Therapeutic antibodies and their uses

Abstract

The present invention relates to antibodies, e.g., full-length antibodies or antigen-binding fragments thereof, that specifically bind to BCMA (B-Cell Maturation Antigen) and CD3 (Cluster of Differentiation 3). The invention also relates to antibody conjugates (e.g., antigen-drug-conjugates) comprising the BCMA antibodies, compositions comprising the BCMA antibodies, and methods of using the BCMA antibodies and their conjugates to treat a disease condition associated with a cell expressing BCMA (e.g., cancer or an autoimmune disease). The 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). Also provided are compositions comprising such heteromultimeric antibodies, methods of making and purifying such heterodimeric antibodies, and their use in diagnosis and therapy.

Description

[0001] This application is a divisional application of a Chinese patent application filed on March 30, 2016, with application number 202210629743.X, and invention name “Therapeutic antibodies and their uses”. Field of the Invention

[0002] The present invention relates to antibodies, such as full-length antibodies or antigen-binding fragments thereof, which specifically bind to BCMA (B-cell maturation antigen) and / or CD3 (differentiation cluster 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. Background of the Invention

[0004] 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 and contains the t(4;16) translocation. The gene is selectively expressed in the B-cell lineage, with the highest expression in antibody-secreting cells plasmablasts and plasma cells. BCMA binds to 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. APRIL or BAFF binding to BCMA promotes a signaling cascade including NF-κB, Elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, which generates signals for cell survival and proliferation.

[0005] 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.

[0006] In the case of multiple myeloma, approximately 24,000 new cases are diagnosed each year in the United States, and this number is equivalent to approximately 15% of 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 plasma cell apoptosis and / or reducing osteoclast activity (e.g., chemotherapy, thalidomide, lenalidomide, bisphosphonates, and / or proteasome inhibitors such as bortezomib or carfilzomib). However, multiple myeloma remains an incurable disease, and almost 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), would be better therapeutic agents. SUMMARY OF THE INVENTION

[0008] The invention disclosed herein relates to therapeutic antibodies that bind 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 (e.g., bispecific antibodies that specifically bind to CD3 and BCMA) are also provided.

[0009] 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 XXXXXXXXXXXX, 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 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 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 is 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), 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).

[0010] 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 CDR3 of the VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 125, 127, 313, 314, 363 or 365. 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 of the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown inIn 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: NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235, 297 or 299; (ii) a VL CDR2 comprising SEQ ID NO: 221; and (iii) a VL CDR3 comprising SEQ ID NO: 225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298 or 300. In some embodiments, the VH region comprises the sequence shown in SEQ ID NO: 112 or a variant having one or several conservative amino acid substitutions in residues not within the CDRs and / or the VL region comprises the amino acid sequence shown in SEQ ID NO: 38 or a variant thereof having one or several amino acid substitutions in amino acids not within the CDRs. In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain comprising the sequence shown in SEQ ID NO: 357, and the 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.

[0011] In another aspect, the present invention provides an isolated antibody comprising a tag containing an acyl donor glutamine engineered at a specific site of the BCMA antibody of the present invention. In some embodiments, the tag comprises a member 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).

[0012] 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.

[0013] 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.

[0014] 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 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-oxoheptane-4-yl]-N-methyl-L-valinamide).

[0015] In some embodiments, the present invention provides a conjugate comprising the formula: antibody-(tag containing acyl donor glutamine)-(linker)-(cytotoxic agent). In some embodiments, the tag containing 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.

[0016] In another aspect, a method of preparing a BCMA antibody as described herein is provided, 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 the culture.

[0017] 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 use of a BCMA antibody 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 a BCMA antibody 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 a BCMA antibody or BCMA antibody conjugate as described herein in the manufacture of a medicament for inducing tumor regression is provided.

[0018] 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 a 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 a 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; 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 a 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 a 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.

[0019] 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.

[0020] 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 a 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 a 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 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 of a VL CDR1, VL CDR2 and VL CDR3. In some embodiments, the first antibody variable domain comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising a VH sequence set forth 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 a sequence set forth in SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449, or 450; and iii) a VH CDR3 comprising a sequence set forth 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 38.

[0021] 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 XXXXXXXXXXXX, 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 is 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), 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: NO:211, 225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298, 300 or 360.

[0022] 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 set forth in SEQ ID NO: 331, 332, 333, 401, 407 or 408; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 336, 417, 404 or 405; and iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 335 or 406; and / or 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: 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.

[0023] 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 positions 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.

[0024] In another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies described herein (eg, BCMA, CD3, or bispecific) or a conjugate thereof (eg, a BCMA antibody-drug conjugate).

[0025] In another aspect, the 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).

[0026] In another aspect, the invention also 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 invention also provides nucleic acids encoding the heavy chain variable region and / or light chain variable region of any of these antibodies described herein.

[0027] 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).

[0028] The present invention also provides methods of treating a disease condition (e.g., inhibition of tumor growth / development; inhibition of metastasis of malignant cells expressing BCMA; tumor regression in an individual with malignant cells expressing BCMA) in an individual in need thereof, the method comprising providing an isolated antibody (e.g., BCMA) or binding fragment, a bispecific antibody (BCMA-CD3 bispecific) or a conjugate thereof (e.g., BCMA antibody-drug conjugate) described herein and administering the antibody or conjugate to the individual.

[0029] Also provided are methods for treating a disease condition associated with malignant cells expressing a tumor antigen in an individual, the method 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 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 B-cell prolymphocytic leukemia (B-cell NHL). ALL, chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's 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's lymphoma, primary mediastinal (thymic) large B lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, lymph node 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-related 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 intermediate features between diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma with intermediate features 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.

[0030] 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 with increased binding affinity to one or more human Fcγ receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A-Figure 1DDual reference sensorgrams with fitting curves for the interaction between selected anti-BCMA antibodies of the invention and human BCMA are shown.

[0032] Figure 2 In vivo efficacy studies of various anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model are shown, 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.

[0033] Figure 3 In vivo efficacy of anti-BCMA ADCs in the MM1S orthotopic multiple myeloma model is shown, 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.

[0034] Figure 4Also shown is the 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-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.

[0035] Figure 5 The in vivo efficacy of anti-BCMA ADC in the MM1S orthotopic multiple myeloma model is also shown. The anti-BCMA antibody COMBO_Rd4_0.6nM-C29 ("Combo C29 DI) was conjugated to H7c / N297A / K222R-amino-PEG6-C2-131 at a dose range of 0.1 mg / kg, 0.38 mg / kg, 0.75 mg / kg, 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 SEQ ID NO: 454 containing glutamine.

[0036] Figure 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 bispecific antibodies is shown as a percentage of pre-study counts.

[0037] Figure 7A-7F In vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys is shown. CD8+ T cell kinetics were tracked after a single dose of the bispecific antibodies.

[0038] Fig. 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.

[0039] Figure 9A-9DIn vivo efficacy of anti-CD3 / anti-CD20 bispecific antibodies in cynomolgus monkeys is shown. The effect of the affinity of the anti-CD3 arm on B cell depletion was analyzed.

[0040] Fig. 10A and 10B Selected anti-CD3 antibodies are shown with thymidine incorporation readout on human and cynomolgus monkey PBMCs.

[0041] Figures 11A-11D All human anti-EpCam_h2B4 bispecific antibodies were shown to have cell killing activity in an in vitro environment.

[0042] Fig.12 We showed that a single dose of a human anti-BCMA / CD3 bispecific antibody resulted in tumor regression in an orthotopic MM1.S myeloma model in a dose-dependent manner.

[0043] Fig.13 Two doses of a human anti-BCMA / CD3 bispecific antibody were shown to result in increased tumor regression in an orthotopic Molp8 myeloma model.

[0044] Fig.14 It was shown 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.

[0045] Figure 15A-Figure 15C It was shown that carfilzomib, lenalidomide and doxorubicin had no negative impact on the function of OPM2 cells compared with the anti-BCMA / CD3 bispecific antibody alone.

[0046] Fig.16 A synergistic effect was shown on the function of the anti-BCMA / CD3 bispecific antibody when combined with carfilzomib and lenalidomide compared to each molecule alone. DETAILED DESCRIPTION OF THE INVENTION

[0048] 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.

[0049] General techniques

[0050] 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 JDCapra, eds., Harwood Academic Publishers, 1995).

[0051] definition

[0052] "Antibodies" are immunoglobulin molecules that are able to specifically bind to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, 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 have to be of any particular class. According to the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulins can be divided into different classes. There are five major 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 corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0053] 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; Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments (Ward et al., Nature 341: 544-546, 1989), and isolated complementary determining regions (CDRs).

[0054] An antibody, antibody conjugate, or polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., a BCMA protein or a CD3 protein) is a term well understood in the art, and methods of 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 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 a CD3 epitope is an antibody that binds to this 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 may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0055] 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 complementary determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are tightly held together by the FRs, as well as 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 may refer to CDRs defined by either method or by a combination of both methods.

[0056] The "CDRs" of a variable domain are amino acid residues within a variable region identified according to the Kabat, Chothia definitions, 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, e.g., 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, e.g., 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 the "contact definition" of CDRs observed for antigen contacts, is shown in MacCallμM 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. There are other CDR boundary definitions that 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 extended according to predictions or experimental findings that a particular residue or group of residues or even the entire CDR does 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.

[0057] 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 possible naturally occurring mutations that may be present in small amounts. 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 directed only against a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be prepared by any particular method. For example, the monoclonal antibodies 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 phage libraries produced using, for example, the technology described in McCafferty et al., Nature 348:552-554, 1990.

[0058] As used herein, "humanized" antibodies refer to forms of non-human (e.g., mouse) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (receptor antibodies), wherein residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from the CDRs of non-human species (donor antibodies) such as mice, rats, or rabbits with desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include 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 immunoglobulins. Antibodies preferably have modified Fc regions 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 are changed to the original antibody, which are also referred to as "derived from" one or more CDRs from one or more CDRs of the original antibody.

[0059] As used herein, "human antibody" means 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 comprising 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, human antibodies are 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 to replace endogenous loci, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This method is described in US Pat. No. 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 target antigens (such B lymphocytes can be recovered from individuals or single cell clones of cDNA, or can be immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1): 86-95, 1991; and US Pat. No. 5,750,373.

[0060] 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 mouse antibodies and the constant region sequences are derived from human antibodies.

[0061] 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, it 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 may exist as a single chain or associated chains.

[0062] A "monovalent antibody" contains one antigen binding site per molecule (eg, IgG or Fab). In certain cases, a monovalent antibody may have more than one antigen binding site, but the binding sites are from different antigens.

[0063] "Monospecific antibodies" contain two identical antigen binding sites per molecule (e.g., IgG), so that the two binding sites bind to the same epitope on the antigen. Therefore, they compete with each other for binding to one antigen molecule. Most antibodies found in nature are monospecific. In some cases, monospecific antibodies can also be monovalent antibodies (e.g., Fab).

[0064] 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.

[0065] "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.

[0066] "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.

[0067] 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.

[0068] 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.

[0069] As used herein, "hinge region", "hinge sequence" and variations thereof include those known in the art, which are 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.

[0070] As used herein, "immunoglobulin-like hinge region", "immunoglobulin-like hinge sequence" and variations thereof refer to hinge regions and hinge sequences of immunoglobulin-like or antibody-like molecules (e.g., immunoadhesins). In some embodiments, the immunoglobulin-like hinge region can be from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD, or IgM, including chimeric forms thereof, such as chimeric IgG1 / 2 hinge regions.

[0071] 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.

[0072] 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, e.g., Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA, et al, J. MoI. Biol., 373 (4): 924-40, 2007).

[0073] As 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 polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogs. If there are modifications, the modification of the nucleotide structure can be performed before or after chain assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotides 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., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and having charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), containing pendant parts, 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, oxidative metals, etc.), containing alkylating agents, having modified bonds (e.g., alpha 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 a standard protecting group to prepare additional bonds for additional nucleotides, or can be conjugated to a solid support. The 5' and 3' OH can be phosphorylated or partially substituted with an amine or an organic capping group of 1-20 carbon atoms. Other hydroxyl groups may also be derivatized to standard protecting groups. Polynucleotides may also include similar forms of ribose or deoxyribose 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 ribonucleosides. One or more phosphodiester bonds may be replaced with an optional linking group. These optional linking groups include, but are not limited to, embodiments in which the phosphate is replaced with 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 ether (-O-) bonds, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. All bonds in a polynucleotide need not be identical.The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0074] 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.

[0075] 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.

[0076] "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 to the original parent cell (morphologically or in genomic DNA complement) due to natural, accidental or artificial mutations. Host cells include cells transfected in vivo with a polynucleotide of the present invention.

[0077] 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" may 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 generally 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.

[0078] 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 alternative splicing 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; Capelet 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).

[0079] As used herein with respect to antibodies, the term "competition" means that the first antibody or its antigen-binding fragment (or portion) binds to an epitope in a manner very similar to the binding of the second antibody or its antigen-binding portion, so that the result of the 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 in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody is possible, but not necessarily so. 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 another antibody to its cognate epitope or ligand, whether to the same, greater or lesser extent, it is considered that the antibodies "cross-compete" with each other to bind 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 changes, or binding to a common epitope or portion thereof), based on the teachings provided herein, the skilled person will understand that such competing and / or cross-competing antibodies are encompassed and can be used in the methods disclosed herein.

[0080] A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require 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.

[0081] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region in that at least one amino acid is modified, but at least one effector function of a native sequence Fc region is retained. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, such as about 1 to about 10 amino acid substitutions in the native sequence Fc region or the Fc region of a 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 a 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.

[0082] 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, e.g., US Pat No. 6,737,056. Such effector functions generally require the Fc region to be combined 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.

[0083] 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 lysis of the target cells. The ADCC activity of the molecule of interest can be evaluated using an in vitro ADCC assay, as described in U.S. Pat. Nos. 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 disclosed in Clynes et al., 1998, PNAS (USA), 95: 652-656.

[0084] "Complement dependent cytotoxicity" or "CDC" refers to target lysis 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 by Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), can be performed.

[0085] 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 BCMA-related diseases (e.g., cancer or autoimmune diseases), reducing the symptoms caused by BCMA-related diseases (e.g., cancer or autoimmune diseases), increasing the quality of life of patients with BCMA-related diseases (e.g., cancer or autoimmune diseases), reducing the dose of other drugs required to treat BCMA-related diseases (e.g., cancer or autoimmune diseases), delaying the development of BCMA-related diseases (e.g., cancer or autoimmune diseases), curing BCMA-related diseases (e.g., cancer or autoimmune diseases), and / or prolonging the survival of patients with BCMA-related diseases (e.g., cancer or autoimmune diseases).

[0086] "Improvement" means a reduction or improvement of one or more symptoms compared to not administering a BCMA antibody or BCMA antibody conjugate. "Improvement" also includes shortening or reducing the duration of symptoms.

[0087] 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 development 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 development of a patient's BCMA-related disease. The 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 a prophylactic or therapeutic treatment directly or indirectly. As understood in the clinical context, an effective dose of a drug, compound, or pharmaceutical composition can be achieved with or without 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 administering a single drug in an effective amount can achieve the desired result.

[0088] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, competition animals, pets, primates, horses, dogs, cats, mice, and rats.

[0089] As used herein, "vector" means 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.

[0090] 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.

[0091] 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, e.g., 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).

[0092] As used herein, the term "a 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.

[0093] 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.

[0094] 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.

[0095] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0096] Reference herein to "about" a value or parameter includes (and describes) embodiments involving that value or parameter per se. For example, description referring to "about X" includes description of "X." Numerical ranges include the numbers defining the range.

[0097] It should be understood that wherever herein an embodiment is described with the language "comprising," other similar embodiments described according to "consisting of" and / or "consisting essentially of" are also provided.

[0098] 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 present. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In the event 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.

[0100] 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.

[0101] BCMA antibody and preparation method thereof

[0102] 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 features: (a) treating, preventing, ameliorating 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) inhibiting tumor growth or development in an individual (who has a malignant tumor expressing BCMA); (c) inhibiting metastasis of cancer (malignant) cells expressing BCMA in an individual (who has one or more malignant cells expressing BCMA); (f) inducing regression (e.g., long-term regression) of a tumor expressing BCMA; (d) exerting cytotoxic activity in malignant cells expressing BCMA; and (e) blocking the interaction of BCMA with other factors yet to be identified.

[0103] 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); (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 X1X2X3X4X5X6, 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 VSPIX1X2X3X4, 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 YWPMX1X2, 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 is 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), 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).

[0104] 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 CDR3 of the VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 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.

[0105] In some embodiments, an antibody is provided, which has any partial light chain sequence listed in Table 1 and / or any partial heavy chain sequence listed in Table 1.

[0106] Table 1

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] 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 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, A02_Rd4 _0.6nM_C18、A02_Rd4_6nM_C10、A02_Rd4_6nM_C05、A02_Rd4_0.6nM_C10、A02_Rd4_6nM_C04、A02_Rd4_0.6nM_C26、A02_Rd4_0.6nM_C13、 A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C08, P5C1_VHVL, C01_Rd4_6nM_C24, C01_Rd4_6nM_C26, C01_Rd4_6nM_C10, C01_Rd4_0.6nM_C27, C01 _Rd4_6nM_C20, C01_Rd4_6nM_C12, C01_Rd4_0.6nM_C16, C01_Rd4_0.6nM_C09, C01_Rd4_6nM_C09, C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM _C06, C01_Rd4_6nM_C04, COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_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. .

[0130] 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 with 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.

[0131] Table 2

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] In some embodiments, the present invention provides an antibody that binds to 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 / H2.HA, L3.PY / L1.AH / H2.QL, L3.PY / L1.AH / H3.YA, L3.PY / L1.<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.

[0149] 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).

[0150] 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: 2, 32, 42 or 78; and / or a VL region comprising the sequence shown in SEQ ID NO: 6, 16, 43 or 85.

[0151] 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 antibodies that infuse specificity of the antibody to the antigen. In general, CDR contact regions include residue positions in the CDR and Vernier regions that are constrained to maintain appropriate loop structures for antibody binding to specific antigens. See, e.g., Makabe et al., J. Biol. Chem., 283: 1156-1166, 2007. The determination of CDR contact regions is within the skill of the art.

[0152] 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 about 0.002nM to about 6500nM. In some embodiments, the binding affinity is about any of 6500nm, 6000nm, 5986nm, 5567nm, 5500nm, 4500nm, 4000nm, 3500nm, 3000nm, 2500nm, 2134nm, 2000nm, 1500nm, 1000nm, 750nm, 500nm, 400nm, 300nm, 250nm, 200nM, 193nM, 100nM, 90nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 19nm, 18nm, 17nm, 16nm, 15nM, 10nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM or 0.002nM. In some embodiments, the binding affinity is less than about any 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.

[0153] 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 features 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.

[0154] 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. The polypeptides of the antibody, especially shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods for 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.

[0155] 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, 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 ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 112, 125, 127, 313, 314, 363 or 365. In other embodiments, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 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 shown in any sequence pair selected from the following 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 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, 84 and 83, 85 and 78, 86 and 78, 88 and 87, 89 and 78, 90 and 78, 91 and 78, 93 and 92, 94 and 78, 96 and 95, 98 and 97, 38 and 78, 102 and 101, 103 and 78, 105 and 104, 107 and 106, 108 and 78, 109 and 78, 111 and 110, 38 and 112, 113 and 112, 115 and 114, 116 and 76, 117 and 112, 119 and 118, 121 and 120, 123 and 122, 124 and 112, 126 and 125, 128 and 127, 80 and 363, or 364 and 365. In another embodiment, the fusion polypeptide comprises one or more CDRs.In other embodiments, the fusion polypeptide comprises CDRH3 (VH CDR3) and / or CDR L3 (VL CDR3). For the purposes of the present invention, the 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.

[0156] The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides.

[0157] 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 / H2.LT, L3.PY / L1.PS / H2.HA, L3.PY / L1.PS / H2.QL, L3.PY / L1.PS / H3.YA, L3.PY / L1.PS / H3.AE, L3.PY / L1.PS / H3.AQ, L3.PY / L1.PS / H3.TAQ, L3.PY / L1.AH / H2.QR, L3.PY / L1.AH / H2.DY, L3.PY / L1.AH / H2.YQ, L3.PY / L1.AH / H2.LT, L3.PY / L1.AH / H2.HA, L3.PY / L1.AH / H2.QL, L3.PY / L1.AH / H3.YA, L3.PY / L1.AH / H3.<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 sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be amplified and frozen for future use. Vectors (including expression vectors) and host cells are further described herein. .

[0158] The present invention also encompasses the scFv of the antibody of the present invention. Single-chain variable region fragments are prepared by connecting light chain and / or heavy chain variable regions using short connecting peptides (Bird et al., Science 242:423-426, 1988). An example of a connecting peptide is (GGGGS)3 (SEQ ID NO:498), which bridges about 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Joints of other sequences have been designed and used (Bird et al., 1988, supra). The joint should be a short flexible polypeptide and preferably contains less than about 20 amino acid residues. The joint can be modified in turn for additional functions, such as the connection of a drug or connection to a solid support. Single-chain variants can be recombinant or synthetically produced. 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 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 operations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0159] Other forms of single-chain antibodies are also contemplated, 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 without allowing 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, e.g., Holliger, P., et al., Proc. Natl. Acad Sci. USA 90: 6444-6448, 1993; Poljak, RJ, et al., Structure 2: 1121-1123, 1994). Minibodies include VL and VH domains of natural antibodies fused to the hinge region and CH3 domains of immunoglobulin molecules. See, e.g., US5,837,821.

[0160] In another aspect, the present invention provides a composition (e.g., a pharmaceutical composition) comprising any polynucleotide of the present invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any antibody described herein. In other embodiments, the composition comprises any one or both of the polynucleotides shown in the following SEQ ID NO: 486 and SEQ ID NO: 485:

[0161] COMBO_Rd4_0.6nM_C29 heavy chain variable region

[0162] gaagtccaactcctcgaatccggtggcggccttgtccagcctggaggttccttgcgcctgtcatgtgccgccagcggattcaccttctcgtcctacccgatgtcgtgggtccgccaggctccgggaaagggcctggaatgggtgtcagccatcggaggatcggggggctccctg ccctacgccgatatcgtgaagggaaggttcaccattagccgggacaactccaagaacactctgtacctccaaatgaacagcctgagagcggaggacaccgcagtgtactattgcgcccggtactggccaatggacatctggggccaggggactctggtcaccgtctcctca(SEQ ID NO:486)

[0163] COMBO_Rd4_0.6nM_C29 light chain variable region

[0164] Gagatcgtgctgactcagtcccctggaaccctgtccctgtcacctggcgaaagagctaccttgtcctgtcgcgcatcacaatccgtgtcgtcgagctatctcgcgtggtaccagcagaagcccggacaggccccaaggctgcttatgtacgacgcctccatc cgggccactggtatccccgaccgcttctcgggctccggaagcggcaccgacttcaccctgactatttcccggctcgaaccggaggatttcgccgtgtactactgccaacagtaccagagctggccgctgacgtttgggcaggggaccaaggtcgaaatcaaa

[0165] (SEQ ID NO:485)

[0166] In other embodiments, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO:488 and SEQ ID NO:487 below:

[0167] L3.PY / H3TAQ heavy chain variable region

[0168] gaagtgcagctgctggaatctggcggaggactggtgcagcctggcggctctctgagactgtcttgtgccgccagcggcttcaccttcggcagctacgctatgacctgggtgcgccaggcccctggcaaaggactggaatgggtgtccgccatctctggcagcggcggcaataccttct acgccgagagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactattgtacacgggtgtcccctatcgccgcgcagatggattattggggccagggcactctggtcaccgtctcctca

[0169] (SEQ ID NO:488)

[0170] L3.PY / H3TAQ heavy chain variable region

[0171] Gagatcgtgctgacacagagccctggcaccctgagcctgtctccaggcgaaagagccaccctgtcctgcagagccagccagagcgtgtccagcagctacctggcctggtatcagcagaagcccggccaggctccccggctgctgatctatggcgcctcttcta gagccaccggcatccccgatagattcagcggctctggcagcggcaccgacttcaccctgaccatcagcagactggaacccgaggacttcgccgtgtactactgccagcactacccttatccccccagcttcacatttggccagggcaccaaggtggagatcaaa

[0172] (SEQ ID NO:487)

[0173] 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:

[0174] A02_Rd4_0.6nM_C01 Heavy chain variable region

[0175] GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTACTGCGTCTGGTGGTTCTA CTTACTATGCCGATGTGGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGTCGTTGTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

[0176] (SEQ ID NO:490)

[0177] A02_Rd4_0.6nM_C01 light chain variable region

[0178] GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGCGTATTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCGTTTACTACTGTCAACAATATGAGCGTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG

[0179] (SEQ ID NO:489)

[0180] In other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 492 and SEQ ID NO: 491 below:

[0181] A02_Rd4_0.6nM_C16 Heavy chain variable region

[0182] GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTTTGGTGGTTCTA CTTACTATGCCGATATCGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

[0183] (SEQ ID NO:492)

[0184] A02_Rd4_0.6nM_C16 light chain variable region

[0185] GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGATCTGTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGA GCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCGTTTACTACTGTCAACAATATCAGACTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG

[0186] (SEQ ID NO:491).

[0187] Expression vectors and administration of polynucleotide compositions are further described herein.

[0188] In another aspect, the invention provides methods of making any of the polynucleotides described herein.

[0189] The present invention also encompasses polynucleotides complementary to any such sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond 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 in the polynucleotides of the present invention, and polynucleotides may, but need not, be connected to other molecules and / or support materials.

[0190] 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 immune response molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be evaluated as described herein. The variant preferably exhibits at least about 70% homology to a polynucleotide sequence encoding a native antibody or a portion thereof, more preferably, at least about 80% homology, more preferably, at least about 90% homology, and most preferably, at least about 95% homology.

[0191] 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 consecutive 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 consecutive positions after the two sequences are optimally aligned.

[0192] 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.

[0193] 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), usually 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 of the same nucleic acid base or amino acid residue present in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence and multiplying the result by 100 to generate the percentage of sequence identity.

[0194] 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).

[0195] 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.

[0196] As used herein, "high stringency conditions" or "high stringency conditions" are: (1) using low ionic strength and high temperature for washing, for example, 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, for example, 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 0.2x SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1x 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.

[0197] 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 structures or functions. Alleles can be identified using standard techniques (e.g., hybridization, amplification and / or database sequence comparisons).

[0198] Polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. The method of 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 a commercial DNA synthesizer to prepare the desired DNA sequence.

[0199] In order to utilize recombinant methods to prepare polynucleotides, as further discussed herein, the polynucleotides 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. The polynucleotides can be inserted into the host cell by any method known in the art. The exogenous polynucleotides are introduced into the transformed 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.

[0200] Alternatively, PCR allows for the replication of a DNA sequence.PCR techniques are well known in the art and are described in, for example, U.S. 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).

[0201] 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.

[0202] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although selected cloning vectors can be used according to the expected host cell changes, useful cloning vectors generally have the ability to self-replicate, can have a single target of a specific restriction endonuclease, and / or can carry genes that can be used to select the markers of the clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., 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.

[0203] 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 a 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 start sites, and stop codons, are generally also required.

[0204] 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.

[0205] The present invention also provides a host cell comprising any polynucleotide 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. cerevisae), Schizosaccharomyces pombe (S. pombe); or Kluyveromyces lactis (K. lactis)). Preferably, the host cell expresses the cDNA at a level about 5 times higher than the corresponding endogenous antibody or protein of interest in the host cell (if present), more preferably 10 times higher, and even more preferably 20 times higher. 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 antibodies or proteins of interest can be identified.

[0206] Representative materials of the present invention were deposited at the American Type Culture Collection (ATCC) on April 15, 2015. The vector with ATCC accession number PTA-122094 is a polynucleotide encoding the heavy chain variable region of the humanized BCMA antibody, and the vector with ATCC accession number PTA-122093 is a polynucleotide encoding the light chain variable region of the humanized BCMA antibody. Deposit is made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulations (Budapest Treaty). This ensures that the living culture of the deposit is 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, 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 comes 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 C.FR. § 1.14, specifically referenced 886 OG 638).

[0207] 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 license to practice the invention in accordance with rights granted under its patent laws.

[0208] BCMA Antibody Conjugate

[0209] The present invention also provides a conjugate (or immunoconjugate) of a BCMA antibody or antigen-binding fragment thereof as described herein, wherein the antibody or antigen-binding fragment is directly or indirectly conjugated to an agent (e.g., a cytotoxic agent) through a linker 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).

[0210] Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies have been described in various publications. For example, chemical modification can be performed in antibodies via lysine side chain amines or via reduction of interchain disulfide bond activated cysteine ​​sulfhydryl groups for conjugation reactions to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, 2005, and Gentletal., Bioconjugate Chem. 15:658-663, 2004. Reactive cysteine ​​residues with defined stoichiometric amounts for specific drug conjugation at specific sites of antibodies have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. International applications WO2012 / 059882 and WO2015015448 also describe 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 comprising or attached to a reactive amine).

[0211] 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 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).

[0212] 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.

[0213] 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), LLQGSHGG (SEQ ID NO: 466), LLQGSHGG (SEQ ID NO: 467), LLQGSHGG (SEQ ID NO: 468), LLQGSHGG (SEQ ID NO: 469), LLQGSHGG (SEQ ID NO: 470), LLQGSHGG (SEQ ID NO: 471), LLQGSHGG (SEQ ID NO: 472), LLQGSHGG (SEQ ID NO: 473), LLQGSHG (SEQ ID NO: 474), LLQGSHG (SEQ ID NO: 475), LLQGSHG 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 (eg, K222R, K340R or K370R).

[0214] 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.

[0215] In some embodiments, a BCMA antibody or conjugate as described herein comprises a tag containing an 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 an 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 from asparagine (N) to alanine (A) 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.

[0216] 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.

[0217] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatins, combretastatins, duocarmycin, pyrrolebenzodiazepine dimers, indoline-benzodiazepines, dimer, enediyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloid, camptothecin, tubulysin, hemiesterin, spliceostatin, pradienolide and stereoisomers, isosteres, analogs or derivatives thereof.

[0218] Anthracyclines are derived from the bacterium Strepomyces and have been used to treat a wide range of cancers, such as leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0219] 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-methyl valine-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methyl valine-valine-dolaisoleuine-dolaproine-phenylalanine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (such as 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-oxoheptane-4-yl]-N-methyl-L-valinamide) having the following structure:

[0220]

[0221] 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:

[0222]

[0223] 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:

[0224]

[0225] 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-oxoheptane-4-yl]-N-methyl-L-valinamide) having the following structure:

[0226]

[0227] 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:

[0228]

[0229] Camptothecins are cytotoxic quinoline alkaloids that inhibit the enzyme topoisomerase I. Examples of camptothecins and their derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites such as SN-38.

[0230] 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.

[0231] 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:

[0232]

[0233] 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:

[0234]

[0235] 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:

[0236]

[0237] 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.

[0238] 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).

[0239] Hemipteratin and its analogs (eg, HTI-286) bind to tubulin, disrupt normal microtubule dynamics, and depolymerize microtubules in stoichiometric amounts.

[0240] 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 maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives and ansamitocin.

[0241] Pyrrolebenzodiazepine dimer (PBD) and indole-benzodiazepine Dimers (IGNs) are antitumor agents containing one or more immine functional groups or their equivalents that bind to double-stranded DNA. PBD and IGN molecules are based on the natural product athramycin 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.

[0242] 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

[0243]

[0244] (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]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate of the structure. Examples of pradienolides include, but are not limited to, pradienolide B, pradienolide D, or E7107.

[0245] 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

[0246] 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.

[0247] Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0248] 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-oxoheptane-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]

[00136] The invention relates to (1-{[(2R,3R,5S,6S)-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]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate.

[0249] 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, mycophenolgate 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 "S 1 factor", erythropoietin and thrombopoietin, or a combination thereof.

[0250] 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 sulfonylrhodamine (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).

[0251] 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 sulfonylrhodamine (SR) (e.g., SR101).

[0252] 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.

[0253] In some embodiments, the pharmaceutical agent is a therapeutic protein, including but not limited to toxins, hormones, enzymes, and growth factors.

[0254] 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).

[0255] In some embodiments, the agent is a biocompatible polymer. The BCMA antibody or antigen-binding fragment as described herein can be conjugated to a biocompatible polymer to increase serum half-life and biological activity, and / or extend in vivo half-life. Examples of biocompatible polymers include water-soluble polymers such as polyethylene glycol (PEG) or its derivatives and biocompatible polymers containing zwitterions (e.g., polymers containing phosphorylcholine).

[0256] In some embodiments, the agent is an oligonucleotide, such as an antisense oligonucleotide.

[0257] In another aspect, the present 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 chain 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., 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).

[0258] 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.

[0259] 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 comprising N297A; 5) Antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-3377 and comprising 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 (e.g., LLQGA (SEQ ID NO: 463) or LLQGPP (SEQ ID NO: 472)) is engineered at the C-terminus of the antibody heavy chain, wherein the lysine residue at the C-terminus 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. .

[0260] In one variation, the conjugate further comprises an amino acid substitution from lysine to arginine at position 222. Thus, for example, the conjugate is 1) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprises K222R; 2) antibody-AcLys-VC-PABC-0101 comprising N297Q and K222R; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprises N297Q and K222R; 4) antibody-LLQG ( SEQ ID NO: 454)-amino-PEG6-C2-0131 and comprising 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 (e.g., LLQGA (SEQ ID NO: 473) or LLQGPP (SEQ ID NO: 472)) is engineered at the C-terminus of the antibody heavy chain, wherein the lysine residue at the C-terminus 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. .

[0261] CD3 antibody and preparation method thereof

[0262] The present invention further provides antibodies that bind to CD3 (eg, human CD3 (SEQ ID NO: 502; or Accession No.: NM_000733.3).

[0263] 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 a 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 a 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; 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 of a light chain variable (VL) region of VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in FIG.

[0264] In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to 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.

[0265] In some embodiments, an antibody is provided, which has any partial light chain sequence listed in Table 3 and / or any partial heavy chain sequence listed in Table 3.

[0266] Table 3

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] In Table 3, the underlined sequences are CDR sequences according to Kabat, while those in bold are according to Chothia.

[0273] The present invention also provides CDR portions (including Chothia, Kabat CDRs and CDR contact regions) of CD3 antibodies. The determination of CDR regions is within the art. It should be understood that in some embodiments, the CDR can be a combination of Kabat and Chothia CDRs (also referred to as "combined CRs" or "extended CDRs"). In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other words, in embodiments with more than one CDR, the CDR can be any Kabat, Chothia, combined CDR or a combination thereof. Table 4 provides examples of CDR sequences provided herein.

[0274] Table 4

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides.

[0282] In one embodiment, the polynucleotide comprises a polypeptide 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, then the host cell can be amplified and frozen for future use. Carriers (including expression vectors) and host cells are further described herein.

[0283] 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 that comprises 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 shown 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, the fusion protein comprises one or more antibodies and another amino acid sequence that is not connected in the natural molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags" such as FLAG tags or 6His tags. Tags are well known in the art.

[0284] 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 preparing expression polynucleotides encoding them using recombinant methods described herein, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[0285] Representative materials of the CD3 antibody in the present invention were deposited at the American Type Culture Collection (ATCC) on September 11, 2015. The vector with ATCC accession number PTA-122513 is a polynucleotide encoding the heavy chain variable region of the humanized CD3 antibody, and the vector with ATCC accession number PTA-122512 is a polynucleotide encoding the light chain variable region of the humanized CD3 antibody. Deposit is made according to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure and Regulations (Budapest Treaty). This ensures that the living culture of the deposit is 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, 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 comes 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 C.FR. § 1.14, specifically referenced 886 OG 638).

[0286] 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 license to practice the invention in accordance with rights granted under its patent laws.

[0287] Bispecific antibodies and methods of making them

[0288] Bispecific antibodies can be prepared using the antibodies disclosed herein, monoclonal antibodies having binding specificity to at least two different antigens. Methods for preparing bispecific antibodies are known in the art (see, e.g., 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 two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

[0289] According to a method for preparing bispecific antibodies, the antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to the immunoglobulin constant region sequence. 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 cotransfected into a suitable host organism. When the unequal proportions of 3 polypeptide chains used in the construction provide the best yield, 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 leading to high yield or when the ratio has no special meaning, the coding sequence of two or all three polypeptide chains can be inserted into an expression vector.

[0290] In one approach, bispecific antibodies consist 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.

[0291] In another approach, the bispecific antibody consists of amino acid modifications in the first hinge region in one arm, and the substituted / replaced amino acid in the first hinge region has an opposite charge to the corresponding amino acid in the second hinge region in the other arm. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0292] In another approach, the formation of a desired heteromultimer or heterodimer protein (e.g., a bispecific antibody) is enhanced by altering or engineering the interface between the first and second immunoglobulin-like Fc regions (e.g., hinge regions and / or CH3 regions). 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 interact together to form a CH3 interface, wherein one or more amino acids within the CH3 interface destabilize homodimer formation and electrostatically disfavor homodimer formation. This approach is described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0293] In another approach, bispecific antibodies can be generated using a glutamine-containing peptide tag engineered to 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 to 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).

[0294] 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 proteins can recruit human immune effector cell activity by specifically binding to effector antigens on human immune effector cells, and wherein the second antibody variable domains of the heterodimeric proteins can specifically bind to target antigens. In some embodiments, human antibodies have IgG1, IgG2, IgG3, or IgG4 isotypes. In some embodiments, the heterodimeric proteins include immunologically inert Fc regions.

[0295] 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 members of, for example but not limited to, human myeloid system, 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.

[0296] 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 include, but are not limited to, human CD3 (or CD3 (differentiation cluster) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

[0297] Target cells can be natural or exogenous cells of 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.

[0298] 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.

[0299] 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 located on human immune effector cells (e.g., a CD3 antigen), 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)).

[0300] In some embodiments, the first and second antibody variable domains of the heterodimeric protein comprise amino acid modifications at positions 221 and 228 in the hinge region (e.g., (D221R or D221E) and (P228R or P228E)) and at position 409 or 368 in the CH3 region of human IgG1 (SEQ ID NO: 494) (e.g., K409R or L368E (EU numbering scheme)).

[0301] 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).

[0302] In another embodiment, the first antibody variable domain of the heterodimeric protein comprises a VH region comprising 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, ​​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 of the VL sequence shown in VL CDR1, VL CDR2 and VLCDR3, 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, VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in FIG.

[0303] 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 38.

[0304] Antibodies that can be used in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, 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. Antibodies may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies).

[0305] In some embodiments, the BCMA or CD3 antibody as described herein is a monoclonal antibody. For example, the BCMA or CD3 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

[0306] In some embodiments, the antibody comprises a modified constant region, such as, 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 Fcγ receptors such as FcγRI, FcγRIIA or FcγIII.

[0307] In some embodiments, the antibody comprises a modified constant region, such as an immunologically inert constant region, i.e., having a 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 may be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc may 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 a constant region of IgG4 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 a 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.

[0308] In some embodiments, the constant region is aglycosylated by mutating the oligosaccharide attachment residue (e.g., Asn297) and / or the flanking residues of the glycosylation recognition sequence portion of the constant region. In some embodiments, the constant region is enzymatically aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

[0309] 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 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.

[0310] In some embodiments, the constant region has a modified constant region having the sequence set forth in SEQ ID NO:497:

[0311] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0312] One method of determining the binding affinity of an antibody for BCMA or CD3 is by measuring 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 BCMAFab 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 the 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 Fab (concentration of 100 μL / min) was injected for 1 min at 100 μL / min, and a dissociation time of up to 2 hours was allowed. The concentration of Fab protein was determined by ELISA and / or SDS-PAGE electrophoresis using Fab of known concentration (as determined by amino acid analysis) as a standard. The kinetic binding rate (k) was obtained simultaneously by fully fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6.99-110) using the 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 to 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). The binding affinity of the antibody is generally measured at 25°C, but can also be measured at 37°C.

[0313] 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.

[0314] It is contemplated that any mammalian individual, including humans, or its antibody-producing cells may be manipulated to serve as the basis for mammalian production, including human and hybridoma cell lines. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intrapedicularly, and / or intradermally with an amount of an immunogen, including as described herein.

[0315] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using Kohler, B. and Milstein, C., Nature 256: 495-497, 1975 or as Buck, DW, et al., In Vitro, 18: 377-381, 1982 modified general somatic cell hybridization techniques. Available myeloma lines can be used in hybridization, including but not limited to X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA. Typically, the technology includes fusing myeloma cells and lymphoid cells using fusogens such as polyethylene glycol or by electrical methods well known to those skilled in the art. After fusion, cells are separated from the fusion culture medium and grown on selective growth culture media such as hypoxanthine-aminopterin-thymidine (HAT) to eliminate unhybridized parental cells. Any culture medium described herein supplemented with or without serum can be used to culture hybridomas secreting monoclonal antibodies. As an alternative to cell fusion techniques, EBV immortalized B cells can be used to produce the monoclonal antibodies of the invention. The hybridomas are expanded and subcloned, if desired, and the supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0316] Hybridomas that can be used as a source of antibodies encompass all derivatives, progeny cells of a parent hybridoma that produces monoclonal antibodies specific for BCMA, CD3 or portions thereof.

[0317] Hybridomas producing such antibodies can be grown in vitro or in vivo using known methods. 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, for example, by running the product on an adsorbent made from an immunogen connected to a solid phase and eluting or releasing the desired antibody from the immunogen. Bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated to cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1 A population of antibodies (e.g., monoclonal antibodies) can be generated by immunizing with 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).

[0318] 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 amplified 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.

[0319] 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 a human constant region so as to avoid immune responses if the antibody is used in clinical trials and treatments 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.

[0320] There are 4 general steps to humanize monoclonal antibodies. They are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light chain and heavy chain variable domains, (2) designing the humanized antibody, i.e., deciding which antibody framework region to use in the humanization process, (3) the actual humanization method / technique, and (4) transfection and expression of the humanized antibody. See, e.g., 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.

[0321] 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 grafted into human supporting framework regions (FRs) prior to fusion with appropriate human antibody constant regions. See, e.g., Riechmann et al. Nature 332:323-327, 1988, Verhoeyen et al. Science 239:1534-1536, 1988, and Jones et al. Nature 321:522-525, 1986. Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize undesirable immune responses to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of those parts in human recipients. For example, an antibody constant region can be engineered so that it is immunologically inert (e.g., does not trigger complement lysis). See, e.g., 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.

[0322] 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.

[0323] 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 .

[0324] 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 libraries from unimmunized donors. According to this technology, the antibody V domain gene is cloned 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 the selection of genes encoding antibodies that exhibit these properties. Therefore, phages mimic some of the properties of B cells. Phage display can be performed in various forms; 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 anti-oxazolone antibody from a small random combinatorial library of V genes derived from the spleen of immune 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 by Mark et al., J. Mol. Biol. 222: 581-597, 1991 or Griffith et al., EMBO J. 12: 725-734, 1993. In the natural immune response, antibody genes accumulate mutations (somatic hypermutations) at a high speed. Some changes introduced can confer high affinity, and the B cells showing high affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigen attacks. This natural process can be imitated by adopting a technique known as "chain shuffling". (Marks et al., Bio / Technol. 10: 779-783, 1992). In this method, the affinity of the "elementary" human antibodies obtained by phage display can be improved by replacing the heavy chain and light chain V regions with the library order of the naturally occurring variants (library) of the V domains obtained from unimmunized donors. This technology allows the production of antibodies and antibody fragments with an affinity in the pM-nM range.Waterhouse et al., Nucl. Acids Res. 21: 2265-2266, 1993 has 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 leads to the separation of human variable regions that can restore functional antigen binding sites, i.e., the selection of epitope-dominated (imprinted) counterparts. When the 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 without framework or CDR residues of rodent origin.

[0325] Antibodies can be recombinantly produced by first isolating antibodies or antibody-producing cells from a host animal, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in a host cell (e.g., CHO cells). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or 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.

[0326] 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.

[0327] Antibodies as described herein can be combined with 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 combining antibodies, or can determine such carriers using routine experiments. In some embodiments, the carrier comprises a portion that targets the myocardium.

[0328] DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). 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, so as to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, e.g., PCT Publication No. WO 87 / 04462. DNA can also be modified, for example, by replacing the coding sequences of human heavy and light chain constant regions in place of homologous mouse sequences, Morrison et al., Proc. Nat. Acad. Sci. 81: 6851, 1984, or by covalently linking to immunoglobulin coding sequences, all or part of the coding sequences of non-immunoglobulin polypeptides. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the monoclonal antibodies herein.

[0329] BCMA or the infused tumor antigen antibody as described herein can be identified or characterized using methods known in the art to detect and / or measure the reduction in the expression level of BCMA or other tumor antigens. In some embodiments, the BCMA antibody is identified by incubating the candidate substance with BCMA and monitoring the binding and / or accompanying reduction in BCMA expression levels. The binding assay can be performed with purified BCMA polypeptides, or with cells expressing naturally or transfected BCMA polypeptides. In one embodiment, the binding assay is a competitive binding assay, in which the ability of the candidate antibody to compete with a known BCMA antibody for BCMA binding is evaluated. The assay can be performed in various formats, including ELISA formats.

[0330] After initial identification, the activity of candidate BCMA, CD3 or other tumor antigen antibodies can be further confirmed and refined by 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.

[0331] 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 structures 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, 8219PH Lelystad, The Netherlands). An epitope can be a linear epitope, that is, contained in a single amino acid segment, or a conformational epitope formed by the three-dimensional interaction of amino acids that may not necessarily be contained in a single segment. Peptides of different lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinant) and used for binding assays with BCMA, CD3 or other tumor antigen antibodies. In another example, the epitopes bound to BCMA, CD3 or other tumor antigen antibodies can be determined in a system screening by utilizing overlapping peptides derived from BCMA, CD3 or other tumor antigen sequences and by BCMA, CD3 or other tumor antigen antibodies. According to the gene fragment expression assay, the open reading frame encoding BCMA, CD3 or other tumor antigens is randomly or fragmented 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. The gene fragment can be produced, for example, by PCR, and then transcribed and translated into protein 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 in a simple binding assay to bind to the test antibody. 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 BCMA, CD3, or other tumor antigen proteins have been replaced (exchanged) with sequences from BCMA from another species (e.g., mouse) or a closely related but antigenically distinct protein (e.g., Trop-1).By evaluating antibody binding to mutant BCMA, CD3 or other tumor antigens, the importance of specific BCMA, CD3 or other tumor antigen fragments for antibody binding can be evaluated.

[0332] Another method that can be used to characterize BCMA, CD3 or other tumor antigen antibodies is to use competition assays with other antibodies known to bind to the same antigen (i.e., various fragments on BCMA, CD3 or other tumor antigens) to determine whether the BCMA, CD3 or other tumor antigen antibodies bind to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.

[0333] The expression vector can be used to directly express BCMA, CD3 or other tumor antigen antibodies. Those skilled in the art are familiar with the administration of expression vectors to obtain the expression of exogenous proteins in vivo. See, for example, US Pat. No. 6,436,908; 6,413,942; and 6,376,471. The administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and surface administration. In another embodiment, the expression vector is directly administered to the sympathetic trunk or section, or to the coronary artery, atrium, ventricle or pericardium.

[0334] Targeted delivery of therapeutic compositions comprising expression vectors or subgenomic polynucleotides can also be used. For example, 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 describe receptor-mediated DNA delivery techniques. For local administration in gene therapy protocols, therapeutic compositions comprising polynucleotides are administered in the range of about 100ng-about 200mg of DNA. Concentration ranges of about 500ng-about 50mg, about 1μg-about 2mg, about 5μg-about 500μg, and about 20μg-about 100μg 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.

[0335] 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 by Curiel, HμM. Gene Ther., 1992, 3:147 can also be used.

[0336] 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, HμM. Gene Ther., 3: 147, 1992); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264: 16985, 1989); eukaryotic cell delivery vehicle cells (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.

[0337] In some embodiments, the present invention encompasses compositions, including pharmaceutical compositions, comprising antibodies described herein or prepared by the methods and having the features 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.

[0338] 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. The polypeptides of the antibody, especially shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods for 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.

[0339] Heteroconjugate antibodies comprising two covalently linked antibodies are also within the scope of the 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.

[0340] 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.

[0341] In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction with Fcγ receptors as well as complement and the immune system. Techniques for preparing such antibodies are described in WO 99 / 58572. For example, the constant region can be engineered to be more like a human constant region so that if the antibody is used in clinical trials and treatment in humans, an immune response is avoided. See, for example, US Pat. Nos. 5,997,867 and 5,866,692.

[0342] The present invention encompasses modifications to the antibodies and polypeptides of the present 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 may be mutated to obtain an antibody with a desired binding affinity to BCMA and / or CD3. Modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly deleteriously change the functional activity, or the affinity of its mature (enhanced) polypeptide to its ligand, or the use of chemical analogs.

[0343] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 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 antibody molecules 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.

[0344] Substitution variants have at least one amino acid residue removed and a different residue inserted in its place 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 changes can be introduced, named "exemplary substitutions" in Table 5, or as further described below with reference to amino acid classes, and the product is screened.

[0345] Table 5: Amino Acid Substitutions

[0346]

[0347]

[0348] Substantial changes in the biological properties of antibodies can be accomplished 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:

[0349] (1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0350] (2) Polarity has no charge: Cys, Ser, Thr, Asn, Gln;

[0351] (3) Acidic (negatively charged): Asp, Glu;

[0352] (4) Basic (positively charged): Lys, Arg;

[0353] (5) Residues that affect chain direction: Gly, Pro; and

[0354] (6) Aromatic: Trp, Tyr, Phe, His.

[0355] Exchanging a member of one of these classes for another class results in non-conservative substitutions.

[0356] Any cysteine ​​residues not involved in maintaining the correct conformation of the antibody may also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

[0357] 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.

[0358] 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 the glycoprotein parts 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 the specific recognition structure, 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-acetylglucosamine transferase 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).

[0359] Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the sugar moiety being attached 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 the sugar moiety to the asparagine side chain. Therefore, 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 the base amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0360] Glycosylation sites are 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).

[0361] The glycosylation pattern of the antibody can also be altered without changing the underlying nucleotide sequence. Glycosylation depends primarily 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, changes in the glycosylation pattern of the antibody can be expected (see, e.g., Hse et al., J. Biol. Chem. 272: 9062-9070, 1997).

[0362] 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 change the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (US Pat. No. 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.

[0363] 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 an immunoassay. The modified polypeptides are 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.

[0364] 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 unmodified antibodies) 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 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT 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 (with reference to the amino acid numbering of the wild-type IgG2 sequence). Eur. J. Immunol., 1999, 29: 2613-2624. In other embodiments, the constant region is non-glycosylated for N-linked glycosylation. In some embodiments, the constant region is non-glycosylated 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 portion. 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 enzymatically (such as by removal of sugars by the enzyme PNGase) or by expression in a glycosylation-deficient host cell.

[0365] 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 a polypeptide 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.

[0366] 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).

[0367] 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 (e.g., improving) the binding affinity of polypeptides such as antibodies is called "library scanning mutagenesis." Typically, library scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using methods known in the art. 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, libraries also include clones containing natural (unsubstituted) amino acids. A small number 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. When the starting antibody has been bound with a relatively high affinity, e.g., a K of about 10 nM or less, D When combined, Biacore TM Particularly useful. This article describes in the examples the use of Biacore TM Screening by surface plasmon resonance.

[0368] 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.

[0369] In some embodiments, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 natural 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).

[0370] In some embodiments, the library to be screened comprises substitutions in two or more positions, which may be in the same CDR or in two or more CDRs. Therefore, the library may comprise substitutions in two or more positions in one CDR. The library may comprise substitutions in two or more positions in two or more CDRs. The library may comprise substitutions in 3, 4, 5 or more positions, which may be in 2, 3, 4, 5 or 6 CDRs. Substitutions may be prepared using low redundancy codons. See, for example, Table 2 of Balint et al., Gene 137 (1): 109-18, 1993.

[0371] 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.

[0372] 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.

[0373] Multiple rounds of screening can be performed. For example, candidates with improved binding (each comprising an amino acid substitution at one or more positions of one or more CDRs) can also be used in the design of 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). The preparation of such libraries and screening or selection is further discussed below.

[0374] Library screening mutagenesis also provides a method for characterizing CDRs, because the frequency of clones with improved binding, identical binding, reduced binding or no binding also provides information about the importance of each amino acid to the stability of the antibody-antigen complex. For example, if the position of the CDR keeps binding when being changed to all 20 kinds of amino acids, then the position is identified as a position that is unlikely to be required for antigen binding. On the contrary, if the position of the CDR only keeps binding in a small portion of substitutions, then the position is identified as a position that is important to the CDR function. Therefore, the library screening mutagenesis method generates information about the position in the CDR that can be changed to many different amino acids (including all 20 kinds of amino acids) and the position in the CDR that cannot be changed or can only be changed to several amino acids.

[0375] The candidate with improved affinity can be combined in the second library, which includes the improved amino acid, the original amino acid at the position, and can further include additional substitutions at the position, depending on the library complexity allowed by the expected screening or selection method. In addition, if desired, adjacent amino acid positions can be randomized to at least two or more amino acids. The randomization of adjacent amino acids can allow additional conformational flexibility in the sudden change CDR, which in turn can allow or promote the introduction of a large number of improved mutations. The library can also include substitutions at positions that did not show improved affinity in the first round of screening.

[0376] The second library is screened or selected for library members with improved and / or altered binding affinity using any method known in the art, including the use of 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.

[0377] 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 connecting these components as described herein. The connection (which generally fixes these components in a directly associated manner at least for administration) can be implemented in any manner. For example, when each has substituents that can react with each other, a direct reaction between the agent and the antibody is possible. For example, a nucleophilic group such as an amino or thiol group on one may be able to react with a carbonyl-containing group such as an anhydride or acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0378] In another aspect, the invention provides a method of making any of the polynucleotides described herein.

[0379] The present invention also encompasses the polynucleotide complementarity of any such sequence. The polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be a DNA (genomic, cDNA or synthetic) or RNA molecule. 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 in the polynucleotides of the present invention, and the polynucleotides may, but need not, be linked to other molecules and / or support materials.

[0380] 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 immune response molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be evaluated as described herein. The variant preferably exhibits at least about 70% homology to a polynucleotide sequence encoding a native antibody or a portion thereof, more preferably, at least about 80% homology, more preferably, at least about 90% homology, and most preferably, at least about 95% homology.

[0381] 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 consecutive 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 consecutive positions after the two sequences are optimally aligned.

[0382] 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.

[0383] 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), usually 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 of the same nucleic acid base or amino acid residue present in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence and multiplying the result by 100 to generate the percentage of sequence identity.

[0384] 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).

[0385] 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.

[0386] As used herein, "high stringency conditions" or "high stringency conditions" are: (1) using low ionic strength and high temperature for washing, for example, 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, for example, 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 0.2x SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1x 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.

[0387] 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 structures or functions. Alleles can be identified using standard techniques (e.g., hybridization, amplification and / or database sequence comparisons).

[0388] Polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. The method of 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 a commercial DNA synthesizer to prepare the desired DNA sequence.

[0389] In order to utilize recombinant methods to prepare polynucleotides, as further discussed herein, the polynucleotides 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. The polynucleotides can be inserted into the host cell by any method known in the art. The exogenous polynucleotides are introduced into the transformed 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.

[0390] Alternatively, PCR allows for the replication of a DNA sequence.PCR techniques are well known in the art and are described in, for example, U.S. 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).

[0391] 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.

[0392] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although selected cloning vectors can be used according to the expected host cell changes, available cloning vectors generally have the ability to self-replicate, can have a single target of a specific restriction endonuclease, and / or can carry genes that can be used to select the markers of the clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., 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.

[0393] 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 a 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 start sites, and stop codons, are generally also required.

[0394] 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.

[0395] The present invention also provides a host cell comprising any polynucleotide 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 cDNA at a level about 5 times higher than the corresponding endogenous antibody or protein of interest (if present) in the host cell, more preferably 10 times higher, and even more preferably 20 times higher. 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 antibodies or proteins of interest can be identified.

[0396] Methods of using bispecific antibodies [Therapeutic applications]

[0397] 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.

[0398] 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 abnormal BCMA expression, malignant cells expressing BCMA, and proliferative disorders (e.g., cancer) or autoimmune disorders.

[0399] In another aspect, the present invention provides a method for treating a B-cell-related cancer or a malignant cell expressing a tumor antigen. In some embodiments, a method for treating a B-cell-related cancer in an individual in need thereof is provided, the method 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, the method comprising administering to the individual in need thereof an effective amount of a pharmaceutical composition comprising a bispecific antibody as described herein.

[0400] Thus, in some embodiments, a method of treating cancer in an individual is provided, the method comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody as described herein (e.g., BCMA or CD3-BCMA bispecific antibody) or a BCMA antibody conjugate. 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 ... ALL, chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's 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's lymphoma, primary mediastinal (thymic) large B lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, lymph node 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-related 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, 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, unclassified, and other B-cell-related lymphomas.

[0401] 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 cell metastasis 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.

[0402] In some embodiments, a method of treating an autoimmune disorder in an individual is provided, the method 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.

[0403] 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 enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, auto Autoimmune pancreatitis, autoimmune endocrine polyglandular syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Behcet's disease, Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, narcolepsy, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatic fever, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis.

[0404] 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 treating a disease condition (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 development 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.

[0405] 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 disease conditions associated with BCMA expression (e.g., cancer or autoimmune disorders) in individuals 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 individuals 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 individuals 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 individuals with malignant cells expressing BCMA.

[0406] 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.

[0407] 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.

[0408] 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.

[0409] In some embodiments, the additional form of therapy includes 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 antibody, anti-CD33 antibody, anti-CD20 antibody (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.

[0410] For example, in some embodiments, a method of treating multiple myeloma is provided, the method comprising administering to a patient in need thereof an effective amount of a composition comprising an antibody as described herein (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody 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 (eg, ), 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 for 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.

[0411] 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 appreciated by those skilled in the art that the examples described herein are not intended to limit but to illustrate available techniques. 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. A commercially available nebulizer for liquid preparations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid preparations can be directly atomized, and lyophilized powders can be atomized after reconstruction. 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.

[0412] 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 technology 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.

[0413] Various preparations of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be used for administration. In some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be administered neat. In some embodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate and a pharmaceutically acceptable excipient can be in various preparations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that promote the administration of pharmacologically effective substances. For example, an excipient can give form or consistency, or act as a diluent. 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 preparations for parenteral and non-parenteral drug delivery are shown in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005.

[0414] 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.

[0415] As described herein, antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates can be administered by any suitable method, including by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). As described herein, the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate 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 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, a dose 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 continuous until the desired symptom suppression occurs or until sufficient treatment levels are reached, such as inhibition or delay of 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 an increased dose (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 practitioners wish to achieve. For example, in some embodiments, 1-4 doses are considered to be administered per week. In other embodiments, a dose administration once a month, once every month, or once every three months is considered to be administered. 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.

[0416] For purposes of the present invention, the appropriate dosage of the 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 rate 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 contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can 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 can 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, etc. Alternatively, sustained release formulations of antibodies (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugates may be suitable. Various formulations and devices to achieve sustained release are known in the art.

[0417] In one embodiment, the dosage of the antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be determined empirically in an individual who has 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 given to the individual. In order to evaluate efficacy, indicators of disease can be tracked.

[0418] Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate according to the methods of the 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 the skilled artisan. Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate can be substantially continuous over a preselected time, or can be a series of spaced doses.

[0419] 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 an epitope on BCMA or CD3 and a second BCMA or CD3 antibody directed against a different epitope on BCMA or CD3.

[0420] 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 having 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 phosphates, citrates and other organic acids; salts such as sodium chloride; 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 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).

[0421] 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 a filter of defined pore size to produce liposomes with a desired diameter.

[0422] 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.

[0423] 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 (US 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 TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0424] Formulations for in vivo administration must be sterile. This is easily 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 generally placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

[0425] 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.

[0426] In order to prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier to form a solid preformulated composition containing a uniform mixture of the compound of the present invention or its non-toxic pharmaceutically acceptable salt, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum, and other pharmaceutical diluents such as water. When it is mentioned that these preformulated compositions are uniform, it means that the active ingredient is uniformly dispersed throughout the composition, so that the composition can 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-about 500 mg of the active ingredient of the present invention. The tablets or pills of the new composition can be coated or compounded to provide a dosage form with the advantage of a 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 may 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.

[0427] 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.

[0428] Commercially available fat emulsions such as Intralipid TM , Liposyn TM Infonutrol TM 、Lipofundin TM Lipiphysan TMPrepare suitable emulsions. 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 form an emulsion when mixed with a phospholipid (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water. It should be understood 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, such as 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.

[0429] 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).

[0430] Compositions for inhalation or insufflation include pharmaceutically acceptable, aqueous or organic solvent solutions or suspensions, 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 composition is administered by oral or nasal respiratory route. Compositions in preferably sterile pharmaceutically acceptable solvents may be atomized using gases. Atomized solutions may be breathed directly from an atomizing device, or the atomizing device may be connected to a mask, tent, or intermittent positive pressure respirator. Solutions, suspensions, or powder compositions may be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

[0431] Composition

[0432] 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 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.

[0433] It should be understood that the composition may 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 of different species that bind to different epitopes of BCMA (e.g., human BCMA) or CD3 and BCMA (human CD3 and BCMA).

[0434] 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 nontoxic to receptors at dosages and concentrations, and may comprise buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl ammonium 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 metacresol); low molecular weight (less about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM Or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0435] Reagent test kit

[0436] The present invention also provides kits for use in the present method. The kits of the present invention include one or more containers containing BCMA antibodies, CD3-BCMA bispecific antibodies or BCMA antibodies as described herein and instructions for use according to any method of the invention described herein. Typically, these instructions contain a description of administering the BCMA antibodies, CD3-BCMA bispecific antibodies or BCMA antibody conjugates for the above-mentioned therapeutic treatments.

[0437] 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 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.

[0438] The kit of the present invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, cans, soft packages (e.g., sealed polyester films or plastic bags), etc. It is also contemplated that the packaging used in conjunction with a specific device, such as an inhaler, a nasal administration device (e.g., a sprayer), or an infusion device such as a micropump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierced by a hypodermic needle). At least one active agent in the composition is a BCMA antibody, a CD3-BCMA bispecific antibody, or a BCMA antibody conjugate. The container may further comprise a second pharmaceutically active agent.

[0439] 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.

[0440] 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

[0441] Example 1: Determination of the kinetics and affinity of hBCMA / human IgG interactions at 25°C and / or 37°C

[0442] This example determines the kinetics and affinity of various anti-BCMA antibodies at 25°C and 37°C.

[0443] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). An array of anti-BCMA antibodies was prepared 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 fixed analysis temperature was 25°C, and the running buffer was HBS-T+ (10mM HEPES, 150mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) direction by injecting a mixture of 1mM ECD and 0.25mM NHS at a flow rate of 30μL / min for 3 minutes. IgG was fixed to the activation point by injecting 20μg / mL in 10mM acetate pH4.5 buffer for 1.5 minutes (30μg / mL) in the ligand (vertical) direction. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 min in the analyte direction.

[0444] The assay temperature for the hBCMA binding assay was 37°C or 25°C in a running buffer of HBS-T+ supplemented with 1 mg / mL BSA. The kinetic titration method was used for the interaction assay 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 (5-member series with a 5-fold dilution factor and a highest concentration of 50 nM). The binding time for a given analyte dilution was 2 minutes. Dissociation was monitored for 2 hours immediately after the 50 nM hBCMA injection. Prior to the hBCMA analyte injection, buffer was injected 5 times in the hBCMA analyte cycle using the same binding and dissociation times to prepare a buffer blank sensorgram for double reference purposes (such as double reference described in Myszka, J. Mol. Recognit. 12, 279-284 (1999)).

[0445] 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.

[0446] Table 6A

[0447]

[0448] Table 6B

[0449]

[0450]

[0451]

[0452]

[0453]

[0454] Table 6C*

[0455]

[0456]

[0457]

[0458] *Binding assays were performed at 37°C.

[0459] Example 2: Flow cytometry of human anti-BCMA antibodies on BCMA-positive tumor cells

[0460] This example demonstrates that various BCMA antibodies of the invention bind to BCMA-positive tumor cells.

[0461] 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 0.5ug antibody in 100uL binding buffer (PBS (phosphate buffered saline) + 0.2% BSA (bovine serum albumin)) and then incubated with Alex Fluor647 conjugated anti-mouse IgG (Biolegend). Table 7 shows the MFI (mean fluorescence intensity) on BCMA-positive tumor cells by various BCMA antibodies (e.g., Combo_Rd4_0.6nM_C29, A02_Rd4_6nM_C01, A02_Rd4_6nM_C16 and P6E01 / H3TAQ).

[0462] Table 7

[0463]

[0464] Example 3: Cytotoxicity of anti-BCMA ADC in BCMA-positive cells

[0465] This example demonstrates the efficacy of anti-BCMA ADC in BCMA-positive cells.

[0466] 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. Respectively, TG17 corresponds to SEQ ID NO: 472 (LLQGPP); LCQ05 corresponds to SEQ ID NO: 474 (GGLLQGPP), H7c corresponds to SEQ ID NO: 454 (LLQG), and is conjugated with AcLys-Val-Cit-PABC-Aur0101 (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Aur3377 or amino-PEG6-C2-Aur0131 as shown in Table 8. In one example, a transglutaminase tag can be engineered in the light chain, the heavy chain, or a combination of the light chain and the heavy chain. In other examples, a transglutaminase tag (e.g., Q) is engineered at a site of the antibody, such as at 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 antibody 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 and an amine-containing derivative of a payload (e.g., MMAD, Aur0101, Aur3377, or Aur0131) 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). In some cases, the wild-type amino acid lysine at position 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 crosslinking between the antibody and the payload, and / or a significant reduction in interchain crosslinking with the glutamine tag on the C-terminus of the antibody light chain.

[0467] 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 acyl acceptor ranging from 5 μM to 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 TM , Ajinomoto, Japan). The reaction conditions were adjusted for individual acyl acceptor derivatives, and the best 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.

[0468] The target expressing (MM1.S, KMS12BM and L363) cells were then seeded at 3000 cells / well on a clear bottom plate. The cells were treated with 4-fold serial dilutions of the antibody-drug conjugate in triplicate. Cell viability was determined by Luminescent Cell Viability Assay 96 (Promega, Madison WI). Relative cell viability was determined as a percentage of the untreated control. EC50 was calculated by Prism software. Table 8 shows that all human anti-BCMA antibodies of the present invention conjugated to cytotoxic agents 0101, 3377 and 0131 via a transglutaminase tag and a linker exerted potent cell killing activity in BCMA expressing cells.

[0469] Table 8

[0470]

[0471]

[0472] Example 4: Anti-BCMA ADC induces tumor regression in an orthotopic multiple myeloma model

[0473] This example demonstrates the in vivo efficacy of anti-BCMA ADC in the MM1S orthotopic multiple myeloma model.

[0474] The in vivo efficacy of BCMA ADC was studied 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 subsequent whole body bioluminescence imaging (BLI) to enable monitoring of tumor burden. The bioluminescent signal emitted by the interaction between luciferase and luciferin expressed by tumor cells was captured by imaging using IVIS SpectrμM CT (Perkin Elmer, MA) and quantified as total flux (photons / sec) using Living Image4.4 (Caliper LifeSciences, Alameda, CA). When the total flux reached an average of 1-3E6 for all animals, the animals were randomized and a single dose of human anti-BCMA antibody and control conjugate conjugated to 1) LCQ05 / K222R-vc0101 at the C-terminus of the antibody light chain was administered by 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).

[0475] This study demonstrated that treatment with BCMA-ADC inhibited the progression of multiple myeloma.

[0476] Example 5: Anti-BCMA ADC induces tumor regression and inhibition in an orthotopic multiple myeloma model

[0477] This example also demonstrates the in vivo efficacy of anti-BCMA ADC in the MM1.S orthotopic multiple myeloma model.

[0478] 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. 7MM1.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 whole body bioluminescence imaging (BLI) was used to monitor tumor burden. The bioluminescent signal emitted by the interaction between the luciferase and luciferin expressed by tumor cells was captured by imaging using IVIS SpectrμM CT (Perkin Elmer, MA) and was quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 1-3E6 for all animals, the animals were randomized into groups; 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur0101, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugates were administered by bolus tail vein injection. Animals were terminated when they exhibited hind limb paralysis, which is the endpoint of the MM1.S orthotopic model. Figure 3 A single dose of human anti-BCMA L3.PY / P6E01 antibody conjugated to 1) H7c / N297A / K222R-amino-PEG6-C2-0131 and 2) H7c / N297A / K222R-amino-PEG6-C2-3377 was shown to result in tumor regression. A single dose of human anti-BCMA L3.PY / P6E01 antibody 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.

[0479] Thus, this study demonstrates that treatment with BCMA-ADC induces regression and inhibits the progression of multiple myeloma.

[0480] Example 6: Anti-BCMA ADC induces tumor suppression in an orthotopic multiple myeloma model

[0481] This example also demonstrates the in vivo efficacy of anti-BCMA ADC in the KMS12BM orthotopic multiple myeloma model.

[0482] In vivo efficacy studies of BCMA ADC 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. D-luciferin (Regis Technologies, Morton Grove, IL) (15 mg / mL, 200 uL / animal) was injected intraperitoneally, and then 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 using IVIS SpectrμM 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 groups: 1) H7c / N297A / K222R-amino-PEG6-C2-3377, 2) N297Q / K222R-AcLys-Val-Cit-PABC-Aur010, 3) LCQ05 / K222R-AcLys-Val-Cit-PABC-Aur0101, 4) H7c / N297A / K222R-amino-PEG6-C2-0131, 5) N297Q / K222R / LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugate LCQ04 / K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMA ADC and control conjugates were 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 4A 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 was shown to result in tumor inhibition.

[0483] Therefore, this study further demonstrates that treatment with BCMA-ADC induces regression and inhibits the progression of multiple myeloma.

[0484] Example 7: Dose response curve of anti-BCMA ADC in MM1S orthotopic model

[0485] This example further illustrates the in vivo efficacy of anti-BCMA ADC in the MM1S orthotopic multiple myeloma model.

[0486] 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. 7MM1.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 whole body bioluminescence imaging (BLI) was used to monitor tumor burden. The bioluminescent signal emitted by the interaction between the luciferase and luciferin expressed by tumor cells was captured by imaging using IVIS SpectrμM CT (Perkin Elmer, MA) and was quantified as total flux (photons / sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, CA). When the total flux reached an average of 1.2E6 for all animals, the animals were randomized into groups: 1) 0.1 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 2) 0.38 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, 3) 0.75 mg / kg H7c / N297A / K222-amino-PEG6-C2-0131, 4) 1.5 mg / kg H7c / N297A / K222R-amino-PEG6-C2-0131, and 5) 3 mg / kg control conjugate N297Q / K222R-AcLys-VC-0101. A single dose of human anti-BCMA ADC and control conjugate were administered by bolus tail vein injection. Animals were terminated when they exhibited 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 the above 1)-4) groups resulted in tumor regression starting at 0.1 mg / kg and tumor suppression for up to 100 days starting at 0.75 mg / kg.

[0487] Thus, this study demonstrates that treatment with BCMA-ADC induces tumor regression and tumor suppression in multiple myeloma.

[0488] Example 8: Production and purification of heterodimeric antibodies

[0489] This example describes the production and purification of heterodimeric antibodies of the invention.

[0490] 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.

[0491] 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 referred to as hIgG1EEE or hIgG2ΔA-EEEE.

[0492] 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 at 37° C. for 24 hrs as described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545). Heterodimers were purified by ion exchange chromatography as described below.

[0493] All heterodimers were purified by ion exchange chromatography. Briefly, analytical ion exchange separation of Fc-hetero and Fc-homodimers was performed on an Agilent 1100 quaternary pump LC system (Agilent Inc, Santa Clara, CA, USA) equipped with a weak cation exchange DIONEX Propac WCX-10G (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 1ml / min in 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. The protein was 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.

[0494] Example 9: Determination of the kinetics and affinity of hCD3 / human IgG interactions at 25°C and / or 37°C

[0495] This example determines the kinetics and affinity of various anti-CD3 antibodies at 25°C and 37°C.

[0496] All experiments were performed on a Bio-Rad Proteon XPR36 surface plasmon resonance biosensor (Bio-Rad, Hercules, CA). Arrays of anti-CD3 antibodies were prepared 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 fixed assay temperature was 25°C, and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The channel was activated in the analyte (horizontal) direction by injecting a mixture of 1 mM ECD and 0.25 mM NHS at a flow rate of 30 μL / min for 3 minutes. IgG was immobilized on the activation spots by injecting 20 μg / mL in 10 mM acetate pH 4.5 buffer for 1.5 minutes (30 μg / mL) in the ligand (vertical) direction. The activated surface was blocked by injecting 1 M ethanolamine, pH 8.5, at 30 μL / min for 3 min in the analyte direction.

[0497] The assay temperature for hCD3 binding assay was 37°C or 25°C in running buffer of HBS-T+ supplemented with 1 mg / mL BSA. The 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 (5-member series with a 5-fold dilution factor and a highest concentration of 50 nM). The binding time for a given analyte dilution was 2 minutes. Dissociation was monitored for 2 hours immediately after the 50 nM hCD3 injection. Prior to the hCD3 analyte injection, buffer was injected 5 times in the hCD3 analyte cycle using the same binding and dissociation times to prepare a buffer blank sensorgram for double referencing purposes (such as double referencing described in Myszka, J. Mol. Recognit. 12, 279-284 (1999)).

[0498] Sensorgrams were doubly referenced and fit 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 for various anti-CD3 antibodies of the invention are shown in Table 9.

[0499] Table 9

[0500]

[0501] Example 10: Flow cytometry of human anti-CD3 bispecific antibodies on B cells and CD8+ T cells

[0502] This example demonstrates the efficacy of an anti-CD3-anti-CD20 bispecific antibody in CD20+ cells.

[0503] Cynomolgus monkey studies were performed at Charles River Laboratories, Preclinical Services Nevada in accordance with the Institutional Animal Care and Use Committee. Animals (n=2) were dosed with bispecific anti-CD20 / h2B4 antibodies at doses of 500ug / kg, 100ug / kg, 20ug / kg, 2ug / kg, 0.2ug / kg, or 0.02ug / kg by 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 peripheral blood vessels not used for iv dosing into K2EDTA tubes.

[0504] The B cells and T cells in peripheral blood were measured by flow cytometry to determine the efficacy. Whole blood was collected at the indicated time points and maintained at 4 ° C until analysis. Red blood cells were lysed at room temperature with ACK buffer (Gibco) for 5 minutes, and leukocytes were precipitated by centrifugation. Cells were stained for 1 hr at 4 ° C with a mixture of antibodies containing fluorescently labeled recognition cyno CD19 (BeckmanCoulter), 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 then Ki67 intracellular staining was performed. Immediately after staining, cells were obtained on a BD LSRII flow cytometer.

[0505] exist Figures 6A-6F The resulting B cell counts were plotted as a percentage of the pre-study B cell counts. Extended B cell depletion after a single dose was achieved with doses as low as 2 ug / kg. B cell depletion was seen at all doses. The duration of the depletion effect was dose-dependent.

[0506] 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.

[0507] Example 11: Flow cytometry of human anti-CD3 bispecific antibodies on CD8+ T cells

[0508] This example demonstrates the efficacy of monovalent anti-CD3 antibodies on T cell kinetics and activation.

[0509] Cynomolgus monkey studies were performed 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 weekly iv doses of 0.2ug / kg. In contrast to the CD20-targeted 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 for T cell activation.

[0510] exist Fig. 8A and 8B The resulting T cell counts were plotted as a percentage of the 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, Ki67+ T cells did not increase.

[0511] Example 12: Flow cytometry of human anti-CD3 bispecific antibodies on B cells

[0512] This example demonstrates the effect of anti-CD3 arm affinity on B cell depletion.

[0513] Cynomolgus monkey studies were performed as described in Example 10 and efficacy was determined by measuring T cells in peripheral blood by flow cytometry. Bispecific antibodies were prepared with an anti-CD20 arm paired with 4 anti-CD3 antibody arms of varying affinities. Efficacy was determined by measuring B cells in peripheral blood by flow cytometry after a single iv dose of 0.2 ug / kg.

[0514] exist Figures 9A-9D The resulting B cell counts were plotted as a percentage of the pre-study B cell counts. The efficacy of B cell depletion correlated with the affinity of the anti-CD3 arm.

[0515] Example 13: In vitro study of bispecific antibodies on T-cell mediated killing of BCMA-positive cells

[0516] This example demonstrates the in vitro cytotoxicity of the anti-BCMA / CD3 hIgG2ΔA bispecific in BCMA-positive cells.

[0517] 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 IgG2dA engineered with EEEE for bispecific exchange as described in Example 8.

[0518] 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 on transparent U-bottom plates at 20,000 and 100,000 cells / well, respectively. 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 by non-radioactive cytotoxicity assay (Promega, Madison WI). Cytotoxicity was determined as the percentage of untreated effector plus target control wells. EC50 was calculated by Prism software. Table 10 shows that all human anti-BCMA_H2B4 bispecific antibodies exerted cell killing activity in BCMA expressing cells.

[0519] Table 10

[0520]

[0521] UND means undetermined; N / A means EC50 could not be determined.

[0522] Example 14: In vitro characterization of anti-CD3 antibodies cloned from mouse hybridomas

[0523] This example demonstrates in vitro T cell activation / proliferation of anti-CD3 cloned from mouse hybridoma in human / cynomolgus PBMC cells for antibody screening.

[0524] 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 a local blood bank by Ficoll (density: 1.083 g / mL, GE) density gradient centrifugation. Red blood cells were removed by incubation for 3 minutes at room temperature in LCK buffer (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA; Gibco). The cells were centrifuged at 600 g for 5 min. The supernatant containing lysed red blood cells was discarded, and the PBMCs were washed twice in 50 ml 1xPBS / 1% BSA / 1 mM EDTA. The precipitated cells were adjusted to 10 in medium X-VIVO-15 (serum-free medium (Lonza)). 7 cells / ml, and PBMCs 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 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 over 2 days of culture. 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.

[0525] Fig. 10A and 10B Selected anti-CD3 1A4, 1C10, 2B4 and 7A3 antibodies were 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 anti-CD3 1C10 and 2B4 antibodies were similar to the positive control SP34 anti-CD3 antibody (BD Biosciences).

[0526] Table 11 Anti-xCD3e ab / bsc_hCD3ed kinetic results from data fitted from 80 nM-0.64 nM

[0527] 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

[0528] Notice:

[0529] Data are reported only for satisfactory kinetic fits. (+) = positive control

[0530] *Kinetic determinations are rough estimates as the antibodies are heterologous. Only steady state affinity is measured.

[0531] Example 15: In vitro study of bispecific antibodies on T cell-mediated killing

[0532] This example demonstrates the in vitro cytotoxicity of the anti-EpCam / CD3 bispecific in SW480 mixed with Pan T cells isolated from healthy donors.

[0533] A: Anti-CD antibodies h2B4-1d, TK, hnpsTK and yaesTK

[0534] 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.

[0535] 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 3ug-3pg / mL were added to the cells in triplicate. The total reaction volume was 200uL. The reactions were incubated for 48 and 72 hours. For cytotoxicity analysis, the cells were analyzed by CytoTox The 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:

[0536] [% 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%]

[0537] Fig.11A and 11B All human anti-EpCam_h2B4 bispecific antibodies were shown to have cell killing activity in an in vitro setting, 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.

[0538] Table 12A

[0539] Day 2: EC50(nM) h2B4-1d_Ep 537.1 h2B4-TK_Ep 405.3 h2B4-hnpsTK_Ep 424.7 h2B4-yaesTK_Ep 1126

[0540] Table 12B Summary of anti-CD3 hIgG2dA bispecific kinetics at 37°C

[0541] 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

[0542] B: Anti-CD antibodies m25A8, h25A8-B12, and h25A8-B13

[0543] Human anti-CD3 h2B4 (h2B4_1d) and h25A8 (m25A8, h25A8-B12 and h25A8-B13) and human anti-EpCam antibodies were expressed as human IgG2dA engineered with RRRR or EEEE for bispecific exchange as described in Example 8.

[0544] Fig. 11C and 11D All human anti-EpCam_anti-CD3 bispecific antibodies were shown to have cell killing activity in an in vitro setting, 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 format 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).

[0545] Table 12C: EC50 of in vitro cell killing

[0546] Day 1: EC50(nM) h2B4-1d_Ep 52.9 m25A8_Ep 127.6 h25A8-B12_Ep 99.36 h25A8-B13_Ep 57.11

[0547] Table 12D: Summary of anti-CD3 hIgG2dA bispecific kinetics at 37°C

[0548] Sample ID - bschIgG2dA ka(1 / Ms) kd(1 / s) t1 / 2(min) KD(nM) h25A8-B5_Ep 1.29E+06 5.81E-02 0.20 45.0 h25A8-B8_Ep 1.19E+06 2.22E-02 0.52 18.7 h25A8-B12_Ep 1.15E+06 2.41E-02 0.48 21.0 h25A8-B13_Ep 1.20E+06 2.19E-02 0.53 18.3 h25A8-C8_Ep 1.20E+06 3.01E-02 0.38 25.1 h2B4-1d_Ep 5.86E+05 2.37E-02 0.49 40.4

[0549] Table 12E: In vitro characterization

[0550]

[0551] Example 16: In vitro T-cell mediated killing of primary myeloma patient samples by bispecific antibodies Research

[0552] This example demonstrates the in vitro cytotoxicity of an anti-BCMA / CD3 bispecific in primary myeloma cells.

[0553] 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 IgG2dA engineered with EEEE or RRRR for bispecific exchange as described in Example 8.

[0554] Total bone marrow mononuclear cells from myeloma patients are seeded in a transparent U-bottom plate with a total bone marrow mononuclear cell count resulting in 3000-5000 myeloma cells / well. Cells are treated with a bispecific antibody with 10-fold serial dilutions. After treatment, 5 days, total viable cells are determined by flow cytometry using antibodies of CD138 and CD38 (Biolegend, CA). Cells are incubated with antibodies at 4 ° in PBS+0.5% FBS for 30 minutes. Cells are washed, and FixableViability Dye eFluor 780 (eBioscience, Inc., CA) in PBS is added to cells at 4 ° for 30 minutes. Before cell collection on BD flow cytometer, 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.

[0555] Table 13A shows that all human anti-BCMA_h2B4 bispecific antibodies have cell killing activity against myeloma patient samples, and 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.

[0556] Table 13A

[0557]

[0558] Table 13B

[0559]

[0560] Example 17: Antibody-Secreting Cells from Cynomolgus Monkeys Administered with Anti-BCMA / CD3 Bispecific Antibodies ELISPOT.

[0561] Thi...

Claims

1. A bispecific antibody comprising a first antibody variable domain capable of recruiting human immune effector cell activity by specifically binding to an effector antigen located on a human immune effector cell, and comprising a second antibody variable domain 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 a 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 SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449 or 450; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335, 406, 423, 429 or 451; and / or 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: The sequence shown in NO:342, 432, 434, 437, 438, 439, 446 or 453.

2. The bispecific antibody of claim 1, 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: 324 or 388; and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 323 or 387; and the second antibody variable domain comprises a heavy chain variable (VH) region comprising VHCDR1, VH CDR2 and VH CDR3 of the VH sequence shown in SEQ ID NO: 112; and / or a light chain variable (VL) region comprising VLCDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO:

38.

3. The bispecific antibody of claim 2, wherein the second antibody variable domain comprises a heavy chain variable (VH) region comprising (i) a VH CDR1 comprising the sequence set forth in SEQ ID NO: 150, 151, 152, 156, 157, 348, 349, 353, 354, or 355; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 203, 204, 350, 351, 356, or 357; and (iii) a VH CDR3 comprising the sequence set forth 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: NO:211, 225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298, 300 or 360.

4. The bispecific antibody of claim 3, wherein said 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 set forth in SEQ ID NO: 331, 332, 333, 401, 407 or 408; (ii) a VH CDR2 comprising the sequence set forth in SEQ ID NO: 336, 417, 404 or 405; and iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 335 or 406; and / or 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; 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 SEQ ID NO: 155; and / or wherein the light chain variable (VL) region comprises (i) a VLCDR1 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.

5. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 4.

6. Use of the bispecific antibody of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating a B-cell-related cancer, wherein the B-cell-related cancer is selected from the group consisting of multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute Myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic 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 lymphoma, Burkitt's lymphoma, primary mediastinal (thymic) large B-cell 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, 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.

7. Use of the bispecific antibody or pharmaceutical composition according to claim 6, wherein the cancer is multiple myeloma.

8. Use of the bispecific antibody or pharmaceutical composition of claim 6 or 7, wherein the antibody or pharmaceutical composition is administered together with one or more other therapeutic agents.

9. Use of the bispecific antibody or pharmaceutical composition according to claim 8, wherein the other therapeutic agent is selected from bortezomib, lenalidomide, carfilzomib and doxorubicin.

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