated killing of primary myeloma patient samples by bispecific antibodies Research

[0547] This example demonstrates the in vitro cytotoxicity of an anti-BCMA / CD3 bispecific in primary myeloma cells.

[0548] Human anti-BCMA (P5A2, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C16, Combo_Rd4_0.6nM_C29, and P6E01 L3PY / H3TAQ) and human anti-CD3 (h2B4) antibodies were expressed as human IgG2dAs engineered with EEEE or RRRR for bispecific exchange as described in Example 8.

[0549] The total bone marrow mononuclear cells from myeloma patients are seeded in a transparent U-bottom plate with the total bone marrow mononuclear cell count resulting in 3000-5000 myeloma cells / well. The cells are treated with the bispecific antibody of 10-times serial dilution. 5 days after treatment, total viable cells are determined by flow cytometry using antibodies to CD138 and CD38 (Biolegend, CA). Cells are incubated for 30 minutes at 4 ° in PBS+0.5% FBS with antibodies. The cells are washed, and FixableViability Dye eFluor 780 (eBioscience, Inc., CA) in PBS is added to the cells at 4 ° for 30 minutes. Before cell collection on a BD flow cytometer, the cells are washed and CountBright absolute counting beads (Molecular Probes, OR) are added. The percentage of viable cells is determined as the viable cell count in the treated vs untreated wells using counting beads. EC50 is calculated by Prism software.

[0550] Table 13A shows that all human anti-BCMA-h2B4 bispecific antibodies have cell killing activity against myeloma patient samples, and that patient T cells are functional effector cells. Table 13B shows that one anti-BCMA bispecific kills multiple myeloma patient samples with different effector to target (E:T) ratios.

[0551] Table 13A

[0552]

[0553] Table 13B

[0554]

[0555] Exa...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to B-cell maturation antigen (BCMA), wherein the antibody comprises: (a) a heavy chain variable (VH) region, wherein the VH region comprises, according to the Kabat numbering system, (i) a VH CDR1 consisting of SEQ ID NO: 156; (ii) a VH CDR2 consisting of SEQ ID NO: 158; and (iii) a VH CDR3 consisting of SEQ ID NO: 155, and a light chain variable (VL) region, wherein the VL region comprises, according to the Kabat numbering system, (i) a VL CDR1 consisting of SEQ ID NO: 209; (ii) a VL CDR2 consisting of SEQ ID NO: 221; and (iii) a VL CDR3 consisting of SEQ ID NO: 225; or (b) a heavy chain variable (VH) region, wherein the VH region comprises, according to the Chothia numbering system, (i) a VH CDR1 consisting of SEQ ID NO: 151; (ii) a VH CDR2 consisting of SEQ ID NO: 159; and (iii) a VH CDR3 consisting of SEQ ID NO: 155, and A light chain variable (VL) region, wherein the VL region comprises, according to the Chothia numbering system, (i) a VL CDR1 consisting of SEQ ID NO: 209; (ii) a VL CDR2 consisting of SEQ ID NO: 221; and (iii) a VL CDR3 consisting of SEQ ID NO:

225.

2. The antibody or antigen-binding fragment of claim 1 , wherein 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 outside the CDRs and / or the VL region comprises the amino acid sequence shown in SEQ ID NO: 38 or a variant having one or several conservative amino acid substitutions in amino acids outside the CDRs.

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VH region produced by an expression vector having ATCC Accession No. PTA-122094.

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VL region produced by an expression vector having ATCC Accession No. PTA-122093.

5. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody of any one of claims 1 to 4.

7. A vector comprising the polynucleotide of claim 6.

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

9. A method of producing an antibody, the method comprising culturing the host cell of claim 8 under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.

10. Use of the antibody or antigen-binding fragment of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating multiple myeloma in an individual.

11. Use of the antibody or antigen-binding fragment of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for inhibiting tumor growth or progression in a subject with multiple myeloma.

12. Use of the antibody or antigen-binding fragment of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for inducing tumor regression in an individual with multiple myeloma.

13. An in vitro binding assay method, wherein the method comprises testing the ability of a candidate antibody to compete with the antibody of any one of claims 1-4 for binding to BCMA.

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