Compositions and methods for BCMA-directed cellular immunotherapy

Abstract

In some embodiments herein, anti-BCMA binding moieties are provided. These anti-BCMA binding moieties may be used in BCMA-directed chimeric antigen receptors (CARs). Also disclosed herein are immune cell-based compositions comprising anti-BCMA binding moieties and BCMA-directed CARs. In some embodiments, the immune cell-based compositions also target additional tumor markers and / or additional epitopes of BCMA. In some embodiments, the BCMA-directed CARs are expressed in natural killer cells. In some embodiments, a combination of BCMA-CAR-expressing NK cells is administered, for example, with CAR-expressing NK cells and / or CAR-expressing T cells against additional cancer markers and / or additional epitopes of BCMA. Also provided herein are methods and uses of chimeric antigen receptors in immunotherapy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 220,842, filed July 12, 2021, the entire contents of which are incorporated herein by reference. Incorporation by reference of material in an ASCII text file

[0002] The Sequence Listing contained in the following ASCII text file has been submitted contemporaneously herewith, has the filename NKT074PR_ST26.xml, was created on July 8, 2022, and is 13,071,224 bytes in size. This Sequence Listing in electronic form is hereby expressly incorporated by reference in its entirety.

[0003] Field Some embodiments of the methods and compositions provided herein relate to cell therapy utilizing B-cell maturation antigen (BCMA)-targeted chimeric antigen receptors (CARs). Some embodiments relate to one or more of such constructs expressed by NK and / or T cells. Antigen binding molecules that bind to BCMA are also disclosed herein. [Background technology]

[0004] As more knowledge is gained about various cancers and the characteristics of cancer cells that specifically distinguish them from healthy cells, therapeutic agents are being developed that exploit the distinct features of cancer cells. Immunotherapy, which utilizes engineered immune cells, is one approach to treat cancer. Summary of the Invention [Means for solving the problem]

[0005] Immunotherapy is a new innovation in the treatment of disease, where immune cells are engineered to express specific targeting and / or effector molecules that specifically identify and react to diseased and damaged cells.This represents a promising advancement, at least in part, due to the possibility of specifically targeting diseased and damaged cells, in contrast to traditional approaches such as chemotherapy, which affect all cells and the desired outcome is that enough healthy cells survive to reduce side effects in patients.One immunotherapy approach is the recombinant expression of chimeric antigen receptors (CARs) in immune cells to achieve targeted recognition and destruction of the desired abnormal cells, such as cancer.

[0006] In certain cancers, patient response to immunotherapy is initially robust and favorable, but temporary. Such a profile is addressed by some embodiments of the cellular immunotherapy compositions provided herein. For example, in some embodiments, natural killer (NK) cells are engineered to express one or more chimeric antigen receptors (CARs). Due to the enhanced cytotoxicity of engineered NK cells combined with the innate rapid immune response of NK cells, some embodiments allow for an enhanced early anti-cancer effect that may substantially reduce or even eliminate tumor burden. In some embodiments, such engineered NK cells are particularly important, at least in part, due to their reduced immunogenicity compared to T cells, because aggressive cancers may not allow enough time for autologous T cell therapy to be generated.

[0007] The anti-BCMA binding portion comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 114, 105, 107, 129, 104, 106, 108-113 or 115-128, and HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 140, 131, 133, 155, 130, 132, 134-139 or comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 141 to 154, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 166, 157, 159, 181, 156, 158, 160 to 165 or 167 to 180. In some embodiments, the anti-BCMA (BMCA) binding moiety further comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 395, 386, 388, 410, 385, 387, 389-392 or 396-409, and LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 421, 412, 414, 436, 411, 41 and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 447, 438, 440, 462, 437, 439, 441-446 or 448-446. In some embodiments, provided herein is a BCMA-directed chimeric antigen receptor (CAR) comprising an anti-BCMA binding moiety.Also provided herein is a population of immune cells engineered to express an anti-BCMA binding moiety, which may be in the form of a BCMA-directed CAR. In some embodiments, provided herein is a method of treating cancer, such as multiple myeloma, comprising administering to a subject a population of immune cells (e.g., natural killer (NK) cells and / or T cells) engineered to express a BCMA-directed CAR. Also provided herein in some embodiments is the use of a BCMA-directed CAR in the manufacture of a medicament and / or for the treatment of cancer.

[0008] In some embodiments HCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:114, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:140, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:166.

[0009] In some embodiments, HCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 105, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 131, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 157.

[0010] In some embodiments, HCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 107, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 133, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 159.

[0011] In some embodiments, HCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 129, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 155, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 181.

[0012] In some embodiments LCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:395, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:421, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:447.

[0013] In some embodiments, LCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:386, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:412, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:438.

[0014] In some embodiments, LCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:388, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:414, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:440.

[0015] In some embodiments, LCDR1 of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:410, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:436, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:462.

[0016] In some embodiments, the VH of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 270, 261, 263, 285, 260, 262, 263-269 or 271-284. In some embodiments, the VL of the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 551, 542, 544, 566, 541, 543, 545-550 or 552-565.

[0017] In some embodiments HCDR1 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 192, 183, 185, 207, 182, 184, 186-191 or 192-206 and HCDR2 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 218, 209, 211, 233, 208, 210, 212-217 or 219-232. and the HCDR3 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 244, 235, 237, 259, 234, 236, 238-243 or 245-258.

[0018] In some embodiments LCDR1 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 473, 464, 466, 488, 463, 465, 467-472 or 474-487, and LCDR2 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 499, 490, 492, 514, 489, 491, 493-498 or 500-513. and the LCDR3 of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 525, 516, 518, 540, 515, 517, 519-524 or 526-539.

[0019] In some embodiments, the VH of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 296, 287, 289, 311, 286, 288, 290-295 or 297-310. In some embodiments, the VL of the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 577, 568, 570, 592, 567, 569, 571-576 or 578-591.

[0020] In some embodiments, the VH and VL (if present) are separated by a linker. In some embodiments, the linker of the anti-BCMA binding moiety comprises a sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1388. In some embodiments, the VH is N-terminal to the VL. In some embodiments, the anti-BCMA binding moiety comprises a sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 603, 594, 596, 618, 593, 595, 597-602 or 604-617. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 629, 620, 622, 644, 619, 621, 623-628, 630-643 or 645-670.

[0021] In some embodiments the VL (if present) is N-terminal to the VH. In some embodiments the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 681, 672, 674, 696, 671, 673, 675-680 or 682-695. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 707, 698, 700, 722, 697, 699, 701-706, 708-721 or 723-748.

[0022] In some embodiments the linker of the anti-BCMA binding moiety comprises a sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2260. In some embodiments the anti-BCMA binding moiety comprises a sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1483, 1474, 1476, 1498, 1473, 1475, 1477-1482 or 1484-1497. In some embodiments the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1509, 1500, 1502, 1524, 1499, 1501, 1503-1508 or 1510-1523.

[0023] Also provided herein is a BCMA-directed chimeric antigen receptor (CAR) comprising the anti-BCMA binding moiety described herein.Also provided herein is an immune cell comprising the anti-BCMA binding moiety described herein.In some embodiments, the immune cell is a natural killer (NK) cell or a T cell.

[0024] In some embodiments, the CAR further comprises an intracellular signaling domain comprising a hinge domain; a transmembrane domain; and a CD3ζ subdomain. In some embodiments, the intracellular signaling domain of the BCMA-directed CAR further comprises an OX40 subdomain. In some embodiments, the OX40 subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1394. In some embodiments, the CD3ζ subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1395. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1392. In some embodiments, the hinge domain is a CD8 hinge domain. In some embodiments, the CD8 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1389. In some embodiments, the hinge, transmembrane, and / or intracellular domains from CD28 may be used in place of one or more of the CD8 domains.

[0025] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3897, 3888, 3890, 3912, 3887, 3889, 3891-3896 or 3898-3911. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3923, 3914, 3916, 3938, 3913, 3915, 3917-3922, 3924-3937 or 3939-3964. In some embodiments, the nucleic acid encoding the BCMA-directed CAR further comprises a sequence encoding membrane-bound interleukin 15 (mbIL15) and having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4001, 3992, 3994, 4016, 3991, 3993, 3995-4000, 4002-4015 or 4017-4042.

[0026] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4053, 4044, 4046, 4068, 4043, 4045, 4047-4052 or 4054-4067. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4079, 4070, 4072, 4094, 4069, 4071, 4073-4078, 4080-4093 or 4095-4120. In some embodiments, the nucleic acid encoding a BCMA-directed CAR further comprises a sequence encoding membrane-bound interleukin 15 (mbIL15) and having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4157, 4148, 4150, 4172, 4147, 4149, 4151-4156, 4158-4171 or 4173-4198.

[0027] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3869, 3867, 3868 or 3870. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3885, 3883, 3884 or 3886. In some embodiments, the nucleic acid encoding the BCMA-directed CAR further comprises a sequence encoding membrane-bound interleukin 15 (mbIL15) and having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3837, 3835, 3836 or 3839.

[0028] Also provided herein is a VHH-BCMA-directed chimeric antigen receptor (CAR) comprising an extracellular anti-BCMA binding portion; a hinge domain; a transmembrane domain; and an intracellular signaling domain, wherein the anti-BCMA binding portion comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 is a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 104-129, 1525-1543 or 3117-3139. wherein HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 130 to 155, 1544 to 1562 or 3140 to 3162, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 156 to 181, 1563 to 1581 or 3163 to 3185.

[0029] In some embodiments the anti-BCMA binding moiety further comprises an additional VH comprising an additional HCDR1, HCDR2 and HCDR3, wherein the additional HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3117-3139 and the additional HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3140-3162. and the additional HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3163 to 3185. In some embodiments, other sequences are used for the additional HCDR1, HCDR2 and / or HCDR3.

[0030] In some embodiments, the intracellular signaling domain comprises a costimulatory subdomain and a CD3 zeta subdomain.

[0031] Also provided herein is a population of engineered immune cells engineered to express a BCMA-directed chimeric antigen receptor (CAR) comprising an extracellular anti-BCMA binding portion comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3; and a hinge domain; a transmembrane domain; and an intracellular signaling domain, wherein HCDR1 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 104-129, 1525-1543 or 3117-3139. HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 130 to 155, 1544 to 1562 or 3140 to 3162, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 156 to 181, 1563 to 1581 or 3163 to 3185. In some embodiments, the CAR expressed by the immune cell further comprises an additional VH comprising an additional HCDR1, HCDR2 and HCDR3, wherein the additional HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3117-3139, and the additional HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3140-3143. 162, and the additional HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3163 to 3185. Depending on the embodiment, other sequences may be used for the additional HCDR1, HCDR2 and / or HCDR3.In some embodiments, HCDR1 is a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-207 or 3186-3208. HCDR2 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 208 to 233 or 3209 to 3231, and HCDR3 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 234 to 259 or 3232 to 3254.

[0032] In some embodiments, the CAR further comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3117-3139, and LCDR2 comprises any one of SEQ ID NOs: 3140-3162. and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3163-3185.

[0033] Also provided herein is a population of immune cells engineered to express a BCMA-directed chimeric antigen receptor (CAR) comprising an anti-BCMA binding portion comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, wherein HCDR1 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 110%, 109%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 140, 131, 133, 155, 130, 132, 134 to 139 or 141 to 154, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 166, 157, 159, 181, 156, 158, 160 to 165 or 167 to 180. LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 395, 386, 388, 410, 385, 387, 389-392 or 396-409; LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 421, 412, 414, 436, 411, 4 13, 415-420 or 422-435, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 447, 438, 440, 462, 437, 439, 441-446 or 448-446.

[0034] In some embodiments, provided herein is a method of treating cancer, the method comprising administering to a subject in need of treatment a population of immune cells comprising a BCMA-directed chimeric antigen receptor (CAR) comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, and an intracellular signaling domain comprising a hinge domain, a transmembrane domain and a CD3ζ subdomain, wherein HCDR1 is selected from the group consisting of SEQ ID NOs: 114, 105, 107, 129, 10 ...5, 107, 129, 104, 105, 105, 105, 105, 105, 106, 107, 108, 109, 110, 111, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 140, 131, 133, 155, 130, 132, 134-139 or 141-154; and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 166, 108-113 or 115-128. , 157, 159, 181, 156, 158, 160-165, or 167-180, and LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 395, 386, 388, 410, 385, 387, 389-392, or 396-409. and LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 421, 412, 414, 436, 411, 413, 415-420 or 422-435; and LCDR3 comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 447, 438, 440, 462, 437, 439, 441-446 or 448-446.In some embodiments, the VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 270, 261, 263, 285, 260, 262, 263-269 or 271-284, and the VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 551, 542, 544, 566, 541, 543, 545-550 or 552-565. In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3897, 3888, 3890, 3912, 3887, 3889, 3891-3896 or 3898-3911. In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4053, 4044, 4046, 4068, 4043, 4045, 4047-4052 or 4054-4067. In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3869, 3867, 3868 or 3870. In some embodiments, the method (and use) of the population of immune cells is for the treatment of cancer, such as multiple myeloma.

[0035] Disclosed herein are anti-BCMA binding moieties comprising a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3. In some embodiments of the anti-BCMA binding moiety, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 104-129, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 130-155, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 156-181. In some embodiments of the anti-BCMA binding moiety, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 385-410, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 411-436, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 437-462. In some embodiments, the VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 260-285. In some embodiments, the VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 541-566.

[0036] Also disclosed herein is a BCMA-directed chimeric antigen receptor comprising an extracellular anti-BCMA binding portion, a hinge domain, a transmembrane domain, and an intracellular signaling domain comprising an OX40 subdomain and a CD3 zeta subdomain. In some embodiments, the anti-BCMA binding portion is any one of the anti-BCMA binding portions disclosed herein. In some embodiments, the OX40 subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1394. In some embodiments, the CD3 zeta subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1395. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the hinge domain is a CD8 hinge domain, an IgG4 hinge domain, or an RQRCD8 hinge domain.

[0037] Also disclosed herein is a BCMA-directed CAR construct comprising a BCMA-directed CAR and membrane-bound IL15 (mbIL15). In some embodiments, the BCMA-directed CAR is any one of the BCMA-directed CARs disclosed herein. In some embodiments, the mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1398.

[0038] Also disclosed herein is an immune cell or population of immune cells comprising any one of the anti-BCMA binding moieties, BCMA-directed CARs, or BCMA-directed CAR constructs disclosed herein. In some embodiments, the immune cell is a NK cell and / or a T cell.

[0039] Also disclosed herein is a method for treating cancer in a subject in need of treatment. In some embodiments, the method comprises administering to a subject any one of the anti-BCMA binding moieties, BCMA-directed CARs, BCMA-directed CAR constructs or engineered immune cells disclosed herein.Also disclosed herein is any one of the anti-BCMA binding moieties, BCMA-directed CARs, BCMA-directed CAR constructs or engineered immune cells disclosed herein for use in treating cancer.Also disclosed herein is any one of the anti-BCMA binding moieties, BCMA-directed CARs, BCMA-directed CAR constructs or engineered immune cells disclosed herein for use in the manufacture of medicaments. [Brief description of the drawings]

[0040] [Figure 1] 1A-1D show a collection of non-limiting embodiments of CAR constructs used, for example, to target BCMA. The shown constructs include a membrane-bound IL15 construct that is separated from the CAR itself by a self-cleaving peptide (e.g., T2A). However, CARs without the IL15 construct (and the separating self-cleaving peptide) are also provided herein. "CD8SP": CD8 signal peptide; "GS linker": linker including glycine and serine; "VH": heavy chain variable region; "VL": light chain variable region; "CD8TM": CD8 transmembrane domain; "CD8IC": CD8 intracellular domain; "OX40": OX40 intracellular domain; "CD3ζ": CD3ζ domain. FIG. 1A shows a VH-VL format. FIG. 1B shows a VL-VH format. FIG. 1C shows a VH-VL format with a shorter hinge (IgG4). Figure ID shows a VH-VL format with a longer hinge (RQRCD8). These represent nucleotide sequences, and it is understood that the encoded CAR construct does not include T2A and mbIL15 when encoded, and is expressed separately on cells.

[0041] [Diagram 2]Figure 2 shows non-limiting combinations of heavy chain variable region (VH) complementarity determining region (CDR) 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) sequences. Embodiments of anti-BCMA binding moieties, BCMA-directed CARs and cells expressing them may use any of the non-limiting combinations shown herein.

[0042] [Diagram 3] Figure 3 shows non-limiting combinations of light chain variable region (VL) CDR1, 2 and 3 (LCDR1, LCDR2 and LCDR3) sequences. Embodiments of anti-BCMA binding moieties, BCMA-directed CARs and cells expressing them may use any of the non-limiting combinations shown herein.

[0043] [Figure 4] Figure 4 shows non-limiting combinations of VH and VL sequences. Embodiments of the anti-BCMA binding moieties, BCMA-directed CARs and cells expressing them may use any of the non-limiting combinations shown herein.

[0044] [Diagram 5] Figures 5A-5D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 5A shows a non-limiting schematic diagram of a VH-GS linker-VL scFv with a CD8α hinge, a CD8α transmembrane domain, a CD8α intracellular domain, an OX-40 costimulatory domain, and a CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 5B shows the encoded amino acids. Figures 5C and 5D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0045] [Figure 6]Figures 6A-6D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 6A shows a non-limiting schematic diagram of a VH-Whitlow linker-VL scFv, AAA spacer with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, OX-40 costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 6B shows the encoded amino acids. Figures 6C and 6D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0046] [Figure 7] Figures 7A-7D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 7A shows a non-limiting schematic diagram of VL-Whitlow linker-VH scFv, AAA spacer with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, OX-40 costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 7B shows the encoded amino acids. Figures 7C and 7D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0047] [Figure 8]Figures 8A-8D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example to target BCMA. Figure 8A shows a non-limiting schematic diagram of a camelid VHH domain with a CD8α hinge, a CD8α transmembrane domain, a CD8α intracellular domain, an OX-40 costimulatory domain, and a CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 8B shows the encoded amino acids. Figures 8C and 8D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0048] [Figure 9] Figures 9A-9D show schematics of various non-limiting embodiments of CAR constructs provided herein, for example to target BCMA. Figure 9A shows a non-limiting schematic of camelid VHH domain-GS linker-VHH domain with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, OX-40 costimulatory domain and CD3ζ signaling domain. The VHH domains may be the same or different domains. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 9B shows the encoded amino acids. Figures 9C and 9D show corresponding schematics of constructs without the FLAG tag-linker complex.

[0049] [Figure 10]Figures 10A-10D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 10A shows a non-limiting schematic diagram of VL-Whitlow linker-VH scFv, AAA spacer with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, OX-40 costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 10B shows the encoded amino acids. Figures 10C and 10D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0050] [Figure 11] Figures 11A-11D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 11A shows a non-limiting schematic diagram of VL-Whitlow-VH, AAA spacer with CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 11B shows the encoded amino acids. Figures 11C and 11D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0051] [Figure 12]Figures 12A-12D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 12A shows a non-limiting schematic diagram of a VH-GS linker VL scFv, AAA spacer, with a CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain, and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 12B shows the encoded amino acids. Figures 12C and 12D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0052] [Figure 13] Figures 13A-13D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 13A shows a non-limiting schematic diagram of a VH-GS linker-VL scFv, AAA spacer, with a CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 13B shows the encoded amino acids. Figures 13C and 13D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0053] [Figure 14]Figures 14A-14D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 14A shows a non-limiting schematic diagram of VL-Whitlow-VH scFv, AAA spacer, with CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 14B shows the encoded amino acids. Figures 14C and 14D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0054] [Figure 15] Figures 15A-15D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 15A shows a non-limiting schematic diagram of VL-Whitlow-VH scFv, AAA spacer, with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 15B shows the encoded amino acids. Figures 15C and 15D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0055] [Figure 16]Figures 16A-16D show schematic diagrams of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 16A shows a non-limiting schematic diagram of a VH-GS linker-VL scFv, AAA spacer, with a CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 16B shows the encoded amino acids. Figures 16C and 16D show corresponding schematic diagrams of constructs without the FLAG tag-linker complex.

[0056] [Figure 17] Figures 17A-17D show schematics of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 17A shows a non-limiting schematic of a VL-Whitlow linker-VH scFv, (AAA)2 spacer with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Also included in the schematics, although not used in all embodiments provided herein, is a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 17B shows the encoded amino acids. Figures 17C and 17D show corresponding schematics of constructs without the FLAG tag-linker complex.

[0057] [Figure 18]Figures 18A-18D show schematics of various non-limiting embodiments of CAR constructs provided herein, for example, for targeting BCMA. Figure 18A shows a non-limiting schematic of a multi-domain camelid BCMA binder of VHH-GS linker-VHH structure, AAA spacer, with CD8α hinge, CD8α transmembrane domain, CD8α intracellular domain, 4-1BB costimulatory domain and CD3ζ signaling domain. Although not used in all embodiments provided herein, the schematic also includes a T2A self-cleaving peptide followed by additional nucleotides encoding mbIL15. Figure 18B shows the encoded amino acids. Figures 18C and 18D show corresponding schematics of constructs without the FLAG tag-linker complex.

[0058] [Figure 19] FIG. 19 shows dot plots depicting expression in Jurkat cells of various non-limiting examples of CAR formats.

[0059] [Figure 20] Figure 20 shows data related to the function of non-limiting embodiments of BCMA-directed CARs. The figure shows data related to the assessment of BCMA-binding induced activation (X-axis) versus tonic activation (Y-axis) when target MM.1S cells are co-cultured with Jurkat cells engineered to express a BCMA-directed CAR provided herein. The effector to target (E:T) cell ratio used was 1:1 (Jurkat:MM.1S).

[0060] [Figure 21] FIG. 21 shows data relating to the ratio of BCMA-induced activation to tonic activation of various non-limiting embodiments of the CARs provided herein.

[0061] [Figure 22]Figure 22 shows additional data relating to expression of various clones of CAR (left panel), where the sequence of each individual clone differs but shares a common CAR structure. The right panel of Figure 22 shows BCMA binding of each clone.

[0062] [Figure 23] Figures 23A-23B show scatter plots of CAR expression versus activity. Figure 23A shows a scatter plot of CAR expression versus BCMA binding-induced CAR activation. Figure 23B shows a scatter plot of CAR expression versus tonic signaling.

[0063] [Figure 24] FIG. 24 shows a scatter plot of CAR expression versus BCMA binding.

[0064] [Diagram 25] FIG. 25 shows a scatter plot of the activation / tonic signaling ratios of various CAR constructs provided herein.

[0065] [Figure 26-1]Figures 26A-26F show data related to the function of the CARs provided herein based on the linker, which is one aspect of their structure. Figure 26A shows data related to the activation / tonic signaling ratio of a CAR using a GS linker versus a CAR using a Whitlow linker. Figure 26B shows expression data of a CAR using a GS linker versus a CAR using a Whitlow linker. Figure 26C shows data related to the activation / tonic signaling ratio of a CAR provided herein using a scFv utilizing a VL-VH format versus a CAR using a scFv utilizing a VH-VL format. Figure 26D shows data related to the expression of a CAR provided herein using a scFv utilizing a VL-VH format versus a CAR using a scFv utilizing a VH-VL format. Figure 26E shows data related to the activation / tonic signaling ratio of a CAR using a CD8 domain with an OX-40 costimulatory domain versus a CAR provided herein using a CD28 (hinge, transmembrane and intracellular) domain. [Figure 26-2] Same as above [Figure 26-3] Same as above

[0066] [Figure 27] FIG. 27 shows summary data relating to expression and BCMA binding function of monovalent or bivalent CARs using one or more camelid VHH domains.

[0067] [Figure 28] Figures 28A-28B show data relating to expression and BCMA binding function of monovalent and bivalent CARs using one or more camelid VHH domains. Figure 28A shows expression data of monovalent CARs versus bivalent CARs using one or more camelid VHHs (Y-axis). Figure 28B shows BCMA binding data of monovalent VHH-CARs versus bivalent VHH-CARs (Y-axis).

[0068] [Figure 29-1]Figures 29A-29D show the evaluation of various characteristics of cells expressing CARs utilizing one or more VHH domains. Figure 29A shows a scatter plot of the tonic signaling activity of monovalent CARs (X-axis) versus bivalent CARs (Y-axis) comprising one or more VHHs. Figure 29B shows a scatter plot of the activation / tonic signaling ratio of monovalent VHH-CARs (X-axis) versus bivalent VHH-CARs (Y-axis). Figure 29C is a histogram showing the activation / tonic signaling ratio of selected bivalent VHH-CARs (black bars) and monovalent VHH-CARs (white bars). Figure 29D shows data related to the tonic signaling detected from selected bivalent VHH-CARs (black bars) and monovalent VHH-CARs (white bars). [Figure 29-2] Same as above

[0069] [Diagram 30] Figures 30A-30B show data comparing different CAR constructs: Figure 30A shows a scatter plot of expression versus tonic signaling for VHH-CAR (triangles) versus CAR using scFv (circles) and corresponding controls (scFv squares, VHH Xs). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description Some embodiments of the methods and compositions provided herein relate to anti-BCMA binding moieties. Also disclosed herein are BCMA-directed chimeric antigen receptors (CARs) comprising any of the anti-BCMA binding moieties disclosed herein. In some embodiments, the CARs are expressed on cells described herein. Some embodiments include the use of the methods or compositions or cells in immunotherapy. Some embodiments relate to the use of anti-BCMA CARs expressed on natural killer (NK) cells.

[0071] The term "anti-cancer effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" may also be manifested by the ability of the CAR in preventing the development of cancer in the first place.

[0072] cell type Some embodiments of the methods and compositions provided herein relate to cells, such as immune cells. For example, immune cells may be engineered to contain a chimeric antigen receptor, such as a BCMA-directed CAR, or may be engineered to contain a nucleic acid encoding said CAR, as described herein.

[0073] Conventional anti-cancer therapies rely on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. As research has led to a deeper understanding of some of the mechanisms of specific cancers, that understanding has been exploited to develop targeted cancer therapies. Targeted therapy is a cancer treatment that utilizes specific drugs to target specific genes or proteins found in cancer cells or cells that support cancer growth (e.g., blood vessel cells) to reduce or prevent cancer cell growth. More recently, genetic engineering has allowed approaches to be developed that utilize specific aspects of the immune system to fight cancer. In some cases, the patient's own immune cells are modified to specifically eradicate the patient's type of cancer. As described in more detail herein, various types of immune cells can be used, such as T cells and / or natural killer (NK) cells.

[0074] Provided herein are polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs) comprising a target binding moiety (e.g., an extracellular binder of a ligand expressed by a cancer cell, e.g., a BCMA-directed chimeric antigen receptor) and a cytotoxic signaling complex to facilitate cancer immunotherapy. Some embodiments include polynucleotides, polypeptides, or vectors encoding BCMA-directed chimeric antigen receptors to facilitate targeting of immune cells to cancer and exerting a cytotoxic effect on cancer cells. Engineered immune cells (e.g., T cells and / or NK cells) expressing such CARs are also provided. In some embodiments herein, polynucleotides, polypeptides, and vectors encoding constructs comprising an extracellular domain comprising two or more subdomains and a cytotoxic signaling complex are also provided. Engineered immune cells (e.g., T cells and / or NK cells) expressing such bispecific constructs are also provided. Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided herein.

[0075] Engineered Cells for Immunotherapy In some embodiments, cells of the immune system are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells. For example, cells of the immune system can be engineered to contain the BCMA-directed chimeric antigen receptors described herein. In some embodiments, white blood cells or leukocytes are used because their innate function is to defend the body against abnormal cell proliferation and infections. There are various types of white blood cells that play specific roles in the human immune system, so they are the preferred starting point for engineering the cells disclosed herein. White blood cells include granulocytes and agranulocytes (the presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils and mast cells. Agranulocytes include lymphocytes and monocytes. Cells as described below or otherwise herein can be engineered to contain chimeric antigen receptors, such as BCMA-directed chimeric antigen receptors, or nucleic acids encoding such chimeric antigen receptors, and / or can be engineered to co-express the membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0076] Monocytes for immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are associated with the adaptive immune system and perform the primary functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of engulfment of cellular material or whole cells followed by digestion and destruction of the phagocytosed cellular material. In some embodiments, monocytes are used in conjunction with one or more additional engineered cells disclosed herein. Some embodiments of the methods and compositions described herein relate to monocytes that comprise a BCMA-directed chimeric antigen receptor or a nucleic acid encoding the BCMA-directed chimeric antigen receptor. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express a BCMA-directed chimeric antigen receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0077] Lymphocytes for immunotherapy Lymphocytes, the other primary subtype of white blood cells, include T cells (cellular cytotoxic adaptive immunity), natural killer cells (cellular cytotoxic innate immunity) and B cells (humoral antibody-driven adaptive immunity). While some embodiments disclosed herein engineer B cells, some embodiments also relate to engineered T cells or engineered NK cells (some embodiments use a mixture of T cells and NK cells). Some embodiments of the methods and compositions described herein relate to lymphocytes that comprise a BCMA-directed chimeric antigen receptor or a nucleic acid encoding the BCMA-directed chimeric antigen receptor. Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express a BCMA-directed chimeric antigen receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0078] T Cells for Immunotherapy T cells are distinguishable from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on the cell surface. T cells can be divided into a variety of different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. In some embodiments, a specific subtype of T cells is engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. In some embodiments, there is no specific selection of the type of T cells to be engineered to express the cytotoxicity receptor complexes disclosed herein. In some embodiments, certain techniques, such as the use of cytokine stimulation, are used to improve the expansion / capture of T cells with specific marker profiles. For example, in some embodiments, activation of certain human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by the use of CD3 and / or CD28 as stimulatory molecules. In some embodiments, methods of treating or preventing cancer or infectious diseases are provided, comprising administering a therapeutically effective amount of T cells expressing a cytotoxicity receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. Some embodiments of the methods and compositions described herein relate to T cells comprising a BCMA-directed chimeric antigen receptor or a nucleic acid encoding the BCMA-directed chimeric antigen receptor. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a BCMA-directed chimeric antigen receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0079] NK Cells for Immunotherapy In some embodiments, methods of treating or preventing cancer or infectious diseases are provided, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, NK cells are preferred because the innate cytotoxicity of NK cells is relatively high. In some embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, resulting in more effective activity against target cells (e.g., tumor or other disease cells). In some embodiments, the high acute cytotoxicity of NK cells (further enhanced by the techniques disclosed herein) is exploited to provide a cell therapy composition that is particularly effective. Some embodiments of the methods and compositions described herein relate to NKs that comprise a BCMA-directed chimeric antigen receptor or a nucleic acid encoding the BCMA-directed chimeric antigen receptor. Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express a BCMA-directed chimeric antigen receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells and lack the major inhibitory receptors presented by normal NK cells, but retain most of the activating receptors. Some embodiments of the NK-92 cells described herein relate to NK-92 cells engineered to silence additional specific inhibitory receptors, such as SMAD3, allowing for upregulation of interferon-gamma (IFNγ), granzyme B, and / or perforin production. Additional information regarding NK-92 cell lines is disclosed in International Publication No. WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entireties. In some embodiments, NK-92 cells are used in combination with one or more of the other cell types disclosed herein.For example, in one embodiment, NK-92 cells are used in combination with the NK cells disclosed herein. In a further embodiment, NK-92 cells are used in combination with the T cells disclosed herein.

[0080] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxicity receptor complex. In some embodiments, HSCs are used to exploit their ability to engraft long-term blood cell production that can provide a sustained source of targeted anti-cancer effector cells, for example, to combat cancer remission. In some embodiments, this ongoing production helps to offset immune unresponsiveness or exhaustion of other cell types, for example, due to the tumor microenvironment. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in conjunction with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein relate to stem cells, e.g., hematopoietic stem cells, that comprise a BCMA-directed chimeric antigen receptor or a nucleic acid encoding the BCMA-directed chimeric antigen receptor. Some embodiments of the methods and compositions disclosed herein relate to stem cells, e.g., hematopoietic stem cells, engineered to express a BCMA-directed chimeric antigen receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0081] Induced pluripotent stem cells for cancer immunotherapy In some embodiments, induced pluripotent stem cells (iPSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, iPSCs are used to exploit their ability to differentiate and induce into non-pluripotent cells, including but not limited to CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells and B cells, or less differentiated cells containing one or several genetic modifications at the selected site, in some embodiments. In some embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In some embodiments, iPSCs are used in conjunction with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein relate to stem cells, e.g., induced pluripotent stem cells, engineered to express CARs targeting tumor markers, e.g., CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, among others, and optionally, membrane-bound interleukin 15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express activating chimeric antibodies targeting ligands on tumors, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally, membrane-bound interleukin 15 (mbIL15) costimulatory domains.

[0082] Genetic engineering of immune cells As discussed above, various cell types can be utilized in cellular immunotherapy. Moreover, as described in more detail below and shown in the Examples, the cells can be genetically modified to improve one or more aspects of their efficacy (e.g., cytotoxicity) and / or survival (e.g., active life span). As discussed herein, in some embodiments, NK cells are used for immunotherapy. In some embodiments provided herein, gene editing of NK cells can advantageously provide the edited NK cells with the ability to resist and / or overcome various inhibitory signals generated in the tumor microenvironment. Tumors are known to produce various signaling molecules intended to reduce the anti-cancer effect of immune cells. As discussed in more detail below, in some embodiments, gene editing of NK cells limits the inhibitory effect of the tumor microenvironment on the NK cells, T cells, combination of NK cells and T cells, or any of the edited / engineered immune cells provided herein. As discussed below, in some embodiments, gene editing is utilized to reduce or knock out the expression of a target protein, for example, by disruption of the underlying gene that codes for that protein. In some embodiments, gene editing can reduce the expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or more (including any amount between the recited amounts). In some embodiments, the gene is completely knocked out so that the expression of the target protein is undetectable. In some embodiments, gene editing is used to "knock in" or otherwise enhance the expression of the target protein. In some embodiments, the expression of the target protein can be enhanced by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or more (including any amount between the recited amounts).

[0083] As a non-limiting example, TGF-beta is such a cytokine released by tumor cells, resulting in immune suppression within the tumor microenvironment. The immune suppression reduces the ability of immune cells, even engineered CAR immune cells, to destroy tumor cells, thus allowing tumor progression. In some embodiments, immune checkpoint inhibitors are disrupted by gene editing, as discussed in detail below. In some embodiments, blockers of immune suppressive cytokines in the tumor microenvironment are used, including blockers of their release, or competitive inhibitors that reduce the ability of signaling molecules to bind and inhibit immune cells. Such signaling molecules include, but are not limited to, TGF-beta, IL10, arginase, inducible NOS, reactive NOS, Arg1, indoleamine 2,3-dioxygenase (IDO), and PGE2. However, in further embodiments, immune cells, such as NK cells, are provided in which the ability of NK cells (or other cells) to respond to certain immune suppressive signaling molecules has been disrupted and / or ablated. For example, in some embodiments, NK cells or T cells are gene edited to have reduced sensitivity to TGF-beta. TGF-beta is an inhibitor of NK cell function, at least in terms of proliferation and cytotoxicity. Thus, according to some embodiments, the expression of TGF-beta receptor is knocked down or knocked out by gene editing, so that the edited NK is resistant to the immunosuppressive effect of TGF-beta in the tumor microenvironment. In some embodiments, the TGFB2 receptor is knocked down or knocked out via gene editing, for example, by using CRISPR-Cas editing. In other embodiments, small interfering RNA, antisense RNA, TALEN, or zinc finger is used. In some embodiments, other isoforms of TGF-beta receptor (e.g., TGF-beta 1 and / or TGF-beta 3) are edited. In some embodiments, the TGF-beta receptor in T cells is knocked down via gene editing.

[0084] According to further embodiments, other regulators of one or more aspects of NK cell (or T cell) function are regulated via gene editing. Various cytokines provide immune cells with negative (similar to TGF-beta above) or positive signals. As a non-limiting example, IL15 is a positive regulator of NK cells and may enhance one or more of NK cell homing, NK cell migration, NK cell expansion / proliferation, NK cell cytotoxicity, and / or NK cell survival, as disclosed herein. In normal physiological situations, cytokine-inducible SH2-containing protein (CIS, encoded by CISH gene) acts as a critical negative regulator of IL-15 signaling in NK cells to keep NK cells under control. As discussed herein, IL15 biology impacts multiple aspects of NK cell functionality, including but not limited to proliferation / proliferation, activation, cytotoxicity, survival, homing, migration, among others. Thus, according to some embodiments, CISH editing improves NK cell functionality across multiple functionalities, resulting in more effective and long-lasting NK cell therapeutics. In some embodiments, the inhibitor of CIS is used in combination with the administration of engineered NK cells.In some embodiments, CIS expression is knocked down or knocked out through gene editing of CISH gene, for example, by using CRISPR-Cas editing.In other embodiments, small interfering RNA, antisense RNA, TALEN or zinc finger is used.In some embodiments, CIS expression is knocked down in T cells through gene editing.

[0085] In some embodiments, CISH gene editing confers improved homing ability to target sites to NK cells. In some embodiments, CISH gene editing confers improved ability to migrate within tissues in response to, for example, chemoattractants or to move away from repellents to NK cells. In some embodiments, CISH gene editing confers improved ability to be activated and thus, for example, to exert anti-tumor effects to NK cells. In some embodiments, CISH gene editing confers enhanced proliferation ability to NK cells, which in some embodiments allows for the generation of stable NK cell numbers from donor blood samples. Furthermore, in such embodiments, NK cells edited for CISH and engineered to express CAR are more easily, robustly, and consistently expanded in culture. In some embodiments, CISH gene editing confers enhanced cytotoxicity to NK cells. In some embodiments, CISH editing synergistically enhances the cytotoxic effect of engineered NK cells and / or engineered T cells expressing CAR.

[0086] In some embodiments, CISH gene editing activates or inhibits a wide variety of pathways. CIS proteins are negative regulators of IL15 signaling, for example, through inhibition of the JAK-STAT signaling pathway. These pathways typically result in the transcription of IL15-responsive genes (including CISH). In some embodiments, knockdown of CISH disinhibits JAK-STAT (e.g., JAK1-STAT5) signaling, and there is enhanced transcription of IL15-responsive genes. In some embodiments, knockout of CISH results in enhanced signaling through mammalian target of rapamycin (mTOR), with a corresponding increase in the expression of genes related to cellular metabolism and respiration. In some embodiments, knockout of CISH results in IL15-induced increased expression of IL-2Rα (CD25), but not IL-15Rα or IL-2 / 15Rβ, improved NK cell membrane binding of IL15 and / or IL2, increased phosphorylation of STAT-3 and / or STAT-5, and increased expression of anti-apoptotic proteins, such as Bcl-2. In some embodiments, CISH knockout results in IL15-induced upregulation of selected genes related to mitochondrial function (e.g., electron transport and cellular respiration) and cell cycle. Thus, in some embodiments, knockout of CISH by gene editing improves NK cell cytotoxicity and / or survival, at least in part through metabolic reprogramming. In some embodiments, negative regulators of cell metabolism, such as TXNIP, are downregulated in response to CISH knockout. In some embodiments, promoters related to cell survival and proliferation, including BIRC5 (survivin), TOP2A, CKS2 and RACGAP1, are upregulated after CISH knockout, whereas antiproliferative or proapoptotic proteins, such as TGFB1, ATM and PTCH1, are downregulated.In some embodiments, the CISH knockout alters the state of signaling (e.g., activation or inactivation) via or through one or more of CXCL-10, IL2, TNF, IFNg, IL13, IL4, Jnk, PRF1, STAT5, PRKCQ, IL2 receptor beta, SOCS2, MYD88, STAT3, STAT1, TBX21, LCK, JAK3, IL& receptor, ABL1, IL9, STAT5A, STAT5B, Tcf7, PRDM1 and / or EOMES.

[0087] In some embodiments, gene editing of immune cells may also provide unexpected improvements in expansion, survival and / or cytotoxicity of edited immune cells. As disclosed herein, engineered cells (e.g., those expressing CARs) can also be edited, the combination providing robust cells for immunotherapy. In some embodiments, editing allows for unexpected improvements in NK cell expansion, survival and / or cytotoxicity. In some embodiments, knocking out CISH expression in NK cells removes a potent negative regulator of IL15-mediated signaling in NK cells, disinhibiting NK cells and allowing for one or more of improved NK cell homing, NK cell migration, NK cell activation, expansion, cytotoxicity and / or survival. Furthermore, in some embodiments, editing may improve NK and / or T cell function in an otherwise suppressive tumor microenvironment. In some embodiments, CISH gene editing results in improved NK cell expansion, survival and / or cytotoxicity without the need to exogenously provide Notch ligand. Additional

[0088] In some embodiments, gene editing is achieved by one or more of various engineered nucleases. In some embodiments, restriction enzymes are used, especially when double-stranded breaks are desired in multiple regions. In some embodiments, bioengineered nucleases are used. Depending on the embodiment, one or more of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and / or clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) systems are used to specifically edit the genes encoding one or more TCR subunits.

[0089] Meganucleases are characterized by their ability to recognize and cleave large DNA sequences (14-40 base pairs). In some embodiments, meganucleases from the LAGLIDADG family are used and mutagenesis and screening are performed to generate meganuclease variants that recognize unique sequences, e.g., specific sites in TCR or CISH, any other target gene disclosed herein. Target sites in TCR can be easily identified. Further information regarding target sites within regions of TCR can be found in U.S. Patent Publication Nos. 2018 / 0325955 and 2015 / 0017136, each of which is incorporated herein by reference in its entirety. In some embodiments, two or more meganucleases or functional fragments thereof are fused to create a hybrid enzyme that recognizes a desired target sequence within a target gene (e.g., CISH).

[0090] In contrast to meganucleases, ZFNs and TALENs function based on a non-specific DNA cleavage catalytic domain linked to a specific DNA sequence recognition peptide, such as a zinc finger or a transcription activation-like effector (TALE). Thus, ZFNs and TALENs advantageously allow sequence-independent cleavage of DNA with a high degree of sequence specificity in target recognition. Zinc finger motifs function in transcription factors in nature, recognizing specific DNA sequences for transcription. The C-terminal part of each finger is responsible for the specific recognition of DNA sequences. Although the sequences recognized by ZFNs are relatively short (e.g., about 3 base pairs), in some embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc fingers with characterized recognition sites are used, thereby allowing targeting of specific sequences, such as a portion of a TCR (or an immune checkpoint inhibitor). To induce target DNA cleavage, the combined ZFNs are then fused to the catalytic domain of an endonuclease, such as FokI (which may be a FokI heterodimer). Additional information regarding the use of ZFNs to edit TCRs and / or immune checkpoint inhibitors can be found in U.S. Pat. No. 9,597,357, which is incorporated herein by reference.

[0091] Transcription activator-like effector nuclease (TALEN) is a specific DNA-binding protein characterized by a series of 33 or 34 amino acid repeats. Similar to ZFN, TALEN is a fusion of the DNA cleavage domain of nuclease with a TALE domain, allowing sequence-independent introduction of double-stranded DNA breaks with highly accurate target site recognition. TALEN can create double-stranded breaks at its target site that can be repaired by error-prone non-homologous end joining (NHEJ), resulting in gene disruption by introducing small insertions or deletions. Advantageously, TALEN is used in some embodiments, at least in part, due to its high specificity in DNA binding, reduced off-target effects, and ease of construction of DNA-binding domain.

[0092] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic element used by bacteria as a defense against viruses. The repeats are short sequences derived from the viral genome and integrated into the bacterial genome. Cas (CRISPR-associated protein) processes the sequence and cuts the matching sequence of the viral DNA. By introducing a plasmid containing the Cas gene and a specifically constructed CRISPR into a eukaryotic cell, the eukaryotic genome can be cut at any desired location. Additional information regarding CRISPR can be found in US Patent Publication No. 2014 / 0068797, which is incorporated herein by reference. In some embodiments, CRISPR is used to manipulate the target gene to be knocked out or knocked in, for example, genes encoding CISH, TGFBR2, TCR, B2M, CIITA, CD47, HLA-E, etc. In some embodiments, CRISPR is used to edit one or more of the genes encoding one or more TCRs and / or one or more immune checkpoint inhibitors of a T cell. In some embodiments, the immune checkpoint inhibitor is selected from one or more of CTLA4 and PD1. In some embodiments, CRISPR is used to shorten one or more of TCRα, TCRβ, TCRγ and TCRδ. In some embodiments, the TCR is shortened without affecting the function of the CD3z signaling domain of the TCR. Depending on the embodiment and depending on which target gene is to be edited, class 1 or class 2 Cas is used. In some embodiments, class 1 Cas is used, and the Cas type is selected from the following types: I, IA, IB, IC, ID, IE, IF, IU, III, IIIA, IIIB, IIIC, IIID, IV, IVA, IVB and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and combinations thereof.In some embodiments, class 2 Cas is used, and the Cas type is selected from the following types: II, IIA, IIB, IIC, V, VI, and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a (previously known as C2c2), Cas13b, Cas13c, CasX, CasY, and combinations thereof. In some embodiments, class 2 CasX is used, and CasX can form a complex with a guide nucleic acid, and the complex can bind to a target DNA, and the target DNA includes a non-target strand and a target strand. In some embodiments, class 2 CasY is used, and CasY can bind to and modify a target nucleic acid and / or a polypeptide associated with the target nucleic acid.

[0093] Extracellular domain (binding site) Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors comprising an extracellular domain, e.g., a BCMA-directed chimeric antigen receptor. In some embodiments, the extracellular domain comprises a binding moiety (also referred to as an antigen-binding protein or antigen-binding domain) that can target a tumor antigen as described herein. In some embodiments, the binding moiety is derived from or comprises a wild-type or non-wild-type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), VH or VL domain, camelid VHH domain, or a non-immunoglobulin scaffold such as DARPIN, affibody, affilin, adnectin, affitin, repebody, finomer, alphabody, avimer, atrimer, sentinin, pronectin, anticalin, Kunitz domain, armadillo repeat protein, autoantigen, receptor, or ligand. In some embodiments, the binding moiety contains two or more antigen-binding domains. In embodiments, the binding moiety is operably linked to the NH2-terminus of a TCR domain (e.g., the constant chain of TCR-alpha, TCR-beta, TCR-beta2, preTCR-alpha, preTCR-alpha-Del48, TCR-gamma, or TCR-delta), either directly or via a linker as appropriate.

[0094] In some embodiments, binding moieties (also referred to as antigen-binding proteins) are provided. The term "binding moiety" as used herein is given its ordinary meaning and also relates to antigen-binding fragments that bind to an antigen, and optionally to proteins that include a scaffold or framework moiety that allows the binding moiety to adopt a conformation that facilitates binding to the antigen. In some embodiments, the antigen is a cancer antigen (e.g., BCMA) or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of the antibody that binds to the antigen, or from the light chain of the antibody that binds to the antigen. Furthermore, in some embodiments, the antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises one, two, three, four, five or six CDRs from the antibody that binds to the antigen, and in some embodiments, the CDRs can be any combination of heavy and / or light chain CDRs. In some embodiments, the antigen-binding fragment is an antigen fragment.

[0095] Non-limiting examples of binding moieties include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy chain antibody; VHH fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity determining region (CDR) fragments. These molecules can be derived from any mammalian source, e.g., human, mouse, rat, rabbit, or pig, dog, or camel. Antibody fragments can compete with intact (e.g., natural) antibodies for binding of a target antigen, and fragments can be produced by modification (e.g., enzymatic or chemical cleavage) of intact antibodies, or can be synthesized de novo using recombinant DNA technology or peptide synthesis. Binding moieties can include, for example, alternative protein scaffolds or artificial scaffolds onto which CDRs or CDR derivatives are grafted. Such scaffolds include, but are not limited to, antibody-derived scaffolds that contain, for example, mutations introduced to stabilize the three-dimensional structure of the binding moiety, as well as fully synthetic scaffolds that contain, for example, biocompatible polymers. Additionally, peptide antibody mimetics ("PAMs") may be used, as well as scaffolds based on antibody mimetics that utilize a fibronectin component as a scaffold.

[0096] In some embodiments, the binding moiety comprises one or more antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For example, the binding moiety may include, but is not limited to, diabodies; intrabodies; domain antibodies (single VL or VH domain or two or more VH domains linked by a peptide linker); maxibodies (two scFvs fused to an Fc region); triabodies; tetrabodies; minibodies (scFvs fused to a CH3 domain); peptibodies (one or more peptides linked to an Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with complementary light chain polypeptides); small modular immunopharmaceuticals; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0097] In some embodiments, the binding moiety has the structure of an immunoglobulin. As used herein, the term "immunoglobulin" is given its ordinary meaning and also refers to tetrameric molecules, with each tetramer containing two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0098] Within light and heavy chains, the variable (V) and constant regions (C) are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

[0099] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0100] Human light chains are classified as kappa and lambda light chains. An antibody "light chain" refers to the smaller of the two types of polypeptide chains in its naturally occurring conformation in an antibody molecule. Kappa (K) and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can comprise a polypeptide that includes, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).

[0101] Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε), and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. An antibody "heavy chain" refers to the larger of the two polypeptide chains in its naturally occurring conformation in the antibody molecule, which usually determines the class to which the antibody belongs. A heavy chain can include a polypeptide that includes, from amino terminus to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4).

[0102] The IgG class is further divided into subclasses, namely IgG1, IgG2, IgG3 and IgG4. The IgA class is further divided into subclasses, namely IgA1 and IgA2. IgM has subclasses including, but not limited to, IgM1 and IgM2. The heavy chains in IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), whereas the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3 and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via interpolypeptide disulfide bonds between the CL and CH1 domains (e.g., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0103] In some embodiments, the binding moiety is an antibody. The term "antibody" as used herein relates to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody may be monoclonal or polyclonal, multi-chain or single-chain, or an intact immunoglobulin, and may be derived from natural or recombinant sources. An antibody may be a tetramer of an immunoglobulin molecule. An antibody may be "humanized," "chimeric," or non-human. An antibody may comprise an intact immunoglobulin of any isotype, including, for example, chimeric, humanized, human, and bispecific antibodies. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains. An antibody sequence may be derived from only a single species, or may be "chimeric," i.e., different portions of the antibody may be derived from two different species, as described further below. Unless otherwise specified, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibody retains the same or similar binding and / or function as an antibody consisting of two full-length light and heavy chains. For example, antibodies with substitutions, insertions or deletions of one, two, three, four or five amino acid residues at the N-terminus and / or C-terminus of the heavy and / or light chains are included within the definition, provided that the antibody retains the same or similar binding and / or function as an antibody comprising two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies and synthetic antibodies. In some embodiments, monoclonal and polyclonal antibodies are provided. The term "polyclonal antibody" as used herein is given its ordinary meaning and also relates to a population of antibodies that typically vary widely in composition and binding specificity. The term "monoclonal antibody" ("mAb") as used herein is given its ordinary meaning and also refers to one or more of a population of antibodies having identical sequence. A monoclonal antibody binds to an antigen at a specific epitope on the antigen.

[0104] In some embodiments, the binding moiety is a fragment or antigen-binding fragment of an antibody. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of a VH domain and a CHI domain, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments can include Fab, Fab', F(ab')2, and / or Fv fragments that contain at least one CDR of an immunoglobulin sufficient to confer specific antigen binding to a cancer antigen. Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0105] In some embodiments, Fab fragments are provided. Fab fragments are monovalent fragments with VL, VH, CL and CH1 domains; F(ab')2 fragments are bivalent fragments with two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments have VH and CH1 domains; Fv fragments have VL and VH domains of a single arm of antibody; and dAb fragments have VH domain, VL domain, or antigen-binding fragments of VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single domain antibodies, single chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. In some embodiments, the antibody comprises at least one CDR described herein.

[0106] In some embodiments herein, single chain variable fragments are also provided. The term "single chain variable fragment" ("scFv") as used herein is given its ordinary meaning and also relates to a fusion protein in which the VL and VH regions are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, which linker is long enough to allow the protein chain to fold and form a monovalent antigen binding site. For clarity, unless specified as such, a "single chain variable fragment" is not an antibody as defined herein. A diabody is a bivalent antibody comprising two polypeptide chains, each polypeptide chain comprising a VH and a VL domain linked by a linker configured to reduce or prevent pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain. According to some embodiments, if the two polypeptide chains of a diabody are identical, the diabody resulting from the pairing will have two identical antigen binding sites. Polypeptide chains with different sequences can be used to create diabodies with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, which may be the same or different, and form three and four antigen-binding sites, respectively.

[0107] In some embodiments, the binding moiety comprises one or more CDRs. The term "CDR" as used herein is given its usual meaning and also relates to the complementarity determining regions (also called "minimal recognition units" or "hypervariable regions") in antibody variable sequences. CDRs allow the binding moiety to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (HCDR1, HCDR2 and HCDR3) and three light chain variable region CDRs (LCDR1, LCDR2 and LCDR3). The CDRs in each of the two chains are typically positioned by framework regions to form a structure that specifically binds to a specific epitope or domain on the target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically follow the following order of their elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). One or more CDRs may be incorporated into a molecule, either covalently or non-covalently, to make it a binding moiety.

[0108] Anti-BCMA binding moiety Disclosed herein are anti-BCMA binding moieties comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDRs) 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) and a light chain variable region (VL) comprising light chain CDRs 1, 2 and 3 (LCDR1, LCDR2 and LCDR3).

[0109] In some embodiments, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 104-129. In some embodiments, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 130-155. In some embodiments, HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 156-181. In some embodiments, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 104-129, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 130-155, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 156-181. In some embodiments, the VH comprises a combination of HCDR1, HCDR2 and HCDR3 as shown in FIG.

[0110] In some embodiments, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 385-410. In some embodiments, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 411-436. In some embodiments, LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 437-462. In some embodiments, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 385-410, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 411-436, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 437-462. In some embodiments, the VL comprises a combination of LCDR1, LCDR2 and LCDR3 as shown in FIG.

[0111] In some embodiments, 1) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 104, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 130, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 156; 2) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 105, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 131, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 157; 3) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 106, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 132, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 158; 4) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 107, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 133, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 159; 5) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 108, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 134, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 160; 6) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 109, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 135, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 161; 7) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 110, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 136, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 162; 8) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 111, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 137, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 163; 9) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 112, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 138, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 164; 10) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 113, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 139, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 165; 11) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 114, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 140, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 166; 12) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 115, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 141, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 167; 13) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 116, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 142, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 168; 14) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 117, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 143, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 169; 15) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 118, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 144, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 170; 16) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 119, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 145, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 171; 17) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 120, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 146, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 172; 18) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 121, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 147, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 173; 19) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 122, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 148, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 174; 20) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 123, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 149, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 175; 21) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 124, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 150, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 176; 22) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 125, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 151, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 177; 23) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 126, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 152, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 178; 24) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 127, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 153, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 179; 25) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 128, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 154, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 180; or 26) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 129, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 155, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 181.

[0112] In some embodiments, 1) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 385, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 411, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 437; 2) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 386, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 412, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 438; 3) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 387, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 413, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 439; 4) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 388, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 414, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 440; 5) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 389, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 415, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 441; 6) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 390, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 416, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 442; 7) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 391, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 417, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 443; 8) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 392, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 418, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 444; 9) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 393, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 419, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 445; 10) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 394, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 420, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 446; 11) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 395, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 421, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 447; 12) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 396, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 422, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 448; 13) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 397, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 423, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 449; 14) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 398, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 424, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 450; 15) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 399, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 425, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 451; 16) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 400, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 426, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 452; 17) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 401, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 427, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 453; 18) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 402, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 428, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 454; 19) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 403, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 429, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 455; 20) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 404, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 430, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 456; 21) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 405, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 431, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 457; 22) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 406, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 432, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 458; 23) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 407, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 433, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 459; 24) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 408, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 434, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 460; 25) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 409, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 435, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 461; or 26) LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 410, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 436, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 462.

[0113] In some embodiments, 1) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 104, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 130, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 156. wherein LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:385, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:411, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:437; 2) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 105, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 131, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 157; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:386, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:412, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:438; 3) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 106, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 132, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 158; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:387, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:413, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:439; 4) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 107, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 133, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 159; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:388, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:414, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:440; 5) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 108, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 134, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 160; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:389, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:415, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:441; 6) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 109, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 135, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 161; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:390, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:416, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:442; 7) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 110, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 136, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 162; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:391, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:417, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:443; 8) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 111, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 137, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 163; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:392, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:418, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:444; 9) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 112, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 138, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 164; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:393, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:419, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:445; 10) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 113, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 139, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 165; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:394, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:420, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:446; 11) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 114, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 140, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 166; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:395, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:421, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:447; 12) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 115, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 141, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 167; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:396, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:422, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:448; 13) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 116, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 142, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 168; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:397, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:423, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:449; 14) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 117, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 143, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 169; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:398, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:424, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:450; 15) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 118, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 144, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 170; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:399, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:425, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:451; 16) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 119, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 145, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 171; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:400, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:426, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:452; 17) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 120, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 146, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 172; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:401, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:427, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:453; 18) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 121, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 147, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 173; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:402, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:428, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:454; 19) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 122, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 148, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 174; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:403, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:429, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:455; 20) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 123, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 149, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 175; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:404, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:430, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:456; 21) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 124, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 150, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 176; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:405, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:431, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:457; 22) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 125, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 151, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 177; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:406, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:432, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:458; 23) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 126, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 152, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 178; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:407, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:433, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:459; 24) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 127, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 153, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 179; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:408, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:434, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:460; 25) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 128, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 154, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 180; CDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:409, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:435, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:461; or 26) HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 129, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 155, and HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 181; LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:410, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:436, and LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:462.

[0114] In some embodiments, the VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 260-285. In some embodiments, the VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 541-566. In some embodiments, the anti-BCMA binding moiety comprises a VH and VL combination as shown in FIG.

[0115] In some embodiments, HCDR1 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-207. In some embodiments, HCDR2 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 208-233. In some embodiments, HCDR3 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 234-259.

[0116] In some embodiments, LCDR1 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 463-488. In some embodiments, LCDR2 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 489-514. In some embodiments, LCDR3 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 515-540.

[0117] In some embodiments, the VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 286-311. In some embodiments, the VH is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 286-311.

[0118] In some embodiments, the VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 567-592. In some embodiments, the VL is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 567-592.

[0119] In some embodiments, the VH further comprises a heavy chain signal peptide (H-SP). In some embodiments, the heavy chain signal peptide comprises any one of the sequences of SEQ ID NOs: 1-9. In some embodiments, the heavy chain signal peptide is encoded by a nucleic acid having any one of the sequences of SEQ ID NOs: 10-19. In some embodiments, the VH further comprises a framework region. In some embodiments, the VH comprises framework regions 1, 2, 3 and 4 (H-FR1, H-FR2, H-FR3 and H-FR4). In some embodiments, H-FR1 comprises any one of the sequences of SEQ ID NOs: 20-32. In some embodiments, H-FR2 comprises any one of the sequences of SEQ ID NOs: 33-38. In some embodiments, H-FR3 comprises any one of the sequences of SEQ ID NOs: 39-53. In some embodiments, H-FR4 comprises the sequence of SEQ ID NO: 54. In some embodiments, H-FR1 is encoded by a nucleic acid having any one of the sequences of SEQ ID NOs: 55-73. In some embodiments, H-FR2 is encoded by a nucleic acid having any one of the sequences of SEQ ID NOs: 74-84. In some embodiments, H-FR3 is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 85 to 102. In some embodiments, H-FR4 is encoded by a nucleic acid having the sequence of SEQ ID NO: 103.

[0120] In some embodiments, the VL further comprises a light chain signal peptide (L-SP). In some embodiments, the light chain signal peptide comprises the sequence of any one of SEQ ID NOs: 312-317. In some embodiments, the light chain signal peptide is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 318-324. In some embodiments, the VL further comprises a framework region. In some embodiments, the VL comprises framework regions 1, 2, 3 and 4 (L-FR1, L-FR2, L-FR3 and L-FR4). In some embodiments, L-FR1 comprises the sequence of any one of SEQ ID NOs: 325-331. In some embodiments, L-FR2 comprises the sequence of any one of SEQ ID NOs: 332-337. In some embodiments, L-FR3 comprises the sequence of any one of SEQ ID NOs: 338-347. In some embodiments, L-FR4 comprises the sequence of any one of SEQ ID NOs: 348-353. In some embodiments, L-FR1 is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 354-357 or 1416-1420. In some embodiments, L-FR2 is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 358-366. In some embodiments, L-FR3 is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 367-377. In some embodiments, L-FR4 is encoded by a nucleic acid having the sequence of any one of SEQ ID NOs: 378-384.

[0121] Embodiments of the anti-BCMA binding moiety include various arrangements of VH and VL as disclosed herein. In some embodiments, the VH and VL are separated by a linker. In some embodiments, the linker comprises the sequence of SEQ ID NO: 1388.

[0122] In some embodiments of the anti-BCMA binding moiety, the VH is N-terminal to the VL. In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 593-618. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 619-670.

[0123] In some embodiments of the anti-BCMA binding moiety, the VL is N-terminal to the VH. In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 671-696. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 697-748.

[0124] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1421-1426. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1447-1472.

[0125] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1473-1498. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1499-1524.

[0126] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1582-1600. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1601-1619.

[0127] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1677-1695. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1696-1714.

[0128] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1715-1733. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1734-1771.

[0129] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1772-1790. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1791-1828.

[0130] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1829-1847. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1848-1866.

[0131] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1867-1885. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1886-1923.

[0132] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3255-3277. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3278-3323.

[0133] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3324-3346. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3347-3392.

[0134] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5457-5479. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5480-5502.

[0135] In some embodiments, the anti-BCMA binding moiety comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3859-3862. In some embodiments, the anti-BCMA binding moiety is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3875-3878.

[0136] In some embodiments, the anti-BCMA binding moiety comprises a VHH domain having a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5457-5479. In some embodiments, the VHH domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5480-5502.

[0137] In some embodiments, the binding moieties provided herein comprise one or more CDRs as part of a larger polypeptide chain. In some embodiments, the antigen binding protein covalently links one or more CDRs to another polypeptide chain. In some embodiments, the antigen binding protein incorporates one or more CDRs non-covalently. In some embodiments, the antigen binding protein may comprise at least one of the CDRs described herein incorporated within a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support or framework or scaffold capable of presenting one or more sequences of amino acids (e.g., CDRs, variable regions, etc.) that bind antigens in a localized surface area. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs) or may have one or more modifications, e.g., additions, deletions and / or substitutions, of amino acids relative to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffold may be derived from polypeptides of a variety of different species (or more than one species), such as human, non-human primate or other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0138] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. In some such embodiments, the framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and / or tendamistat domain.

[0139] In some embodiments, binding moieties are provided that have two or more binding sites. In some embodiments, the binding sites are identical to each other, and in some embodiments, the binding sites are different from each other. For example, an antibody typically has two identical binding sites, whereas a "bispecific" or "bifunctional" antibody has two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes that may be present on the same or different protein targets. In some embodiments, this is particularly advantageous. Because bispecific chimeric antigen receptors can provide engineered cells with the ability to target multiple tumor markers, such as BCMA, and additional tumor markers, such as CD19, CD38, CS1, FCRL5, GPR5CD, CD229, NKG2D, or any other marker disclosed herein or valued in the art as a tumor-specific or tumor-associated antigen.

[0140] Additional anti-BCMA binding moieties are known in the art, such as those disclosed in, e.g., U.S. Pat. Nos. 9,765,342, 10,294,304, 10,174,095, European Patent Application Publication No. 3230321, U.S. Patent Publication Nos. 2018 / 0118842, 2019 / 0153061, and PCT Patent Publication No. WO 2019 / 149269, each of which is incorporated by reference in its entirety.

[0141] The term "chimeric antibody" as used herein is given its ordinary meaning and also relates to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In some embodiments, one or more of the CDRs are derived from an anti-cancer antigen (e.g., BCMA) antibody. In some embodiments, all of the CDRs are derived from an anti-cancer antigen antibody (e.g., anti-BCMA). In some embodiments, CDRs from two or more anti-cancer antigen antibodies are mixed and matched in a chimeric antibody. For example, a chimeric antibody may contain CDR1 from a light chain of a first anti-cancer antigen antibody, CDR2 and CDR3 from a light chain of a second anti-cancer antigen antibody, and CDRs from a heavy chain of a third anti-cancer antigen antibody. Furthermore, the framework regions of the antigen binding proteins disclosed herein may be derived from one of the same anti-cancer antigen (e.g., BCMA) antibodies, one or more different antibodies, such as a human antibody, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies that exhibit the desired biological activity are also provided herein.

[0142] Cytotoxicity Signaling Complex Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors, such as BCMA-directed CARs, that include a cytotoxic signaling complex. As disclosed herein, according to some embodiments, the provided cytotoxic receptor complexes include one or more transmembrane domains and / or intracellular domains that initiate a cytotoxic signaling cascade when the extracellular domain binds to a ligand on the surface of a target cell. Certain embodiments disclosed herein relate to chimeric antigen receptor constructs in which a tumor targeting domain (e.g., an anti-BCMA binding moiety) is linked to a cytotoxic signaling complex.

[0143] In some embodiments, the cytotoxic signaling complex comprises at least one transmembrane domain, at least one costimulatory domain, and / or at least one signaling domain. In some embodiments, two or more components make up a given domain - for example, a costimulatory domain may comprise two subdomains. Furthermore, in some embodiments, a domain may be used for multiple functions. For example, a transmembrane domain may also be used to provide a signaling function.

[0144] Hinge Domain Some embodiments of the CARs disclosed herein, such as BCMA-directed CARs, include a hinge domain. The hinge domain is typically used to separate the extracellular binding portion from the remainder of the CAR component, which includes an intracellular component bridged to the extracellular binding portion by a transmembrane domain. Any hinge domain disclosed herein or generally known in the art can be used in the BCMA-directed CARs disclosed herein.

[0145] In some embodiments, the hinge domain is a CD8 hinge domain. In some embodiments, the CD8 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1389. In some embodiments, the CD8 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1400. In some embodiments, the CD8 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1389.

[0146] In some embodiments, the hinge domain is an IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1390. In some embodiments, the IgG4 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1401. In some embodiments, the IgG4 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1390.

[0147] In some embodiments, the hinge domain is a RQRCD8 hinge domain. As understood in the art, an "RQRCD8 hinge domain" is a CD8 hinge domain with additional CD20 and CD34 epitopes. The presence of these additional epitopes allows for selective removal of immune cells engineered with CARs that have these epitopes, as well as allowing alternative detection methods using these epitopes (e.g., using antibodies specific for CD20 or CD34). In some embodiments, the RQRCD8 hinge domain comprises a sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1391. In some embodiments, the RQRCD8 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1402. In some embodiments, the RQRCD8 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having a sequence of SEQ ID NO: 1391. The RQRCD8 hinge domain (also referred to as "RQR8") is explored in Philip et al. "A highly compact epitope-based marker / suicide gene for easier and safer T-cell therapy" Blood 124(8):1277-1287 and PCT Publication WO 2013 / 153391, each of which is expressly incorporated herein by reference in its entirety.

[0148] Transmembrane domain Some embodiments of the CARs disclosed herein, such as BCMA-directed CARs, include a transmembrane domain. As conventionally understood, a transmembrane domain is used to act as a region that spans the plasma membrane of a cell, connecting the extracellular domain and the intracellular domain of a CAR. Any transmembrane domain disclosed herein or generally known in the art can be used in the BCMA-directed CARs disclosed herein.

[0149] In some embodiments, the transmembrane domain is a CD8 transmembrane domain (CD8TM). In some embodiments, the CD8 transmembrane domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1392. In some embodiments, the CD8 transmembrane domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1403. In some embodiments, the CD8 transmembrane domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1392.

[0150] In some embodiments, the CD8 transmembrane domain may also include a CD8 intracellular domain (CD8IC). In some embodiments, the CD8 intracellular domain includes the sequence of SEQ ID NO: 1393. In some embodiments, the CD8 intracellular domain is encoded by a nucleic acid that includes the sequence of SEQ ID NO: 1404. In some embodiments, the CD8 transmembrane domain may be considered, for purposes of this disclosure, to include the CD8 intracellular domain such that the CD8 transmembrane domain includes the sequences of SEQ ID NO: 1392 and SEQ ID NO: 1393 in an N-terminal to C-terminal orientation.

[0151] In some embodiments, the transmembrane domain is a CD28 transmembrane domain (CD28TM). In some embodiments, the CD28 transmembrane domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1414. In some embodiments, the CD8 transmembrane domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1415. In some embodiments, the CD8 transmembrane domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1414.

[0152] Signaling domains Some embodiments of the CAR disclosed herein, for example, BCMA-directed CAR, include an intracellular signaling domain.As is generally understood in the art, the use of multiple signaling domains whose activities act synergistically can achieve unexpectedly enhanced signaling.Any intracellular signaling domain disclosed herein or generally known in the art can be used in the BCMA-directed CAR disclosed herein.

[0153] In some embodiments, the intracellular signaling domain comprises an OX40 subdomain and a CD3zeta subdomain. In some embodiments, the OX40 subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1394. In some embodiments, the OX40 subdomain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1405. In some embodiments, the OX40 subdomain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having a sequence of SEQ ID NO: 1394. In some embodiments, the CD3 zeta subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1395. In some embodiments, the CD3 zeta subdomain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1406. In some embodiments, the CD3 zeta subdomain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having a sequence of SEQ ID NO: 1395. In some embodiments, the OX40 subdomain is N-terminal to the CD3 zeta subdomain. In some embodiments, the CD3 zeta subdomain is N-terminal to the OX40 subdomain.

[0154] Costimulatory domain In some embodiments, the CAR disclosed herein is expressed with a costimulatory domain, and the additional coactivator molecule improves aspects of CAR activity, including but not limited to cytotoxic activity, CAR stability, and cell life span. In some embodiments, the additional coactivator molecule may be a cytokine, including but not limited to certain interleukins, such as interleukin 2 (IL2) and / or interleukin 15 (IL15). In some embodiments, the immune cells expressing the CAR are also engineered to express such additional coactivator molecules in secreted form. In some embodiments, the immune cells expressing the CAR are also engineered to express such additional coactivator molecules in membrane-bound form, acting as autocrine stimulatory molecules (or even as paracrine stimulators for neighboring cells). Membrane-bound IL15 is explored in WO2015 / 174928, which is expressly incorporated herein by reference in its entirety.

[0155] In some embodiments, immune cells are engineered to express IL15, optionally as membrane-bound IL15 (mbIL15). In such embodiments, mbIL15 expression on immune cells enhances the cytotoxic effect of engineered immune cells by improving the proliferation and / or lifespan of the immune cells. In some embodiments, IL15 is human IL15. In some embodiments, IL15 comprises the sequence of SEQ ID NO: 1397. In some embodiments, IL15 is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1408. In some embodiments, mbIL15 is assembled by fusing IL15 with one or more of CD8 signal peptide (CD8SP), CD8 hinge (CD8h), CD8 transmembrane domain (CD8TM) and CD8 intracellular domain (CD8IC). In some embodiments, mbIL15 is assembled according to the order from N-terminus to C-terminus: CD8SP-IL15-CD8h-CD8TM-CD8IC. However, other methods of producing membrane-bound IL15 are envisaged.

[0156] In some embodiments, the CD8SP of mbIL15 comprises the sequence of SEQ ID NO: 1396. In some embodiments, the CD8h of mbIL15 comprises the sequence of SEQ ID NO: 1400. In some embodiments, the CD8TM of mbIL15 comprises the sequence of SEQ ID NO: 1392. In some embodiments, the CD8IC of mbIL15 comprises the sequence of SEQ ID NO: 1393. In some embodiments, the CD8SP of mbIL15 is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1407. In some embodiments, the CD8h of mbIL15 is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1409. In some embodiments, the CD8TM of mbIL15 is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1410. In some embodiments, the CD8IC of mbIL15 is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1411. In some embodiments, mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 1398. In some embodiments, mbIL15 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 1412. In some embodiments, mbIL15 may be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 1398.

[0157] In some embodiments, CAR and mbIL15 are simultaneously expressed in a bicistronic configuration as a polypeptide comprising an autocleaving peptide. In some embodiments, CAR and mbIL15 are simultaneously expressed in a bicistronic configuration from a polynucleotide comprising a sequence encoding an autocleaving peptide. In some embodiments, the autocleaving peptide is a T2A autocleaving peptide, a P2A autocleaving peptide, an E2A autocleaving peptide, or an F2A autocleaving peptide. In this manner, CAR and mbIL15 can be delivered to immune cells as a single vector, if necessary. In some embodiments, a T2A autocleaving peptide is used. In some embodiments, the T2A autocleaving peptide comprises the sequence of SEQ ID NO: 1399. In some embodiments, the T2A autocleaving peptide is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1413.

[0158] BCMA-directed chimeric antigen receptor BCMA-directed CARs are disclosed herein. In some embodiments, the BCMA-directed CARs comprise an extracellular anti-BCMA binding moiety, a hinge domain, a transmembrane domain, and an intracellular signaling domain comprising an OX40 subdomain and a CD3ζ subdomain. In some embodiments, the anti-BCMA binding moiety can be any one of the anti-BCMA binding moieties disclosed herein. In some embodiments, the hinge domain can be any one of the hinge domains disclosed herein. In some embodiments, the transmembrane domain can be any one of the transmembrane domains disclosed herein. In some embodiments, the intracellular signaling domain can be any one of the intracellular signaling domains disclosed herein.

[0159] In some embodiments, the OX40 subdomain (OX40) comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1394. In some embodiments, the OX40 subdomain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1405. In some embodiments, the OX40 subdomain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having a sequence of SEQ ID NO: 1394. In some embodiments, the CD3 zeta subdomain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1395. In some embodiments, the CD3 zeta subdomain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1406. In some embodiments, the CD3 zeta subdomain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having a sequence of SEQ ID NO: 1395. In some embodiments, the OX40 subdomain is N-terminal to the CD3 zeta subdomain. In some embodiments, the CD3 zeta subdomain is N-terminal to the OX40 subdomain.

[0160] In some embodiments, the transmembrane domain is a CD8 transmembrane domain (CD8TM). In some embodiments, the CD8 transmembrane domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1392. In some embodiments, the CD8 transmembrane domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1403. In some embodiments, the CD8 transmembrane domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1392.

[0161] In some embodiments, the transmembrane domain is a CD28 transmembrane domain (CD28TM). In some embodiments, the CD28 transmembrane domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1414. In some embodiments, the CD8 transmembrane domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1415. In some embodiments, the CD8 transmembrane domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1414.

[0162] In some embodiments, the hinge domain is a CD8 hinge domain (CD8h). In some embodiments, the CD8 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1389. In some embodiments, the CD8 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1400. In some embodiments, the CD8 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1389.

[0163] In some embodiments, the CD8 hinge domain, transmembrane domain and intracellular signaling domain constitute the portion of the BCMA-directed CAR other than the anti-BCMA binding portion. In some embodiments, the BCMA-directed CAR comprises a CD8 hinge domain (CD8h), a CD8 transmembrane domain (CD8TM), a CD8 intracellular domain (CD8IC), an OX40 subdomain (OX40) and a CD3ζ subdomain (CD3ζ). In some embodiments, the CD8h, CD8TM, CD8IC, OX40 and CD3ζ are in the following order from N-terminus to C-terminus: CD8h-CD8TM-CD8IC-OX40-CD3ζ. In some embodiments, the CD8 hinge domain, transmembrane domain and intracellular signaling domain are represented by a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1375. In some embodiments, the CD8 hinge domain, transmembrane domain and intracellular signaling domain are encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1376.

[0164] In some embodiments, where the BCMA-directed CAR comprises CD8h, CD8TM, CD8IC, OX40 and CD3zeta together with an anti-BCMA binding moiety, the BCMA-directed CAR may comprise a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 749-774. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid having a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 775-800. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that is codon-optimized for human and has a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 801-826. In some embodiments, the BCMA-directed CAR comprises a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 905-930. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid having a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 931-956. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that is codon-optimized for human and has a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 957-982.

[0165] In some embodiments, the hinge domain is an IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1390. In some embodiments, the IgG4 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1401. In some embodiments, the IgG4 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1390.

[0166] In some embodiments, the IgG4 hinge domain, transmembrane domain and intracellular signaling domain constitute the part of the BCMA-directed CAR other than the anti-BCMA binding portion. In some embodiments, the BCMA-directed CAR comprises an IgG4 hinge domain (IgG4h), CD8TM, CD8IC, OX40 and CD3zeta. In some embodiments, the IgG4h, CD8TM, CD8IC, OX40 and CD3zeta are in the following order from N-terminus to C-terminus: IgG4h-CD8TM-CD8IC-OX40-CD3zeta. In some embodiments, the IgG4 hinge domain, transmembrane domain and intracellular signaling domain are represented by a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1377. In some embodiments, the IgG4 hinge domain, transmembrane domain and intracellular signaling domain are encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1378.

[0167] In some embodiments, where the BCMA-directed CAR comprises IgG4, CD8TM, CD8IC, OX40 and CD3zeta together with an anti-BCMA binding moiety, the BCMA-directed CAR may comprise a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1061-1086. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid having a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1087-1112. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that is codon-optimized for human and has a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1113-1138.

[0168] In some embodiments, the hinge domain is a RQRCD8 hinge domain. In some embodiments, the RQRCD8 hinge domain comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1391. In some embodiments, the RQRCD8 hinge domain is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1402. In some embodiments, the RQRCD8 hinge domain is truncated or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a peptide having the sequence of SEQ ID NO: 1391.

[0169] In some embodiments, the RQRCD8 hinge domain, transmembrane domain and intracellular signaling domain constitute the portion of the BCMA-directed CAR other than the anti-BCMA binding portion. In some embodiments, the BCMA-directed CAR comprises the RQRCD8 hinge domain (RQRCD8h), CD8TM, CD8IC, OX40 and CD3ζ. In some embodiments, the RQRCD8h, CD8TM, CD8IC, OX40 and CD3ζ are in the following order from N-terminus to C-terminus: RQRCD8h-CD8TM-CD8IC-OX40-CD3ζ. In some embodiments, the RQRCD8 hinge domain, transmembrane domain and intracellular signaling domain are represented by a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1379. In some embodiments, the RQRCD8 hinge domain, transmembrane domain and intracellular signaling domain are encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1380.

[0170] In some embodiments, where the BCMA-directed CAR comprises RQRCD8, CD8TM, CD8IC, OX40 and CD3zeta together with an anti-BCMA binding moiety, the BCMA-directed CAR may comprise a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1217-1242. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid having a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1243-1268. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that is codon-optimized for human and has a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1269-1294.

[0171] BCMA-directed chimeric antigen receptor constructs In some embodiments, a BCMA-directed CAR construct is provided that includes a BCMA-directed CAR. Figure 1 shows an exemplary BCMA-directed CAR construct embodied in the present disclosure. As shown in Figure 1, an embodiment of a BCMA-directed CAR construct includes an anti-BCMA binding moiety, a hinge domain, a CD8 transmembrane domain, a CD8 intracellular domain, an OX40 subdomain, a CD3ζ subdomain, a T2A self-cleaving peptide, and a membrane-bound IL15. In some embodiments, the CD8 transmembrane domain may be replaced with a CD28 transmembrane domain. Any one or more of these components may be any of the corresponding component embodiments disclosed herein. In some embodiments, the BCMA-directed CAR is any one of the BCMA-directed CARs disclosed herein, and the BCMA-directed CAR construct includes the BCMA-directed CAR and membrane-bound IL15 in a bicistronic configuration.

[0172] In some embodiments, the BCMA-directed CAR construct comprises a BCMA-directed CAR comprising an anti-BCMA binding moiety, CD8h, CD8TM, CD8IC, OX40 and CD3zeta, and membrane-bound IL15 (mbIL15), where the BCMA-directed CAR and mbIL15 are separated by a self-cleaving peptide (e.g., a T2A self-cleaving peptide). In some embodiments, the BCMA-directed CAR construct is arranged in the following order from N-terminus to C-terminus: anti-BCMA binding moiety-CD8h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15. In some embodiments, CD8h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1381. In some embodiments, CD8h-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1382. In some embodiments, the anti-BCMA binding moiety comprises a VH and a VL arranged such that the VH is N-terminal to the VL. In some embodiments, the BCMA-directed CAR construct comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 827-852. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 853-878. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence that is codon-optimized for human and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 879-904.

[0173] In some embodiments, the BCMA-directed CAR construct comprises a BCMA-directed CAR comprising an anti-BCMA binding moiety, CD8h, CD8TM, CD8IC, OX40 and CD3zeta, and mbIL15, and the BCMA-directed CAR and mbIL15 are separated by a self-cleaving peptide (e.g., a T2A self-cleaving peptide). In some embodiments, the BCMA-directed CAR construct is arranged in the following order from N-terminus to C-terminus: anti-BCMA binding moiety-CD8h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15. In some embodiments, CD8h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1381. In some embodiments, CD8h-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1382. In some embodiments, the anti-BCMA binding moiety comprises a VH and a VL arranged such that the VL is N-terminal to the VH. In some embodiments, the BCMA-directed CAR construct comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 983-1008. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1009-1034. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence that is codon-optimized for human and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1035-1060.

[0174] In some embodiments, the BCMA-directed CAR construct comprises a BCMA-directed CAR comprising an anti-BCMA binding moiety, IgG4h, CD8TM, CD8IC, OX40 and CD3zeta, and mbIL15, and the BCMA-directed CAR and mbIL15 are separated by a self-cleaving peptide (e.g., a T2A self-cleaving peptide). In some embodiments, the BCMA-directed CAR construct is arranged in the following order from N-terminus to C-terminus: anti-BCMA binding moiety-IgG4h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15. In some embodiments, the IgG4h-CD8TM-CD8IC-OX40-CD3zeta-T2A-mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1383. In some embodiments, IgG4h-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1384. In some embodiments, the BCMA-directed CAR construct comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1139-1164. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1165-1190. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence that is codon-optimized for human and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1191-1216.

[0175] In some embodiments, the BCMA-directed CAR construct comprises a BCMA-directed CAR comprising an anti-BCMA binding moiety, RQR8CDh, CD8TM, CD8IC, OX40 and CD3ζ, and mbIL15, and the BCMA-directed CAR and mbIL15 are separated by a self-cleaving peptide (e.g., a T2A self-cleaving peptide). In some embodiments, the BCMA-directed CAR construct is arranged in the following order from N-terminus to C-terminus: anti-BCMA binding moiety-RQR8CDh-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15. In some embodiments, RQRCD8h-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1385. In some embodiments, RQRCD8h-CD8TM-CD8IC-OX40-CD3ζ-T2A-mbIL15 is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1386. In some embodiments, the BCMA-directed CAR construct comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1295-1320. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1321-1346. In some embodiments, the BCMA-directed CAR construct is encoded by a nucleic acid comprising a sequence that is codon-optimized for human and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1347-1372.

[0176] In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3851-3854.

[0177] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3867-3870. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3883-3886.

[0178] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3887-3912. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3913-3964.

[0179] In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 3991-4042.

[0180] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4043-4068. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4069-4120. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4147-4198.

[0181] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4199-4224. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4225-4276. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4303-4354.

[0182] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4355-4380. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4381-4432. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4459-4510.

[0183] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4513-4531. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4531-4569. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4589-4626.

[0184] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4627-4645. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4646-4683. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4703-4740.

[0185] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4741-4759. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4760-4797. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4817-4854.

[0186] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4855-4873. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4874-4911. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4931-4968.

[0187] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4969-4994. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 4995-5046. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5073-5124.

[0188] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5125-5150. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5151-5176. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5203-5228.

[0189] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5229-5247. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5248-5285. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5305-5342.

[0190] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5343-5361. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5362-5399. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5419-5456.

[0191] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5503-5525. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5526-5571. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5595-5640.

[0192] In some embodiments, the BCMA-directed CAR comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5641-5663. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5664-5709. In some embodiments, the BCMA-directed CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5733-5778.

[0193] In some embodiments, the BCMA-directed CAR comprises a VHH domain and comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5779-5801. In some embodiments, the BCMA-directed VHH-CAR is encoded by a nucleic acid comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5802-5847. In some embodiments, the BCMA-directed VHH-CAR is encoded by a nucleic acid that also encodes mbIL15 and has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5871-5916.

[0194] With respect to any of the receptor constructs described herein, it is understood that certain sequence variations, extensions, and / or shortenings of the disclosed sequences may occur, for example, when combining sequences as a result of ease of cloning or efficiency of cloning (e.g., for creation of restriction sites).

[0195] Treatment and Methods of Administration Disclosed herein is a population of immune cells comprising any one of the anti-BCMA binding moieties, BCMA-directed CARs or BCMA-directed CAR constructs disclosed herein. In some embodiments, the immune cells are NK cells and / or T cells. In some embodiments, the population of immune cells further comprises an additional extracellular moiety or CAR that binds to a non-BCMA cancer marker. In some embodiments, the non-BCMA cancer marker comprises one or more of CD138, SLAMF7, CD38, GPRC5D or CD19.

[0196] Also provided herein are embodiments relating to methods of treating, ameliorating, inhibiting or preventing cancer using an immune cell or population of immune cells comprising any one of the anti-BCMA binding moieties, BCMA-directed CARs or BCMA-directed CAR constructs disclosed herein. In some embodiments, the method comprises administering a therapeutically effective amount of an immune cell or population of immune cells comprising any one of the anti-BCMA binding moieties, BCMA-directed CARs or BCMA-directed CAR constructs disclosed herein.

[0197] In certain embodiments, treatment of a subject with the genetically engineered cells described herein achieves one, two, three, four or more of the following effects, including, for example: (i) reducing or ameliorating the severity of a disease or a symptom associated therewith; (ii) reducing the duration of a symptom associated with a disease; (iii) protecting against progression of a disease or a symptom associated therewith; (iv) regressing a disease or a symptom associated therewith; (v) protecting against the onset or onset of a symptom associated with a disease; (vi) protecting against recurrence of a symptom associated with a disease; (vii) reducing hospitalization of the subject; (viii) reducing the length of hospitalization; (ix) increasing survival of a subject with a disease; (x) reducing the number of symptoms associated with a disease; (xi) enhancing, improving, supplementing, complementing or increasing the prophylactic or therapeutic effect of another treatment. Each of these comparisons is with a different treatment for the disease, including, for example, a cell-based immunotherapy for the disease using cells that do not express the constructs disclosed herein.

[0198] Administration may be by a variety of routes, including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue. The dose of immune cells, such as NK and / or T cells, can be readily determined for a given subject based on their body weight, the type and condition of the disease, and the desired aggressiveness of the treatment, but may range from about 10 per kg depending on the embodiment. 5 Approximately 10 per kg of cells 12 cells (e.g., 10 5 ~10 7 , 10 7 ~10 10 , 10 10 ~10 12 and overlapping ranges therein). In one embodiment, a dose escalation regimen is used. In some embodiments, for example, about 1x10 6 Cells / kg to approximately 1x10 8Immune cells such as NK and / or T cells are administered in the range of between 100 and 150 cells / kg. Depending on the embodiment, various types of cancers may be treated. In some embodiments, hepatocellular carcinoma is treated. Further embodiments provided herein include the treatment or prevention of the following non-limiting examples of cancers: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytoma, spinal tumor, brain stem glioma, glioblastoma, craniopharyngioma, ependymomas, ependymomas, medulloblastomas, medulloepitheliomas), breast cancer, bronchial tumors, Burkitt's lymphoma, cervical cancer, colon cancer, and ovarian cancer. Cancer, including but not limited to chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer and vaginal cancer.

[0199] Also provided is the use of engineered NK cells disclosed herein for the treatment of cancer and / or for the preparation of a medicament for the treatment of cancer. In some embodiments, the cancer is multiple myeloma.

[0200] In some embodiments, the polynucleotide encoding the disclosed chimeric antigen receptor (including but not limited to a BCMA-directed chimeric antigen receptor) is an mRNA. In some embodiments, the polynucleotide is a DNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expression of a cytotoxic receptor complex.

[0201] According to some embodiments, there is further provided a vector comprising a polynucleotide encoding any of the polynucleotides provided herein, the polynucleotide being operably linked to at least one regulatory element for expression of a cytotoxic receptor complex. In some embodiments, the vector is a retrovirus.

[0202] Further provided herein are engineered immune cells (e.g., NK and / or T cells) that contain the polynucleotides, vectors, or cytotoxic receptor complexes disclosed herein. Further provided herein are compositions that include a mixture of engineered immune cells (e.g., NK cells and / or engineered T cells), each population containing a polynucleotide, vector, or cytotoxic receptor complex disclosed herein.

[0203] Cancer type Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a chimeric antigen receptor, e.g., a BCMA-directed chimeric antigen receptor, to a subject with cancer. Various embodiments provided herein include the treatment or prevention of the following non-limiting examples of cancer. Examples of cancer include multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytoma, spinal tumor, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumor, Burkitt's lymphoma, cervical cancer, colon cancer, chronic lymphocytic leukemia (CLL), including, but not limited to, chronic myeloid leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cavity cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer and vaginal cancer.

[0204] Cancer Targeting Some embodiments of the compositions and methods described herein relate to immune cells that contain one or more chimeric antigen receptors that target cancer antigens. Non-limiting examples of target antigens include: BCMA, CD19, CD38, CD138 (also known as syndecan 1), G protein-coupled receptor, class C group 5 member D (GPRC5D), SLAMF7, CD229 (SLAMF3), CD123, DLL3, epidermal growth factor receptor (EGFR), prostate specific membrane antigen (PSMA), Fms-like tyrosine kinase 3 (FLT3); KREMEN2 (kringle-containing transmembrane protein 2), placenta-like 2 alkaline phosphatase (ALPPL2), ... rhodin 4, claudin 6, CD5, CD22; CD30; CD171; CS1 (CD2 subset 1, also known as CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen [(Tn Ag) or (GalNAca-Ser / Thr)]; prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 ( CD276; KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific fetal antigen-4 (SSEA-4); CD20;Folate receptor alpha (FRa or FR1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macrophages) in) subunit beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDCla1p(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-Glycine D2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen ( NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1);ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCT Al or galectin 8), melanoma antigen recognized by T cell 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG [transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene]; N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 IB1 (CYPIB1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites; squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal universal 1 (RU1); renal universal 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89);Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR alpha 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen); fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL1 1Ra, IL13Ra2, CD179b-IGL11, TCR gamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1- / HVCR1, CSF2RA (GM-CSFR-alpha), TGF beta R2, Lewis Ag, TCR-beta 1 chain, TCR-beta 2 chain, TCR-gamma chain, TCR-delta chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLVl-Tax, CMV pp65, EBV-EBNA3c, KSHV Antigens recognized by K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), autoantibodies to desmoglein 3 (Dsg3), autoantibodies to desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Liv1, nectin-4, Crypto, gpA33, BST1 / CD157, small conductance chloride channel, and TNT antibodies; EXAMPLES

[0205] The materials and methods disclosed herein are non-limiting examples that may be utilized with specific embodiments disclosed herein.

[0206] [Example 1] Anti-BCMA CAR expression in NK cells According to some embodiments, NK cells are isolated from peripheral blood mononuclear cells and expanded through the use of feeder cell lines. In some embodiments, the feeder cells are engineered to express certain stimulatory molecules (e.g., interleukins, CD3, 4-1BBL, etc.) to promote immune cell expansion and activation. Engineered feeder cells and related methods for expanding NK cells are disclosed, for example, in U.S. Pat. Nos. 7,435,596 or 8,026,097, International Patent Application No. PCT / SG2018 / 050138, International Patent Application No. PCT / US2020 / 044033, and U.S. Provisional Patent Application No. 63 / 073,671, each of which is expressly incorporated herein by reference in its entirety.

[0207] NK cells isolated from PBMCs are co-cultured with K562 cells expressing membrane-bound IL15 and 4-1BBL using medium supplemented with IL2. Viral transduction with a vector encoding an anti-BCMA-directed chimeric antigen receptor construct, such as any one of those disclosed herein, is performed on approximately day 7. Different populations of NK cells are transduced with various anti-BCMA CAR constructs. Any combination of one or more transmembrane domains, one or more hinge domains, one or more costimulatory domains and one or more signaling domains disclosed herein may be used. In some embodiments, the anti-BCMA CAR comprises an OX40 domain and a CD3ζ signaling domain. In some embodiments, the viral vector also encodes interleukin 15, which may be membrane-bound, that is expressed by NK cells together with the anti-BCMA CAR. The resulting engineered NK cells are evaluated on day 14 or more of total culture time.

[0208] Expression of the anti-BCMA CAR construct is assessed by detecting the CAR construct, for example, by assessing the percentage of NK cells in the test population that express a tag sequence incorporated within the CAR (e.g., a FLAG epitope tag or the CD34 and CD20 epitopes of the RQRCD8 hinge) (although embodiments disclosed herein also provide epitope tag-free CAR constructs). At least about 75% or more of the NK cells are expected to stably express the CAR (e.g., for at least 2-3 weeks or more in culture).

[0209] This is a logical example.

[0210] [Example 2] Anti-BCMA CAR expression in T cells According to some embodiments, T cells are isolated from peripheral blood mononuclear cells and expanded through the use of commercially available T cell expansion products (e.g., beads coupled to anti-CD3 and anti-CD28 antibodies).

[0211] Viral transduction of T cells with various vectors encoding anti-BCMA-directed chimeric antigen receptor constructs, such as any one of those disclosed herein, is performed on approximately day 7. Different populations of T cells are transduced with various anti-BCMA CAR constructs. Any combination of one or more transmembrane domains, one or more hinge domains, one or more costimulatory domains and one or more signaling domains disclosed herein may be used. In some embodiments, the anti-BCMA CAR comprises an OX40 domain and a CD3ζ signaling domain. In some embodiments, the viral vector also encodes interleukin 15, which may be membrane-bound, which is expressed by T cells together with the anti-BCMA CAR. The resulting engineered T cells are evaluated on day 14 or more of total culture time.

[0212] Expression of the anti-BCMA CAR construct is assessed by detecting the CAR construct, for example, by assessing the percentage of T cells in the test population that express a tag sequence incorporated within the CAR (e.g., a FLAG epitope tag or CD34 and CD20 epitopes of the RQRCD8 hinge) (although embodiments disclosed herein also provide epitope tag-free CAR constructs). At least about 75% or more of the T cells are expected to stably express the CAR (e.g., for at least 2-3 weeks or more in culture).

[0213] This is a logical example.

[0214] [Example 3] In Vitro Evaluation of Cytotoxicity of Anti-BCMA CAR-Expressing NK and T Cells NK cells and / or T cells expressing various anti-BCMA CARs such as those disclosed herein are co-cultured with tumor cells expressing BCMA, as well as with cells expressing little or no BCMA as a control. Non-transduced NK and / or T cells may also be used as a negative control. Tumor cells may be tagged with a fluorescent detection tag (e.g., GFP) for detection / quantification by flow cytometry. Various effector:target (E:T) ratios, such as 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and / or 1:8, are evaluated. After co-culture, the culture medium is collected and the levels of various cytotoxic or proinflammatory cytokines are evaluated. Tumor cell survival is quantified.

[0215] NK and / or T cells expressing an anti-BCMA CAR and co-cultured with BCMA-expressing tumor cells are seen to result in increased release of cytotoxic effector molecules (e.g., granzyme B, perforin and / or interferon gamma) compared to release of these effectors by non-transduced NK and / or T cells, and BCMA-CAR-expressing NK and / or T cells cultured with tumor cells expressing reduced levels of BCMA.

[0216] NK and / or T cells expressing anti-BCMA CARs and co-cultured with BCMA-expressing tumor cells are expected to exhibit cytotoxic effects against tumor cells in a manner that is dependent on the E:T ratio of a given experiment. The engineered NK and / or T cells are expected to exhibit anti-tumor cell effects that are durable in nature (e.g., may exhibit cytotoxicity for at least 2-3 weeks following transduction with an anti-BCMA CAR).

[0217] This is a logical example.

[0218] [Example 4] In Vivo Evaluation of Cytotoxicity of Anti-BCMA CAR-Expressing NK and T Cells NK and T cells are isolated and expanded from PBMC as described herein. NK and T cells are engineered to express anti-BCMA CARs, such as any one of those disclosed herein, by viral transduction of NK or T cells. Viral transduction is performed approximately 7 days after isolation. Different anti-BCMA CAR constructs are transduced into different populations of NK and T cells. Any combination of one or more transmembrane domains, one or more hinge domains, one or more costimulatory domains and one or more signaling domains disclosed herein may be used. In some embodiments, the anti-BCMA CAR comprises an OX40 domain and a CD3ζ signaling domain. In some embodiments, the viral vector also encodes interleukin 15, which may be membrane-bound, that is expressed by NK cells along with the anti-BCMA CAR. The resulting engineered NK and engineered T cells are evaluated at or above 14 days of total culture time.

[0219] On day 0, immunodeficient NSG mice are inoculated with BCMA-positive tumor cells expressing a luminal marker (e.g., multiple myeloma cells such as NCI-H929, U266-B1 or RPMI-8226) at an appropriate dose (e.g., 1x10 5 Mice are injected intravenously with 100-fold increased NK and / or T cells (cells). On day 1, mice are injected with either PBS control, non-transduced NK and / or T cells, or NK and / or T cells expressing one of the various anti-BCMA CARs disclosed herein. Bioluminescence imaging data is collected at various time points, e.g., days 0, 8, 11, 16, 20, 28, 32, and 40. Blood samples are taken at various time points, e.g., days 5, 15, 20, 25, 30, 35, and 40.

[0220] Blood samples are analyzed for the presence and number of tumor cells using flow cytometry to detect BCMA or another distinguishing cell surface protein. Bioluminescence images are examined for signal intensity over time. Bioluminescence signals are expected to increase over time for the PBS control group and non-transduced NK and / or T cells, indicating an increase in tumor cells. Injection of NK cells expressing an anti-BCMA CAR is expected to result in a reduction in tumor cell proliferation progression. Injection of T cells expressing an anti-BCMA CAR is expected to result in a reduction in tumor cell proliferation progression. Injection of a combination of NK cells expressing an anti-BCMA CAR and T cells expressing an anti-BCMA CAR is expected to result in a significant, even synergistic, reduction in tumor cell proliferation progression. In embodiments targeting additional epitopes of BCMA (e.g., by bispecific CARs or a second CAR expressed by the NK and / or T cells), further enhanced cytotoxicity is expected.

[0221] Mice receiving NK cells and / or T cells expressing the anti-BCMA CAR (or multiple CARs) are expected to show improved survival over control groups.

[0222] This is a logical example.

[0223] [Example 5] Combining anti-BCMA CAR-expressing NK and / or T cells with other cancer targets NK and T cells are isolated and expanded from PBMC as described herein. NK and T cells are engineered to express anti-BCMA CARs, such as any one of those disclosed herein, by viral transduction of NK or T cells. Viral transduction is performed approximately 7 days after isolation. Different anti-BCMA CAR constructs are transduced into different populations of NK and T cells. Any combination of one or more transmembrane domains, one or more hinge domains, one or more costimulatory domains and one or more signaling domains disclosed herein may be used. In some embodiments, the anti-BCMA CAR comprises an OX40 domain and a CD3ζ signaling domain. In some embodiments, the viral vector also encodes interleukin 15, which may be membrane-bound, which is expressed by NK cells together with the anti-BCMA CAR.

[0224] NK cells and / or T cells are engineered to express CARs against additional, e.g., non-BCMA tumor markers. The additional tumor markers are one or more of CD19, CD38, CD138, SLAM-F7 or GPRC5D, or other tumor markers generally known in the art. In some embodiments, a single CAR is engineered to target both BCMA and one or more of CD19, CD38, CD138, SLAM-F7 or GPRC5D, or other tumor markers generally known in the art. As with BCMA-targeting CARs, any combination of one or more transmembrane domains, one or more hinge domains, one or more costimulatory domains, and one or more signaling domains disclosed herein may be used. In some embodiments, the CAR against non-BCMA markers comprises an OX40 domain and a CD3ζ signaling domain. In some embodiments, the viral vector used to transduce the non-BCMA CAR into NK and / or T cells also encodes interleukin 15, which may be membrane bound, that is expressed by the NK cells along with the non-BCMA CAR (or bispecific CAR). The resulting engineered NK and engineered T cells are evaluated at or above day 14 of total culture time.

[0225] On day 0, inoculate immunodeficient NSGs with tumor cells (at an appropriate dose, e.g., 1x10) that are positive for one or more of BCMA-positive, non-BCMA tumor markers (e.g., CD19, CD38, CD138, SLAM-F7 or GPRC5D) and express a luminescent marker. 5Mice are injected intravenously (with BCMA cells). On day 1, mice are injected with either PBS control, non-transduced NK and / or T cells, or NK and / or T cells expressing one of the various anti-BCMA CARs disclosed herein, expressing one of the various non-BCMA CARs disclosed herein, or expressing a bispecific BCMA / non-BCMA CAR. Bioluminescence imaging data is collected at various time points, e.g., days 0, 8, 11, 16, 20, 28, 32, and 40. Blood samples are taken at various time points, e.g., days 5, 15, 20, 25, 30, 35, and 40.

[0226] Blood samples are analyzed for the presence and number of tumor cells using flow cytometry to detect BCMA and non-BCMA cell surface proteins. Bioluminescence images are examined for signal intensity over time. Bioluminescence signals are expected to increase over time for PBS control groups and non-transduced NK and / or T cells, indicating an increase in tumor cells. Injection of NK cells, T cells, and / or a combination of NK and T cells expressing an anti-BCMA CAR and a CAR against a non-BCMA target is expected to result in a reduction in tumor cell proliferation progression. This reduction is expected to result in a greater reduction in tumor growth than cells expressing either anti-BCMA or non-BCMA CARs alone. In some embodiments, a similar reduction in tumor cell proliferation is expected with bispecific CARs targeting BCMA and non-BCMA targets, whether expressed on NK cells, T cells, or a combination of both NK cells and T cells.

[0227] Mice receiving NK cells and / or T cells expressing an anti-BCMA CAR and a non-BCMA targeted CAR (or a single bispecific CAR) are expected to show improved survival versus control groups, as well as versus groups treated with cells expressing only one CAR (either against BCMA or against non-BCMA).

[0228] This is a logical example.

[0229] [Example 6] Screening for target binding-induced signaling versus tonic signaling As mentioned above, the CARs provided herein utilize various structural components and / or use the same components in different structural configurations. To select CARs for immunotherapy, a series of experiments were performed to evaluate how the various CAR components and CAR formats disclosed herein affect expression, target binding, and target binding-induced activation compared to tonic signaling (e.g., signaling from the CAR in the absence of a target). As shown in the figures (as non-limiting embodiments) and described herein, the CARs utilize various structures depending on the embodiment. For example, in some embodiments, different hinge domains are used to operably link the tumor-binding portion of the CAR with the remainder of the CAR (e.g., the transmembrane and signaling regions). In some embodiments, a CD8 alpha-derived domain is used. For example, in some embodiments, CD8 hinge, transmembrane and intracellular domains are used in the CAR. In some embodiments, CD28 is used. In some embodiments, longer or shorter hinge domains are used. Similarly, for scFv-containing CARs, VH-linker-VL or VL-linker-VH formats may be utilized, with the linker noted to vary between constructs. Figure 19 shows the results of the generation of four non-limiting CAR structures: VH-linker-VL-CD8 alpha hinge, VL-linker-VH-CD8 alpha hinge, VH-linker-VL-IgG4 hinge (short), and VH-linker-VL-RQRCD8 hinge (long). Expression was measured by detection of a FLAG tag embedded in the nucleotide sequence encoding the construct. It is understood that any construct presented herein utilizing a FLAG (or other detection tag) is also contemplated in the absence of the tag (and any corresponding additional sequences, e.g., associated linkers). The expression data in Figure 19 is presented as mean fluorescence intensity (MFI).

[0230] As shown in Figure 19, each of these formats was expressed by Jurkat cells, regardless of the linker utilized. Expression did not appear to be significantly affected by the choice of linker in this experiment, whereas the VH-linker-VL scFv format appeared to result in increased expression levels. However, in some embodiments, the VL-linker-VH scFv format may still be expressed well enough to allow CARs utilizing such scFvs to bind to their respective targets (e.g., BCMA) and induce cytotoxicity against such target-expressing tumor cells.

[0231] Cancer immunotherapy requires that engineered cells can bind to tumor cells while avoiding targeting and acting mainly or completely on non-tumor cells. Certain CARs are mainly or only responsive when they bind to the tumor markers that they are engineered to recognize. This is referred to as activation herein. Other CARs that are less desirable for use in immunotherapy show signaling even in the absence of their corresponding tumor markers. This is referred to as tonic signaling herein. Certain experiments discussed herein concern the ratio of activation to tonic signaling, with higher values ​​of the ratio representing higher signaling and / or lower tonic signaling for a given CAR.

[0232] Activation and tonic signaling are evaluated in a model system for evaluating signaling using Jurkat cells (as an alternative to other cells used in cell therapy). Jurkat cells are an immortal human leukemia T cell line that has been widely used in evaluating T cell activation and signaling mechanisms. Jurkat cells do not secrete the full cytokine repertoire secreted by primary T cells, and Jurkat cells lack significant cytolytic activity, but Jurkat cells produce IL-2 and upregulate CD69 upon activation. Due to the ease of detecting cell surface CD69 expression using fluorescent antibodies (e.g., via flow cytometry), we used staining to evaluate the induced expression of CD69 in the presence and absence of BCMA-expressing target cells (here, MM.S1 cells) to determine the activation of Jurkat cells expressing CARs provided herein.

[0233] Figure 20 shows data plotted based on the off signaling of engineered Jurkat cells in the absence of target cells resulting from a 1:1 E:T ratio (Jurkat:MM.1S). Compared to similar experiments where fewer tumor cells were present (e.g., 10:1 E:T, data not shown), an increase in the number of target tumor cells results in an increase in the expression of CD69. As shown in the scatter plot, some of the clones show high activity as well as elevated tonic signaling (top right). Similarly, some clones show low activation and elevated tonic signaling or low activation and low signaling (left part of the scatter plot). However, selected clones show high activation and low tonic signaling, making them attractive for potential use in cancer immunotherapy.

[0234] Such data relating to tonic signaling and BCMA binding can be used to generate a ratio of BCMA binding-induced signaling to tonic signaling, which can be used as a data point to compare a given CAR with others in terms of on-target performance. Figure 21 shows selected non-limiting CAR constructs compared in terms of their activation / tonic signaling ratio. Each of construct A, construct B, and construct C has a different architecture (e.g., VH / VL domain order, linker, etc.). Within a given architecture, each of clones 1, 2, 3, and 4 differs in terms of their binder sequence. Two different control CAR architectures were used as controls. The sequence of tumor marker binders within a given CAR architecture results in some differences in terms of activation / tonic signaling ratio. For example, clone A1 appears to have an elevated ratio compared to clone A4. Similar results are seen, for example, with clone C1 compared to clone C4. Comparison of clones A1 or C1 compared to clones A3 or C3 shows significant differences. While the entire group of clones 1 and 3 share a similar pattern, clones A3 and C3 show significantly higher activation / tonic signaling ratios. These data demonstrate that the architecture of the CAR can have a significant effect on the activation / tonic signaling ratio. Thus, according to some embodiments, the CAR architecture (e.g., VH-linker A-VL, VL-linker A-VH, VH-linker B-VL, or VL-linker B-VH) can be optimized to improve the degree of activity resulting from the binding of the CAR to a tumor marker, e.g., BCMA, while reducing / eliminating tonic signaling that occurs even when the tumor marker is minimally expressed or not expressed at all.

[0235] [Example 7] Evaluation of CAR components for expression and function Based on the results of the previous examples, further experiments were performed to evaluate BCMA-directed CAR expression and function. As provided herein, two series of CARs that differ in terms of their architecture were designed and constructed as outlined below. [Table 1]

[0236] Based on the various structures described above, assays were performed to determine whether specific CAR architectures, and specific sequences within a given architecture, result in improved expression and / or activity (e.g., improved activation and reduced tonic signaling).

[0237] FIG. 22 shows a series of histograms for the expression of each of the clones within a given CAR architecture (architecture F is shown in this figure as non-limiting data). The left panel of FIG. 22 shows the expression of CAR by Jurkat cells, measured by detection of the FLAG tag incorporated within the nucleic acid encoding the CAR. The data is shown as MFI, which represents the extent to which a particular cell expresses the Car (e.g., "copies" of CAR per cell). As can be seen, even within a given architecture, certain clone sequences are expressed more robustly than other clone sequences. The right panel of FIG. 22 relates to the ability of each of the clones within a given architecture to bind to their intended target, BCMA. Interestingly, while some robustly expressing cells achieved elevated BCMA binding, even some that were low expressers still showed BCMA binding comparable to other CARs that had higher average expression. This data indicates that not only is sequence important for expression, but that binding may be expression independent. Therefore, in some embodiments, analysis of both aspects of a CAR is important insofar as a low-expressing CAR may overcome its low expression level by improving binding activity.

[0238] Another feature that can be evaluated is the ratio of activation (based on target binding) to tonic signaling, as discussed above. This ratio helps evaluate the potential "on-target" and "off-target" potential of a particular CAR. Figures 23A-23B show scatter plots for BCMA binding-induced activation (23A) and undesired tonic signaling (23B). As seen in Figure 23A, several of the constructs (from different architectures, even within the architecture) achieve CAR activation based on binding to BCMA, which is shown as circles above a horizontal threshold line of values ​​10,000. Those below the threshold line were considered to have insufficient activation to pass the screen (at least based solely on activation). Figure 23B shows the corresponding tonic signaling data. Again, CAR-expressing cells that are activated even in the absence of all or substantially all target tumor markers are considered to exhibit some degree of tonic signaling. Circles above the 10,000 unit threshold are considered to exhibit some degree of tonic signaling. Those below the threshold are not. However, the evaluation of only tonic signaling or activation is a partial analysis. Looking at clones that show activation above the threshold but not tonic signaling, five CARs meet these criteria in this particular screen. They are identified by arrows in Figure 23A.

[0239] Based on this analysis, Figure 24 shows a scatter plot of BCMA binding as a function of CAR expression, with labels indicating various groupings of CARs. The bottom left of the plot shows CARS that do not express well enough and do not show sufficient BCMA binding. The bottom right shows a subpopulation of CARs that express well but show poor BCMA binding. The two clones in the center of the plot show good expression and good BCMA binding. However, as discussed above, for a more robust understanding of a given CAR, expression and activation can be considered along with an assessment of tonic signaling (or lack thereof).

[0240] Figure 25 shows a scatter plot of the activation / tonic signaling ratio in a screen of a portion of the CARs provided herein. The activation / tonic signaling ratio is shown on the Y-axis, and expression is shown on the X-axis. As in the previous figure, the lower left portion shows low expression or a low activation / tonic signaling ratio (showing a higher than desired degree of tonic signaling, which lowers the ratio). The lower right portion shows clones with reasonable expression but undesirable activation / tonic signaling ratios. A number of clones in the center of the graph show various amounts of acceptable expression levels and high activation / tonic signaling ratios.

[0241] Further evaluation of the impact of CAR architecture was performed to determine whether a particular feature of the CAR architecture has a dominant effect on expression, target binding, or activation / tonic signaling. Figures 26A and 26B show data related to activation / tonic signaling ratio (26A) and expression (26B) when using the GS linker to Whitlow. While certain individual clones appear to behave uniquely, the majority of clones do not appear to deviate from the norm in terms of activation / tonic signaling or expression. A somewhat broader distribution of expression and activation / tonic signaling ratios was observed when comparing the orientation of the heavy and light chains in the scFv. Figure 26C shows the activation / tonic signaling ratio when comparing the VL-linker-VH scFv structure (X-axis) versus the VH-linker-VL scFv structure (Y-axis). Certain clones show improved activation with VH-VL order, while others show better activity with VL-VH order. Similarly, clone-specific results with respect to expression are shown in Figure 26D.

[0242] Because certain CARs in this screen differ in subcomponents of the transmembrane and signaling domains, additional comparisons were made to compare the activation / tonic signaling ratio in CARs with CD28-derived sequences for the hinge, transmembrane and intracellular domains compared to those using the corresponding domains from CD8 alpha (including the OX40 costimulatory domain). Here, the data seems to suggest a general trend toward improved activation / tonic signaling ratios when using the CD8-OX40 domain versus the CD28 domain, as shown in FIG. 26E. This also seems to be the case, and perhaps even more evident, with respect to the expression of the CAR constructs, as shown in FIG. 26F. These data indicate that both structural and sequence-based changes can affect CAR expression, binding and activation. According to some embodiments disclosed herein, BCMA-directed CARs can achieve appropriate expression in immune cells (e.g., NK cells), as well as desirable levels of activation and tonic signaling, making them suitable candidates for BCMA-directed cancer immunotherapy.

[0243] [Example 8] Evaluation of VHH-containing CARs Also provided herein are CARs that utilize camelid-based tumor binders rather than scFv tumor binder formats. In some embodiments, monovalent (single VHH) CARs are used, whereas in other embodiments, bivalent (VHH-linker-VHH) CAR formats are used. As with the examples discussed above, experiments were performed to determine whether structural or sequence-based changes to the VHH-CAR architecture affect expression, target binding, and / or activation of cells expressing the VHH-CAR. Monovalent or bivalent VHH-CARs were expressed in Jurkats to assess expression, BCMA binding, and activation / tonic signaling ratios.

[0244] Figure 27 shows data plotting the degree of expression of monovalent and bivalent VHH-CARs against BCMA binding. These data show relatively similar expression for both monovalent (circles) and bivalent (triangles) VHH-CARs, with variation between individual clones. These data also seem to show a trend of increased BCMA binding for bivalent VHH-CARs, which is not entirely unexpected given the dual binding domain format of bivalent VHH-CARs.

[0245] Figures 28A-28B further support this initial suggestion. Figure 28A shows data on the expression of bivalent versus monovalent VHH-CARs. As the scatter plot shows, the majority of the data points are above the equivalence threshold line, indicating that expression is higher for bivalent VHH-CARs. Similarly, Figure 28B shows results in which BCMA binding appears to be more robust, albeit less significant, when using bivalent VHH-CARs compared to monovalent VHH-CARs.

[0246] However, as discussed above, there is a need to understand the potential interplay between binding of target tumor markers and activation (e.g., tonic activation) of cells expressing CARs in the absence of target tumor markers. Figure 29A shows data related to detection of tonic signaling of bivalent and monovalent VHH-CARs. The data suggests that tonic signaling is more prevalent in the bivalent VHH-CAR format. Figure 29B evaluates the activation / tonic signaling ratio and shows a fairly even distribution of ratios between bivalent versus monovalent formats. This may be explained, in part, by the apparent increase in BCMA binding exhibited by the bivalent constructs. Thus, both the numerator (binding / activation) and denominator (tonic signaling) are elevated in the bivalent format, making the ratio-based evaluation more similar to the monovalent ratio.

[0247] Figures 29C-29D retrieve a portion of the data using six VHH-CARs that exhibit the most favorable characteristics for monovalent and bivalent formats. Figure 29C is a histogram comparing the activation / tonic signaling ratio. The data suggests that the monovalent formats exhibit a generally higher ratio than the bivalent formats. The underlying mechanism appears to be supported by Figure 29B, which shows that the tonic signaling of the bivalent formats is much higher than the monovalent constructs.

[0248] Taking each of these examples into account, an analysis was performed to compare scFv-CARs to VHH-CARs. The data are shown in Figures 30A and 30B. Figure 30A shows the degree of tonic signaling as a function of expression. Candidate VHH-CARs show significantly higher tonic signaling than candidate scFvs (as well as control VHH-CARs). Figure 30B shows data related to cell activation, revealing that while VHH-CARs have generally higher activation than scFvs, the typically low activation of scFv CARs when bound to BCMA, combined with the dramatically lower tonic signaling, makes the scFv CAR format appear to operate in a favorable manner and therefore a promising candidate for use in cellular immunotherapy. While this particular data suggests that scFv-CARs are preferred, in some embodiments / formats, VHH-CARs are also available. Nonetheless, these data suggest that independent evaluation of each of these parameters may be warranted to fully understand the characteristics of a given CAR.

[0249] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within the scope of one or more of the present inventions. Moreover, disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc. in the context of one embodiment may be used in all other embodiments described herein. It is therefore understood that the various features and aspects of the disclosed embodiments may be combined or substituted to form various modes of the disclosed invention. Thus, it is not intended that the scope of the invention disclosed herein should be limited by the specific embodiments disclosed above. Moreover, the invention is susceptible to various modifications and alternative forms, examples of which are shown in the drawings and described in detail herein. However, the invention is not limited to the specific forms and methods disclosed, but on the contrary, it is understood that the invention covers all modifications, equivalents, and alternatives that fall within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein need not be performed in the order recited. The methods disclosed herein include specific actions taken by a practitioner; however, the methods may also include third-party instructions of any of those actions, whether explicit or implied. Moreover, where features or aspects of the disclosure are described in terms of Markush groups, those of skill in the art will recognize that the disclosure is then also described in terms of individual members or subgroups of members of the Markush group.

[0250] Ranges disclosed herein include any overlaps, subranges, and combinations thereof. Terms such as "up to," "at least," "greater than," "less than," "between," and the like, include the recited number. Numbers preceded by terms such as "about" or "approximately" include the recited number. For example, "about 90%" includes "90%." In some embodiments, sequences with at least 95% sequence identity include sequences with 96%, 97%, 98%, 99%, and 100% sequence identity to a reference sequence. Furthermore, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference also includes that the sequence "comprises," "consists of," or "consists essentially of" the recited sequence, unless otherwise specified.

[0251] In some embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein are provided, accounting for the degeneracy of the nucleic acid code. Additionally, those sequences (whether nucleic acid or amino acid) that differ from the sequences explicitly disclosed herein, but have functional similarity or equivalence, are also contemplated within the scope of the present disclosure. This includes mutations, truncations, substitutions, or other types of modifications.

[0252] Any names or subheadings used herein are for organizational purposes only and should not be used to limit the scope of the embodiments disclosed herein.

[0253] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein in their entirety by reference and made a part of this specification. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification supersede and / or take precedence over any such conflicting material.

Claims

1. An anti-BCMA binding moiety comprising a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3, wherein the VH contains HCDR1, HCDR2, and HCDR3 contained within the VH amino acid sequence described in any one of SEQ ID NOs: 270, 268, 260-267, 269, 271-285, and 1582-1600, and the VL contains LCDR1, LCDR2, and LCDR3 contained within the VL amino acid sequence described in any one of SEQ ID NOs: 551, 549, 541-548, 550, 552-566, and 1677-1695.

2. The anti-BCMA binding moiety according to claim 1, wherein the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in any one of SEQ ID NOs: 270, 268, 260-267, 269, 271-285 and 1582-1600, and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in any one of SEQ ID NOs: 551, 549, 541-548, 550, 552-566 and 1677-1695.

3. The anti-BCMA binding moiety according to claim 1, wherein the VH comprises an amino acid sequence described in any one of SEQ ID NOs: 270, 268, 260-267, 269, 271-285 and 1582-1600, and the VL comprises an amino acid sequence described in any one of SEQ ID NOs: 551, 549, 541-548, 550, 552-566 and 1677-1695.

4. (a) The VH includes HCDR1, HCDR2, and HCDR3 contained in the VH amino acid sequence described in SEQ ID NO: 270, and the VL includes LCDR1, LCDR2, and LCDR3 contained in the VL amino acid sequence described in SEQ ID NO: 551; (b) The VH comprises HCDR1, HCDR2, and HCDR3 contained within the VH amino acid sequence described in SEQ ID NO: 268, and the VL comprises LCDR1, LCDR2, and LCDR3 contained within the VL amino acid sequence described in SEQ ID NO: 549; (c) The VH comprises HCDR1, HCDR2, and HCDR3 contained in the VH amino acid sequence described in SEQ ID NO: 1600, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL amino acid sequence described in SEQ ID NO: 1695; or (d) The anti-BCMA binding moiety according to claim 1, wherein the VH comprises HCDR1, HCDR2, and HCDR3 contained in the VH amino acid sequence described in SEQ ID NO: 1586, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL amino acid sequence described in SEQ ID NO: 1681.

5. (a) The VH comprises an amino acid sequence having at least 90% sequence identity with the VH amino acid sequence described in SEQ ID NO: 270, and the VL comprises an amino acid sequence having at least 90% sequence identity with the VL amino acid sequence described in SEQ ID NO: 551; (b) The VH comprises an amino acid sequence having at least 90% sequence identity with the VH amino acid sequence described in SEQ ID NO: 268, and the VL comprises an amino acid sequence having at least 90% sequence identity with the VL amino acid sequence described in SEQ ID NO: 549; (c) The VH comprises an amino acid sequence having at least 90% sequence identity with the VH amino acid sequence described in SEQ ID NO: 1600, and the VL comprises an amino acid sequence having at least 90% sequence identity with the VL amino acid sequence described in SEQ ID NO: 1695; or (d) The anti-BCMA binding moiety according to claim 1, wherein the VH comprises an amino acid sequence having at least 90% sequence identity with the VH amino acid sequence described in SEQ ID NO: 1586, and the VL comprises an amino acid sequence having at least 90% sequence identity with the VL amino acid sequence described in SEQ ID NO: 1681.

6. (a) The VH comprises the amino acid sequence described in SEQ ID NO: 270, and the VL comprises the amino acid sequence described in SEQ ID NO: 551; (b) The VH comprises the amino acid sequence described in SEQ ID NO: 268, and the VL comprises the amino acid sequence described in SEQ ID NO: 549; (c) The VH comprises the amino acid sequence described in SEQ ID NO: 1600, and the VL comprises the amino acid sequence described in SEQ ID NO: 1695; or (d) The anti-BCMA binding moiety according to claim 1, wherein VH comprises the amino acid sequence described in SEQ ID NO: 1586, and VL comprises the amino acid sequence described in SEQ ID NO: 1681.

7. The anti-BCMA binding moiety is a single-chain variable fragment (scFv), Fv, Fab, or F(ab)'. ２ The anti-BCMA binding portion according to claim 1.

8. The anti-BCMA bonding portion according to claim 1, wherein VH and VL are connected by a linker.

9. The anti-BCMA binding moiety according to claim 8, wherein the linker comprises the amino acid sequence described in SEQ ID NO: 1388 or SEQ ID NO: 2388.

10. The anti-BCMA binding portion according to claim 1, wherein the anti-BCMA binding portion is a single-chain variable fragment (scFv).

11. The anti-BCMA binding portion according to claim 10, wherein the VH is located at the N-terminus of the VL.

12. The anti-BCMA binding moiety according to claim 11, wherein the scFv comprises the amino acid sequence described in any one of sequence numbers 593 to 618.

13. The anti-BCMA binding portion according to claim 10, wherein the VL is located at the N-terminus of the VH.

14. The anti-BCMA binding moiety according to claim 13, wherein the scFv comprises the amino acid sequence described in any one of sequence numbers 671 to 696.

15. An anti-BCMA binding moiety comprising a first heavy chain variable region (VHH1), wherein the VHH1 comprises HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in any one of SEQ ID NOs. 3255 to 3277.

16. The anti-BCMA binding moiety according to claim 15, wherein the VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in any one of SEQ ID NOs. 3255 to 3277.

17. The anti-BCMA binding moiety according to claim 15, wherein the VHH1 comprises the amino acid sequence described in any one of SEQ ID NOs: 3255 to 3277.

18. (a) The VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in Sequence ID No. 3262; (b) The VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in Sequence ID No. 3263; (c) The VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in Sequence ID No. 3264; (d) The VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in Sequence ID No. 3265; (e) The VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in Sequence ID No. 3271; or (f) The anti-BCMA binding moiety according to claim 15, wherein the VHH1 includes HCDR1, HCDR2, and HCDR3 contained in the VHH1 amino acid sequence described in SEQ ID NO: 3273.

19. (a) The VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3262; (b) The VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3263; (c) The VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3264; (d) The VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3265; (e) The VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 3271; or (f) The anti-BCMA binding moiety according to claim 15, wherein the VHH1 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 3273.

20. (a) The VHH1 comprises the amino acid sequence described in Sequence ID No. 3262; (b) The VHH1 comprises the amino acid sequence described in Sequence ID No. 3263; (c) The VHH1 comprises the amino acid sequence described in Sequence ID No. 3264; (d) The VHH1 comprises the amino acid sequence described in Sequence ID No. 3265; (e) The VHH1 comprises the amino acid sequence described in Sequence ID No. 3271; or (f) The anti-BCMA binding moiety according to claim 15, wherein the VHH1 comprises the amino acid sequence described in SEQ ID NO: 3273.

21. The anti-BCMA binding moiety according to claim 15, further comprising a second heavy chain variable region (VHH2), wherein the VHH2 comprises HCDR1, HCDR2, and HCDR3 contained within the VHH2 amino acid sequence described in any one of SEQ ID NOs. 3255 to 3277.

22. The anti-BCMA binding moiety according to claim 21, wherein the VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in any one of SEQ ID NOs: 3255 to 3277.

23. The anti-BCMA binding moiety according to claim 21, wherein the VHH2 comprises the amino acid sequence described in any one of sequence numbers 3255 to 3277.

24. (a) The VHH2 includes HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in Sequence ID No. 3262; (b) The VHH2 includes HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in Sequence ID No. 3263; (c) The VHH2 includes HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in Sequence ID No. 3264; (d) The VHH2 includes HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in Sequence ID No. 3265; (e) The VHH2 includes HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in Sequence ID No. 3271; or (f) The anti-BCMA binding moiety according to claim 21, wherein the VHH2 comprises HCDR1, HCDR2, and HCDR3 contained in the VHH2 amino acid sequence described in SEQ ID NO: 3273.

25. (a) The VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3262; (b) The VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3263; (c) The VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3264; (d) The VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in Sequence ID No. 3265; (e) The VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 3271; or (f) The anti-BCMA binding moiety according to claim 21, wherein the VHH2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 3273.

26. (a) The VHH2 comprises the amino acid sequence described in Sequence ID No. 3262; (b) The VHH2 comprises the amino acid sequence described in Sequence ID No. 3263; (c) The VHH2 comprises the amino acid sequence described in Sequence ID No. 3264; (d) The VHH2 comprises the amino acid sequence described in Sequence ID No. 3265; (e) The VHH2 comprises the amino acid sequence described in Sequence ID No. 3271; or (f) The anti-BCMA binding moiety according to claim 21, wherein the VHH2 comprises the amino acid sequence described in SEQ ID NO: 3273.

27. The anti-BCMA bonding portion according to claim 21, wherein the VHH1 and VHH2 are connected by a linker.

28. The anti-BCMA binding moiety according to claim 27, wherein the linker comprises the amino acid sequence described in SEQ ID NO: 1388 or SEQ ID NO: 2388.

29. The anti-BCMA binding moiety according to claim 21, wherein the anti-BCMA binding moiety includes the amino acid sequence described in any one of SEQ ID NOs. 3324 to 3346.

30. (a) The anti-BCMA binding moiety according to any one of claims 1, 15, and 21; (b) Hinged domain; (c) Transmembrane domain; and (d) A BCMA-directed CAR comprising an intracellular signaling domain including a co-stimulatory subdomain and a CD3ζ subdomain.

31. The BCMA-directional CAR according to claim 30, wherein the co-stimulatory subdomain includes the OX40 subdomain, the CD28 subdomain, or the 4-1BB subdomain.

32. The BCMA-directed CAR according to claim 30, wherein the BCMA-directed CAR is encoded by a nucleic acid sequence that also encodes membrane-bound interleukin 15 (mbIL15).

33. The anti-BCMA binding moiety according to any one of claims 1, 15, and 21; or A polynucleotide encoding a BCMA-directed CAR, comprising (a) an anti-BCMA binding moiety according to any one of claims 1, 15, and 21, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain comprising a co-stimulatory subdomain and a CD3ζ subdomain.

34. A vector comprising the polynucleotide of claim 33.

35. An immune cell comprising an anti-BCMA binding moiety according to any one of claims 1 to 29; or a BCMA-directed CAR comprising (a) an anti-BCMA binding moiety according to claim 1, 15, or 21, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain comprising a costimulatory subdomain and a CD3ζ subdomain; or a polynucleotide encoding the anti-BCMA binding moiety or the BCMA-directed CAR; or a vector comprising the polynucleotide.

36. The immune cell according to claim 35, wherein the immune cell is a natural killer (NK) cell or a T cell.

37. A composition comprising immune cells as described in claim 35.

38. A pharmaceutical composition comprising the immune cells described in claim 35 for treating cancer.

39. The pharmaceutical composition according to claim 38, wherein the cancer is multiple myeloma.

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