Chimeric antigen receptors specific for B-cell maturation antigen and encoding polynucleotides

This application is a divisional of U.S. patent application Ser. No. 16/178,571, filed Nov. 1, 2018, entitled, “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” which claims priority from U.S. provisional application 62/580,439, filed Nov. 1, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/580,445, filed Nov. 1, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/582,932, filed Nov. 7, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/582,938, filed Nov. 7, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/596,765, filed Dec. 8, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/596,763, filed Dec. 8, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/614,960, filed Jan. 8, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/614,963, filed Jan. 8, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/665,442, filed May 1, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” and U.S. provisional application No. 62/665,447, filed May 1, 2018, entitled “METHOD OF ASSESSING ACTIVITY OF RECOMBINANT ANTIGEN RECEPTORS,” the contents of which are incorporated by reference in their entirety.

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042009910SeqList.txt, created Jun. 21, 2021, which is 606,341 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

The present disclosure relates in some aspects to chimeric antigen receptors (CARs), which contain antibody portions specific to B-cell maturation antigen (BCMA) and polynucleotides that encode CARs specific for BCMA. The disclosure further relates to genetically engineered cells, containing such BCMA-binding receptors, and uses thereof in adoptive cell therapy.

B-cell maturation antigen (BCMA) is a transmembrane type III protein expressed on mature B lymphocytes. Following binding of BCMA to its ligands, B cell activator of the TNF family (BAFF) or a proliferation inducing ligand (APRIL), a pro-survival cell signal is delivered to the B cell which has been found to be required for plasma cell survival. The expression of BCMA has been linked to several diseases including cancer, autoimmune disorders and infectious diseases Due to the role of BCMA in various diseases and conditions, including cancer, BCMA is a therapeutic target. Various BCMA-binding chimeric antigen receptors (CARs), and cells expressing such CARs, are available. However, there remains a need for improved BCMA-binding CARs and engineered BCMA-CAR expressing targeting cells, such as for use in adoptive cell therapy. Provided herein are embodiments that meet such needs.

Provided are polynucleotides encoding a chimeric antigen receptor, containing nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some cases, the spacer is derived from an immunoglobulin. In some embodiments, the spacer includes a sequence of a hinge region, a C H 2 and a C H 3 region. In some embodiments, one of more of the hinge, C H 2 and C H 3 is derived all or in part from IgG4 or IgG2. In some cases, the hinge, C H 2 and C H 3 is derived from IgG4. In some aspects, one or more of the hinge, C H 2 and C H 3 is chimeric and contains sequence derived from IgG4 and IgG2. In some examples, the spacer contains an IgG4/2 chimeric hinge, an IgG2/4 C H 2, and an IgG4 C H 3 region. In some embodiments, the encoded spacer is or contains (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes the sequence set forth in SEQ ID NO: 649.

In some of any embodiments, the spacer has a length of 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the foregoing.

In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the spacer includes at least one modified splice donor and/or splice acceptor site, said modified splice donor and/or acceptor site containing one or more nucleotide modifications corresponding to a reference splice donor site and/or reference splice acceptor site contained in the sequence set forth in SEQ ID NO:621. In some cases, the one or more nucleotide modifications contains an insertion, deletion, substitution or combinations thereof. In some instances, the reference splice acceptor and/or reference splice donor sites are canonical, non-canonical, or cryptic splice sites. In some examples, the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

In some embodiments of any of the polynucleotides described herein, the reference splice donor site includes the sequence aatctaagtacggac (SEQ ID NO: 705), tcaactggtacgtgg (SEQ ID NO:706), acaattagtaaggca (SEQ ID NO:707) and/or accacaggtgtatac (SEQ ID NO:708); and/or the reference splice acceptor site includes the sequence aagtttctttctgtattccaggctgaccgtggataaatctc (SEQ ID NO:742) and/or gggcaacgtgttctcttgcagtgtcatgcacgaagccctgc (SEQ ID NO:743). In some embodiments, the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, the reference splice donor site contains the sequence tcaactggtacgtgg (SEQ ID NO:706); and/or the reference splice acceptor site contains the sequence aagtttctttctgtattccaggctgaccgtggataaatctc (SEQ ID NO:742).

In some embodiments of any of the polynucleotides described herein, at least one of the one or more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the reference splice acceptor and/or reference splice donor site. In some aspects, the one or more nucleotide modifications is silent and/or results in a degenerate codon compared to SEQ ID NO:621 and/or does not change the amino acid sequence of the encoded spacer. In some embodiments, the modified splice donor site is set forth in agtctaaatacggac (SEQ ID NO:661), tcaactggtatgtgg (SEQ ID NO:662), accatctccaaggcc (SEQ ID NO:663) and/or gccccaggtttacac (SEQ ID NO:664); and/or the modified splice acceptor site is set forth in cagtttcttcctgtatagtagactcaccgtggataaatcaa (SEQ ID NO:672) gggcaacgtgttcagctgcagcgtgatgcacgaggccctgc (SEQ ID NO: 673) and/or aagtttctttctgtattccagactgaccgtggataaatctc (SEQ ID NO:854). In some cases, the modified splice donor site is set forth in tcaactggtatgtgg (SEQ ID NO:662) and/or the modified acceptor site is set forth in cagtttcttcctgtatagtagactcaccgtggataaatcaa (SEQ ID NO:672). In some of any such embodiments, the spacer is encoded by a sequence of nucleotide set forth in SEQ ID NO:622 or a portion thereof.

Provided is a polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide includes nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is or includes the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.

Also provided is a polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide including nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid includes or mostly includes the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.

In some of any of the embodiments, following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments, following expression in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide exhibits reduced heterogeneity compared to the heterogeneity of the mRNA transcribed from a reference polynucleotide, said reference polynucleotide encoding the same amino acid sequence as the polynucleotide, wherein the reference polynucleotide differs by the presence of one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer and/or includes one or more nucleotide modifications compared to the polynucleotide. In some instances, the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the transcribed RNA, optionally messenger RNA (mRNA), from the reference polynucleotide exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some of any such embodiments, the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography. In some of any such embodiments, the polynucleotide is codon-optimized.

In some embodiments of any of the polynucleotides described herein, the antigen is associated with the disease or condition or expressed in cells of the environment of a lesion associated with the disease or condition. In some cases, the disease or condition is a cancer. In some examples, the disease or condition is a myeloma, leukemia or lymphoma. In some embodiments, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR viii, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen. In some cases, the antigen is B cell maturation antigen (BCMA).

In some of any such embodiments, the antigen-binding domain is an antibody fragment containing a variable heavy chain (V H ) and a variable light chain (V L ) region. In some aspects, the V H region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609 617, 772-774, or 814-832; and/or the V L region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some cases, the V H region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the V L region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.

In some embodiments of any of the polynucleotides described herein, the V H region is or contains a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the V L region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some embodiments, the V H region is or contains a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the V L region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849. In some embodiments, the V H region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295,507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or the V L region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.

In some embodiments of any of the polynucleotides described herein, the V H region is or contains (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or the V L region is or contains (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603.

In some embodiments of any of the polynucleotides described herein, the V H region contains a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively.

In some embodiments of any of the polynucleotides described herein, the V H region contains a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;

In some embodiments of any of the polynucleotides described herein, the V H region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832. In some aspects, the V H region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617. In some embodiments, the V H region contains a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or the V H region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively. In some embodiments, the V H region is or includes the amino acid sequence set forth in SEQ ID NO: 617.

In some embodiments of any of the polynucleotides described herein, the V L region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.

In some embodiments of any of the polynucleotides described herein, the V L region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.

In some of any such embodiments, the V L region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some aspects, the V L region is or contains the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.

In some embodiments of any of the polynucleotides described herein, the V L region contains a CDR-L1, CDR-L2, and CDR-L3 including the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or the V L region contains a CDR-L1, CDR-L2, and CDR-L3 including the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively. In some cases, the V L region is or includes the amino acid sequence set forth in SEQ ID NO:618.

In some of any embodiments, the V H region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region sequence of any of SEQ ID NOs:617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the V L region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.

In some of any embodiments, the V H region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the V L region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.

In some of any embodiments, the V H region is or comprises (a) a CDR-H1 comprising the sequence selected from any one of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289, 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 comprising the sequence selected from any one of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 comprising the sequence selected from any one of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517, or 606; and the V L region is or comprises (a) a CDR-L1 comprising the sequence selected from any one of SEQ ID NOs: 601, 614, 26-36, 174-178, 302, 303, 380-392, 394-398, 589, or 607; (b) a CDR-L2 comprising the sequence selected from any one of SEQ ID NOs: 602, 615, 37-46, 179-183, 304, 305, 399-409, 411-414, 590, or 608; and (c) a CDR-L3 comprising the sequence selected from any one of SEQ ID NOs: 603, 47-58, 184-194, 306, 307, 415-427, 429-433, or 591.

In some of any such embodiments, the V H region and the V L regions includes the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 111 and 117, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 114 and 126, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 115 and 536, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 115 and 555, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:772 and 775, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:773 and 776, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:774 and 777, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:815 and 833, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:816 and 834, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:817 and 835, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:818 and 836, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:819 and 837, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:820 and 838, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:821 and 839, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:822 and 840, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:823 and 841, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:824 and 842, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:825 and 843, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:826 and 844, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:827 and 845, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:828 and 846, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:829 and 847, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:830 and 847, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:831 and 848, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:832 and 849, respectively.

In some embodiments of any of the polynucleotides described herein, the V H region and the V L regions encoded by the polynucleotides include the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 112 and 124, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:772 and 775, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:773 and 776, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:774 and 777, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:815 and 833, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:816 and 834, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:817 and 835, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:818 and 836, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:819 and 837, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:820 and 838, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:821 and 839, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:822 and 840, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:823 and 841, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:824 and 842, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:825 and 843, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:826 and 844, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:827 and 845, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:828 and 846, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:829 and 847, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:830 and 847, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:831 and 848, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:832 and 849, respectively.

In some of any embodiments, the V H region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the V L region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.

In some embodiments of any of the polynucleotides described herein, the fragment includes an scFv. In some embodiments, the V H region and the V L region are joined by a flexible linker. In some embodiments, the scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:361). In some embodiments, the V H region is amino-terminal to the V L region.

In some embodiments of any of the polynucleotides described herein, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771. In some embodiments, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.

In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the antigen-binding domain includes (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen-binding domain includes (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 352, 647, 648, 716, or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 352, 647, 648, 716, or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen-binding domain is codon-optimized. In some embodiments, the nucleic acid encoding the antigen-binding domain includes the sequence of nucleotides set forth in any of SEQ ID NO: 440, 460, 715, 717 or 719. In some embodiments, the nucleic acid encoding the antigen-binding domain includes the sequence of nucleotides set forth in SEQ ID NO:460.

In some embodiments of any of the polynucleotides described herein, the V H region is carboxy-terminal to the V L region. In some embodiments, the scFv includes the amino acid sequence set forth in SEQ ID NOs:328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:328 or 586.

Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (V H ) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V H region sequence of SEQ ID NO: 617; and (ii) a variable light chain (V L ) region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region sequence of any of SEQ ID NO: 618; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3) chain and an intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some of any embodiments, the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.

Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: a variable heavy (V H ) region comprising a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and a variable light (V L ) region comprising a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a human CD3-zeta (CD3) chain and an intracellular signaling domain of a human 4-1BB or a human CD28. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some of any embodiments, the extracellular antigen-binding domain comprises the V H region sequence of SEQ ID NO:617 and the V L region sequence of SEQ ID NO:618.

In some embodiments, the receptor includes an antigen-binding domain that binds to the same or substantially the same epitope on BCMA, or competes for binding to BCMA with, any of the antibodies and fragments, or antibodies having the provided combinations of V H /V L or CDR sequences, described herein including in any of the foregoing embodiments. In some embodiments, the binding domain recognizes an epitope comprising a portion of one or more amino acid sequences within a BCMA polypeptide. In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), YFDSL (SEQ ID NO:779), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), YFDSLL (SEQ ID NO:641), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), QNEYFDSLL (SEQ ID NO:780), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: QNEYF (SEQ ID NO:637), CIPCQL (SEQ ID NO:638), and CQRYC (SEQ ID NO:639). In some aspects, such one or more amino acid sequences are or comprise: CSQNEYF (set forth in SEQ ID NO:410) and LLHACIPCQLR (set forth in SEQ ID NO:428).

In some embodiments of any of the polynucleotides described herein, the intracellular signaling region includes an activating cytoplasmic signaling domain. In some embodiments, the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the activating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3) chain or a functional variant or signaling portion thereof. In some embodiments, the activating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628.

In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:627 or is a codon-optimized sequence and/or degenerate sequence thereof. In other embodiments, the nucleic acid encoding the activating cytoplasmic signaling domain is or includes the sequence set forth in SEQ ID NO:652.

In some embodiments of any of the polynucleotides described herein, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In other embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626.

In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the costimulatory region is or includes the sequence set forth in SEQ ID NO:625 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the costimulatory signaling region includes the sequence set forth in SEQ ID NO:681. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In other embodiments, the transmembrane domain is or includes the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO: 624.

In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the transmembrane domain is or includes the sequence set forth in SEQ ID NO:623 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the transmembrane domain includes the sequence set forth in SEQ ID NO:688. In some embodiments of any of the polynucleotides described herein, the encoded chimeric antigen receptor includes from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.

In some of any of the embodiments, the polynucleotide comprises the sequence set forth in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 751-756 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 755 and 756 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NOs:755 or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NOs:755 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity.

In some embodiments, the polynucleotide further encodes a truncated receptor

Also provided are vectors comprising any of the polynucleotides described herein. In some of any embodiments, the vector is a viral vector. In some of any embodiments, the viral vector is a retroviral vector. In some of any embodiments, the viral vector is a lentiviral vector.

Provided in some aspects are chimeric antigen receptors encoded a polynucleotide of any of the embodiments described herein. In some embodiments, the chimeric antigen receptor includes: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.

In some embodiments of any of the chimeric antigen receptors described herein, the spacer is derived from an immunoglobulin. In some embodiments, the spacer includes a sequence of a hinge region, a C H 2 and C H 3 region. In some embodiments of any of the chimeric antigen receptors described herein, one of more of the hinge, C H 2 and C H 3 is derived all or in part from IgG4 or IgG2. In some embodiments, the hinge, C H 2 and C H 3 is derived from IgG4. In some embodiments, one or more of the hinge, C H 2 and C H 3 is chimeric and includes sequence derived from IgG4 and IgG2. In some embodiments, the spacer includes an IgG4/2 chimeric hinge, an IgG2/4 C H 2, and an IgG4 C H 3 region.

In some embodiments of any of the chimeric antigen receptors described herein, the spacer is or includes (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes the sequence set forth in SEQ ID NO: 649.

Provided in other aspects are chimeric antigen receptors that include (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments of any of the chimeric antigen receptors described herein, the antigen-binding domain is an antibody fragment containing a variable heavy chain (V H ) and a variable light chain (V L ) region.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the V L region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the V L region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the V L region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the V L region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or the V L region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or the V L region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) alight chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603. In some embodiments of any of the chimeric antigen receptors described herein, the V H region includes a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;

In some embodiments of any of the chimeric antigen receptors described herein, the V H region includes a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832. In some embodiments of any of the chimeric antigen receptors described herein, the V H region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832. In some embodiments, the V H region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or the V H region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively. In some embodiments, the V H region is or includes the amino acid sequence set forth in SEQ ID NO:617.

In some embodiments of any of the chimeric antigen receptors described herein, the V L region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.

In some embodiments of any of the chimeric antigen receptors described herein, the V L region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.

In some embodiments of any of the chimeric antigen receptors described herein, the V L region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some embodiments of any of the chimeric antigen receptors described herein, the V L region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.

In some embodiments of any of the chimeric antigen receptors described herein, the V L region includes a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or the V L region includes a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;

In some embodiments of any of the chimeric antigen receptors described herein, the V L region is or includes the amino acid sequence set forth in SEQ ID NO:618. In some embodiments of any of the chimeric antigen receptors described herein, the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:114 and 126, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 555, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO: 112 and 124, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the V H region and the V L regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.

In some embodiments of any of the chimeric antigen receptors described herein, the fragment includes an scFv. In some embodiments, the V H region and the V L region are joined by a flexible linker. In some embodiments, the scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 361). In some embodiments, the V H region is amino-terminal to the V L region.

In some embodiments of any of the chimeric antigen receptors described herein, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771. In some embodiments, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.

In some embodiments of any of the chimeric antigen receptors described herein, the V H region is carboxy-terminal to the V L region. In some embodiments, the scFv includes the amino acid sequence set forth in SEQ ID NOs: 328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 328 or 586.

In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region includes an activating cytoplasmic signaling domain. In some embodiments of any of the chimeric antigen receptors described herein, the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the activating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3) chain or a functional variant or signaling portion thereof. In some embodiments, the activating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO: 628.

In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In some embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In some embodiments of any of the chimeric antigen receptors described herein, the transmembrane domain is or includes the sequence set forth in SEQ ID NO: 624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.

In some embodiments of any of the chimeric antigen receptors described herein, the encoded chimeric antigen receptor includes from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.

In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 751-756. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 755 and 756. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 755 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 755.

Provided in some embodiments are engineered cells that contain a polynucleotide of any of the embodiments described herein. In some embodiments of any of the engineered cells described herein, the engineered cell contains the chimeric antigen receptor of any of the embodiments described herein.

In some embodiments of any of the engineered cells described herein, the cell is an immune cell. In some embodiments, the immune cell is a primary cell obtained from a subject. In some embodiments, the immune cell is an NK cell or a T cell. In some embodiments, the immune cell is a T cell and the T cell is a CD4+ and/or CD8+ T cell.

In some embodiments of any of the engineered cells described herein, the cell contains transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments, the cell contains transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits reduced heterogeneity compared to the heterogeneity of transcribed mRNA in a cell encoding a reference chimeric antigen receptor, said reference chimeric antigen receptor containing the same amino acid sequence as the chimeric antigen receptor but encoded by a different polynucleotide sequence containing one or more nucleotide differences in the polynucleotide encoding the CARs and/or in which the reference chimeric antigen receptor is encoded by a polynucleotide containing one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer. In some embodiments, the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some embodiments, the cell encoding the reference CAR includes transcribed RNA encoding the reference CAR, optionally messenger RNA (mRNA), that exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some embodiments, the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography.

In some embodiments of any of the engineered cells described herein, among a plurality of the engineered cells, less than or less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2% or 1% of the cells in the plurality contain a chimeric antigen receptor that exhibits tonic signaling and/or antigen independent activity or signaling.

Also provided are compositions comprising any of the engineered cells provided herein. In some of any such embodiments, the composition comprises CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells is from or from about 1:3 to 3:1.

Also provided herein are compositions containing a polynucleotide of any of the embodiments described herein, a chimeric antigen receptor of any of the embodiments described herein, or a engineered cell of any of the embodiments described herein. In some embodiments, the composition further contains a pharmaceutically acceptable excipient. In some of any of these embodiments, the composition is sterile.

Provided in other aspects are methods of treatment that involve administering the engineered cells of any of the embodiments described herein or the composition of any of the embodiments described herein to a subject having a disease or disorder. In some of any embodiments, the method comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells.

Also provided are uses any of the engineered cells or the compositions described herein for the manufacture of a medicament for the treatment of a disease or disorder. Also provided are uses any of the engineered cells or the compositions described herein for treating a disease or disorder. In some of any such embodiments, the engineered cells or the composition are for use in a treatment regimen, wherein the treatment regimen comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells.

In some embodiments of any of the methods described herein, the disease or disorder is associated with expression of B cell maturation antigen (BCMA). In some embodiments, the disease or disorder associated with BCMA is a B cell-related disorder. In some embodiments, the disease or disorder associated with BCMA is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA associated vasculitis, idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), autoimmune thrombocytopenia, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, anti-phospholipid syndrome, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis, or progressive glomerulonephritis.

In some embodiments of any of the methods described herein, the disease or disorder associated with BCMA is a cancer. In some embodiments, the cancer is a BCMA-expressing cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is a lymphoma, a leukemia, or a plasma cell malignancy. In some embodiments, the cancer is a lymphoma and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). In some embodiments, the cancer is a leukemia and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL). In some embodiments, the cancer is a plasma cell malignancy and the plasma cell malignancy is multiple myeloma (MM) or plasmacytoma. In some embodiments, the cancer is multiple myeloma (MM).

In some of any embodiments, the dose of engineered T cells comprises between at or about 1×10 7 CAR-expressing T cells and at or about 2×10 9 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise between at or about 2.5×10 7 CAR-expressing T cells and at or about 1.2×10 9 CAR-expressing T cells, between at or about 5.0×10 7 CAR-expressing T cells and at or about 4.5×10 8 CAR-expressing T cells, or between at or about 1.5×10 8 CAR-expressing T cells and at or about 3.0×10 8 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise at or about 2.5×10 7 , at or about 5.0×10 7 , at or about 1.5×10 8 , at or about 3.0×10 8 , at or about 4.5×10 8 , at or about 8.0×10 8 or at or about 1.2×10 9 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise at or about 5.0×10 7 , at or about 1.5×10 8 , at or about 3.0×10 8 or at or about 4.5×10 8 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprises a combination of CD4 + T cells and CD8 + T cells, at a defined ratio of CD4 + CAR-expressing T cells to CD8 + CAR-expressing T cells and/or of CD4 + T cells to CD8 + T cells, that is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

In some of any embodiments, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express a marker of apoptosis, optionally Annexin V or active Caspase 3. In some of any embodiments, less than 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express Annexin V or active Caspase 3.

In some of any embodiments, prior to the administration, the subject has received a lymphodepleting therapy comprising the administration of fludarabine at or about 20-40 mg/m 2 body surface area of the subject, optionally at or about 30 mg/m 2 , daily, for 2-4 days, and/or cyclophosphamide at or about 200-400 mg/m 2 body surface area of the subject, optionally at or about 300 mg/m 2 , daily, for 2-4 days.

In some of any embodiments, the subject has received a lymphodepleting therapy comprising the administration of fludarabine at or about 30 mg/m 2 body surface area of the subject, daily, and cyclophosphamide at or about 300 mg/m 2 body surface area of the subject, daily, for 3 days.

In some of any embodiments, at or prior to the administration of the dose of cells, the subject has received three or more therapies selected from among: autologous stem cell transplant (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody.

In some of any embodiments, the immunomodulatory agent is selected from among thalidomide, lenalidomide or pomalidomide. In some of any embodiments, the proteasome inhibitor is selected from among bortezomib, carfilzomib or ixazomib. In some of any embodiments, the anti-CD38 antibody is or comprises daratumumab.

In some of any embodiments, at the administration of the dose of cells, the subject has not had active or history of plasma cell leukemia (PCL).

In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving objective response (OR), in at least 50%, 60%, 70%, 80%, 90%, or 95% of subjects that were administered. In some of any embodiments, the OR includes subjects who achieve stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimal response (MR). In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving stringent complete response (sCR), complete response (CR), very good partial response (VGPR) or partial response (PR), in at least 50%, 60%, 70%, 80%, or 85% of subjects that were administered. In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving stringent complete response (sCR) or complete response (CR) at least 20%, 30%, 40% 50%, 60% or 70% of subjects that were administered.

In some of any embodiments, the dose of engineered T cells comprise at or about 5.0×10 7 , at or about 1.5×10 8 , at or about 3.0×10 8 or at or about 4.5×10 8 CAR-expressing T cells. In some of any embodiments, the dose of the engineered T cells comprise at or about 5.0×10 7 CAR-expressing T cells.

Also provided herein are methods of determining the heterogeneity of a transcribed nucleic acid of a transgene, the method comprising: a) amplifying a transcribed nucleic acid using at least one 5′ and 3′ primer pair, wherein at least one pair comprises a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR) of the transcribed nucleic acid to generate one or more amplified products; and b) detecting the amplified products, wherein the presence of two or more amplified products from at least one 5′ and 3′ primer pair indicates heterogeneity in the amplified products.

In some embodiments of the method, the detected differences in b) are different lengths of the amplified transcripts. In some embodiments, the differences in b) are differences in chromatographic profiles of the amplified transcripts.

In some embodiments, the differences in the amplified products are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or chromatography. In some embodiments, the 5′ primer is specific to sequence transcribed from the promoter region of the transcribed nucleic acid. In some embodiments, the transcribed nucleic acid is amplified using a 3′ primer specific to a sequence within the amino acid-coding sequence of the polynucleotide, and/or the 3′ untranslated region, on of the transcribed pre-mRNA. In some embodiments, the 3 primer is specific to the polyadenylation sequence or enhancer region of the 3′ untranslated region of the transcribed pre-mRNA.

In some embodiments, step a) is effected by a single amplification reaction, using a single 5′ and 3′ primer pair comprising a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR). In some embodiments, step a) is effected by parallel or subsequent amplification reactions using a first 5′ and 3′ primer pair, a second 5′ and 3′ primer pair, and optionally additional 5′ and 3′ primer pairs, wherein: the first 5′ and 3′ primer pair contains a 5′ primer that is complementary to a nucleic acid sequence within the 5′ UTR of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ UTR of the transcribed nucleic acid; the second 5′ and 3′ primer pair contains a 5′ primer whose sequence is complementary to a portion of the translated sequence of the nucleic acid transcript and a 3′ primer whose sequence is complementary to a nucleic acid sequence within the 3′ UTR of the transcript; and the optionally additional 5′ and 3′ primer pairs each contain sequences complementary to sequences within the translated region of the transcript. In some embodiments, the parallel or subsequent amplification reactions amplify overlapping portions of the transcript.

In some embodiments, the amplified products are predicted to be about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 kilobases, 4.5 kilobases, 5 kilobases, 5.5 kilobases, 6 kilobases, 7 kilobases, or 8 kilobases in length.

In some of any embodiments, a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more splice site. In some of any embodiments, the transcribed nucleic acid of the transgene candidate exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity following expression in a cell.

Also provided are methods of reducing the heterogeneity of an expressed transgene transcript, the method comprising: a) identifying a transgene candidate for the removal of splice sites according to any of the methods for determining the heterogeneity of a transcribed nucleic acid provided herein; b) identifying one or more potential splice donor and/or splice acceptor sites; and c) modifying the nucleic acid sequence at or near the one or more potential splice donor and/or splice acceptor sites identified in b), thereby generating a modified polynucleotide.

In some of any such embodiments, the method also involves d) assessing the transgene candidacy for the removal of splice sites as in step a). In some of any such embodiments, the method also involves e) repeating steps b)-d) until the heterogeneity of the transcript in step d) is reduced compared to the heterogeneity of the transcript as determined in step a).

In some of any such embodiments, the one or more potential splice donor and/or splice acceptor sites exhibit a score about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 of a splice event, and/or is/are predicted to be involved in a splice event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

In some of any such embodiments, splice donor sites and splice acceptor sites are identified independently. In some of any such embodiments, the splice acceptor and/or donor site(s) is/are canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s).

In some of any such embodiments, the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor. In some of any such embodiments, the CAR polypeptide comprises an antigen-binding domain comprising an antibody fragment, optionally a single chain antibody fragment (scFv), comprising a variable heavy chain (V H ) and a variable light chain (V L ), a spacer (e.g., a spacer region located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor), a transmembrane region, and an intracellular signaling region.

In some of any such embodiments, the modified polynucleotide is not modified within the coding sequence for the antigen-binding domain of the encoded CAR polypeptide. In some of any such embodiments, the encoded amino acid sequence of the transgene is unchanged following modification of the polynucleotide. In some of any such embodiments, the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80%, 85%, 90%, or 95% homogeneity following expression of the unmodified polynucleotide in a cell.

In some of any such embodiments, the cell is a human cell. In some of any such embodiments, the cell is a T-cell.

In some of any such embodiments, the method is a computer implemented method, and wherein one or more steps a)-c) occur at an electronic device comprising one or more processors and memory.

Also provided are computer systems comprising a processor and memory, the memory comprising instructions operable to cause the processor to carry out any one or more of steps of the methods of reducing the heterogeneity of an expressed transgene transcript.

FIGS. 1 A and 1 B depict results of an assay assessing RNA heterogeneity as assessed by agarose gel electrophoresis. FIG. 1 A depicts the RNA heterogeneity of several anti-BCMA-CARs, containing a long spacer (LS) region, or a shorter CD28 spacer region. FIG. 1 B depicts RNA heterogeneity of three different anti-BCMA CAR encoding sequences, containing the long spacer (LS) region, before and after coding sequence optimization and splice site elimination (O/SSE).

FIG. 2 depicts results of an assay assessing levels of BCMA-LS CAR expression on the surface of transduced T cells before (Non-SSE) and after (O/SSE) optimization and splice site elimination of the coding sequence.

FIG. 3 depicts the comparison of transduction efficiency of lentiviral vectors encoding BMCA-LS CAR constructs and lentiviral vectors encoding BCMA-LS CAR constructs that have been codon optimized and modified to eliminate predicted splice sites (O/SSE).

FIG. 4 A depicts results of an assay assessing the cytolytic activity of BMCA-LS CAR-expressing T cells against cell lines that express high (K562/BCMA) or low (RPMI 8226) levels of BCMA at several effector:target cell (E:T) ratios. FIG. 4 B depicts the cytolytic activity of several BMCA-LS CAR-expressing T cells against RPMI-8226 cells at an E:T ratio of 3:1.

FIG. 4 C and FIG. 4 D depict the cytolytic activity of non-optimized BCMA-LS CAR-expressing T cells and optimized (O/SSE) BCMA-LS CAR-expressing T cells on various BCMA-expressing cell lines.

FIG. 5 A depicts results of an assay assessing IFNγ, IL-2, and TNFα cytokine release of BMCA-LS CAR-expressing T cells in response to incubation with cell lines that express high (K562/BCMA) or low (RPMI 8226) levels of BCMA at several effector:target cell (E:T) ratios (5:1, 2.5:1, 1.25:1 and 0.6:1 indicated as a, b, c and d, respectively, in the figure). FIG. 5 B depicts the IFNγ, IL-2, and TNFα cytokine release of non-optimized BMCA-LS CAR-expressing T cells and optimized (O/SSE) BCMA-LS CAR-expressing T cells in response to incubation with BCMA-expressing K562/BCMA and RPMI 8226 cells at different E:T ratios (3:1, 1.5:1, 0.75:1 and 0.375:1 indicated as a, b, c and d, respectively, in the figure).

FIG. 6 depicts results of an assay assessing cytolytic activity following incubation of BCMA-55-LS-O/SSE CAR-expressing T cells, from two donors, with BCMA-expressing cells that express varying levels of BCMA.

FIG. 7 depicts results of an assay assessing IFNγ release following incubation of BCMA-55-LS CAR O/SSE-expressing T cells, from two donors, with BCMA-expressing cells that express varying levels of BCMA.

FIG. 8 depicts results of an assay assessing cytolytic activity of anti-BCMA-expressing CAR T cells that express CARs containing different spacer regions, on OPM2 target cells.

FIGS. 9 A and 9 B depict results of an assay assessing cytolytic activity of anti-BCMA CAR-expressing T cells following incubation of anti-BCMA CAR-expressing T cells with OPM2 target cells in the presence of soluble BCMA-Fc.

FIG. 10 A depicts results of an assay assessing cytolytic activity of optimized (O/SSE) anti-BCMA CAR-expressing T cells in the presence of supernatant from the H929 multiple myeloma cell line. FIG. 10 B depicts results of an assay assessing cytolytic activity of optimize (O/SSE) anti-BCMA CAR-expressing T cells in the presence of recombinant B-cell activating factor (BAFF).

FIGS. 11 A and 11 B depict results of an assay assessing IFNγ, IL-2, and TNFα cytokine release following incubation of anti-BCMA CAR-expressing T cells with OPM2 target cells in the presence of soluble BCMA-Fc ( FIG. 11 A ) or supernatant from a multiple myeloma cell line H929 ( FIG. 11 B ) at different concentrations (0 ng/mL, 111 ng/mL, 333 ng/mL and 1000 ng/mL indicated as a, b, c and d, respectively, in the figures).

FIG. 12 A depicts results of an assay assessing tumor growth in an OPM2 human multiple myeloma xenograft mouse model, following a single intravenous injection of CAR T cells expressing optimized (O/SSE) anti-BCMA CARs. FIG. 12 B depicts results of an assay assessing survival in an OPM2 human multiple myeloma xenograft mouse model, following a single intravenous injection of CAR T cells expressing optimized (O/SSE) anti-BCMA CARs.

FIG. 13 A depicts results of an assay assessing tumor growth in an RPMI-8226 (subcutaneous) xenograft mouse model, following a single intravenous injection of CAR T cells expressing optimized (O/SSE) anti-BCMA CARs. FIG. 13 B depicts survival in an RPMI-8226 (subcutaneous) xenograft mouse model, following a single intravenous injection of CAR T cells expressing optimized (O/SSE) anti-BCMA CARs.

FIGS. 14 A and 14 B depict results of an assay assessing the number of CD4+( FIG. 14 A ) and CD8+( FIG. 14 B ) CAR-positive T cells in the blood from RPMI-8226 (subcutaneous) xenograft mice treated with optimized (O/SSE) anti-BCMA CAR T cells derived from a single donor (Donor 2).

FIGS. 15 A and 15 B depict results of an assay assessing the number of CD4+( FIG. 15 A ) and CD8+( FIG. 15 B ) CAR-positive T cells in the blood from RPMI-8226 (subcutaneous) xenograft mice treated with optimized (O/SSE) anti-BCMA CAR T cells derived from a single donor (Donor 1).

FIG. 16 A depicts results of an assay assessing expression level of tdTomato and a truncated receptor (surrogate marker for CAR expression), as detected by flow cytometry, in BCMA-55-LS-O/SSE CAR-expressing cells, incubated for 6 hours in 96-well cell culture plates coated overnight with (0.008 μg/mL, 0.04 μg/mL, 0.2 μg/mL, 1 μg/mL and 5 μg/mL) of BCMA-Fc (soluble human BCMA fused at its C-terminus to an Fc region of IgG) fusion polypeptide. A recombinant Fc polypeptide was used as a control (Fc Control).

FIG. 16 B depicts results of an assay assessing percentage of tdTomato+ cells among cells expressing the truncated receptor, in reporter cells expressing BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, and BCMA-25-LS-O/SSE CAR, incubated with ten (10) 2-fold serial dilution of BCMA-Fc. Cells expressing a CAR specific for a different antigen (anti-CD19 CAR) was used as control.

FIG. 17 depicts the percentage of tdTomato+ cells among reporter cells expressing BCMA-55-LS-O/SSE CAR or BCMA-55-SS CAR, following co-cultured with human BCMA-expressing K562 target cells (BCMA.K562) target cells at various E:T ratios.

FIG. 18 depicts the expression level of tdTomato and GFP (surrogate marker for CAR expression), as detected by flow cytometry, in reporter cells expressing an anti-CD19 CAR, BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-52-LS-O/SSE CAR, incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling for 3 days.

FIGS. 19 A and 19 B depict the expression level of tdTomato and truncated receptor (surrogate marker for CAR expression), as detected by flow cytometry, in reporter cells expressing an anti-CD19 CAR, BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-52-LS-O/SSE CAR that contain intracellular domains derived from 4-1BB or CD28 incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling.

FIG. 20 A depicts the percentage of tdTomato+ cells, as assessed by flow cytometry, among the Nur77-tdTomato reporter cells engineered to express BCMA-55-LS-O/SSE CAR, specific for human BCMA, co-cultured with K562 human myelogenous leukemia cells expressing human BCMA (huBCMA), murine BCMA (muBCMA) or cynomolgus monkey BCMA (cynoBCMA), at an E:T ratio of 2:1 or 5:1. FIGS. 20 B and 20 C depict the percentage ( FIG. 20 B ) and mean fluorescence intensity (MFI; FIG. 20 C ) of tdTomato+ cells, as assessed by flow cytometry, among reporter cells expressing BCMA-55-LS-O/SSE CAR, incubated with increasing concentrations (0, 0.1, 0.25, 1, 2.5, 10, 25 and 100 μg/mL) of huBCMA and cynoBCMA coated on 96-well flat-bottom plates.

FIG. 21 A depicts an exemplary amplification strategy for a transcript and predicted amplified product. FIG. 21 B depicts exemplary amplified products resulting from amplification of a transcript known and unknown (cryptic) splice sites. FIG. 21 C depicts exemplary sliding window amplification of a transcript using nested primer pairs.

Among the provided embodiments are compositions, articles of manufacture, compounds, methods and uses including those targeting or directed to BCMA and BCMA-expressing cells and diseases. It is observed that BCMA is expressed, e.g., heterogeneously expressed, on certain diseases and conditions such as malignancies or tissues or cells thereof, e.g., on malignant plasma cells such as from all relapsed or newly diagnosed myeloma patients, for example, with little expression on normal tissues. Among the provided embodiments are approaches useful in the treatment of such diseases and conditions and/or for targeting such cell types, including nucleic acid molecules that encode BCMA-binding receptors, including chimeric antigen receptors (CARs), and the encoded receptors such as the encoded CARs, and compositions and articles of manufacture comprising the same. The receptors generally can contain antibodies (including antigen-binding antibody fragments, such as heavy chain variable (V H ) regions, single domain antibody fragments and single chain fragments, including scFvs) specific for BCMA. Also provided are cells, such as engineered or recombinant cells expressing such BCMA-binding receptors, e.g., anti-BCMA CARs and/or containing nucleic acids encoding such receptors, and compositions and articles of manufacture and therapeutic doses containing such cells. Also provided are methods of evaluating, optimizing, making and using nucleic acid sequence(s), for example, nucleic acid sequences encoding recombinant BCMA-binding receptors. Also provided are methods of making and using (such as in the treatment or amelioration of BCMA-expressing diseases and conditions) cells (e.g., engineered cells) expressing or containing the recombinant BCMA-binding receptors and recombinant BCMA-binding receptor-encoding polynucleotides or compositions containing such cells.

Adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. In certain contexts, available approaches to adoptive cell therapy may not always be entirely satisfactory. In some aspects, the ability of the administered cells to recognize and bind to a target, e.g., target antigen such as BCMA, to traffic, localize to and successfully enter appropriate sites within the subject, tumors, and environments thereof, to become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states to provide effective and robust recall responses following clearance and re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, terminal differentiation, and/or differentiation into a suppressive state.

In some contexts, optimal response to therapy can depend on the ability of the engineered recombinant receptors such as CARs, to be consistently and reliably expressed on the surface of the cells and/or bind the target antigen. For example, in some cases, heterogeneity of the transcribed RNA from an introduced transgene (e.g., encoding the recombinant receptor) can affect the expression and/or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy. In some contexts, the length and type of spacer in the recombinant receptor, such as a CAR, can affect the expression, activity and/or function of the receptor.

Also, in some contexts, certain recombinant receptors can exhibit antigen-independent activity or signaling (also known as “tonic signaling”), which could lead to undesirable effects, such as due to increased differentiation and/or exhaustion of T cells that express the recombinant receptor. In some aspects, such activities may limit the T cell's activity, effect or potency. In some cases, during engineering and ex vivo expansion of the cells for recombinant receptor expression, the cells may exhibit phenotypes indicative of exhaustion, due to tonic signaling through the recombinant receptor.

In some contexts, properties of particular target antigens that the recombinant receptors specifically bind, recognize or target, can that affect the activity of the receptor. In some contexts, B-cell maturation antigen (BCMA), is typically expressed on malignant plasma cells and is an attractive therapeutic target for cell therapy. In some cases, BCMA is can be cleaved by gamma secretase, generating a soluble BCMA (sBCMA), or “shed” form of BCMA, reducing the BCMA expressed on the surface of target cells. In some cases, the activity of the BCMA-binding molecules, such as anti-BCMA chimeric antigen receptors, can be blocked or inhibited by the presence of soluble BCMA. Improved strategies are needed for optimal responses to cell therapies, in particular, for recombinant receptors that specifically bind, recognize or target BCMA.

The provided embodiments, in some contexts, are based on the observation that particular spacers and optimization of the nucleic acid sequences can lead to consistent and robust expression of the recombinant receptor. The provided BCMA-binding recombinant receptors offer advantages over available approaches for cell therapies, in particular, BCMA-targeting cell therapy. In some embodiments, provided BCMA-binding recombinant receptors are observed to exhibit reduced antigen-independent, tonic signaling and lack of inhibition by soluble BCMA. In various aspects, the provided BCMA-binding recombinant receptors, polynucleotides encoding such receptors, engineered cells and cell compositions, exhibit certain desired properties that can overcome or counteract certain limitations that can reduce optimal responses to cell therapy, for example, cell therapy with engineered cells expressing a BCMA-binding recombinant receptor. In some aspects, compositions containing engineered cells expressing an exemplary BCMA-binding recombinant receptor provided herein was observed to exhibit consistency of cell health of the engineered cells, and was associated with clinical response. In some contexts, the provided embodiments, including the recombinant receptors, polynucleotides encoding such receptors, engineered cells and cell compositions, can provide various advantages over available therapies targeting BCMA, to improve the activity of the recombinant receptors and response to BCMA-targeting cell therapies.

Provided in some aspects are BCMA-binding agents, such as cell surface proteins, such as recombinant receptors or chimeric antigen receptors that bind or recognize BCMA molecules and polynucleotides encoding BCMA-binding cell surface proteins, such as recombinant receptors (e.g, CARs), and cells expressing such receptors. The BCMA-binding cell surface proteins generally contain antibodies (e.g., antigen-binding antibody fragments), and/or other binding peptides that specifically recognize, such as specifically bind to BCMA, such as to BCMA proteins, such as human BCMA protein. In some aspects, the agents bind to an extracellular portion of BCMA.

In some embodiments, the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and/or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor. In some embodiments, polynucleotides, encoding BCMA-binding cell surface proteins, are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, encoding BCMA-binding cell surface proteins, are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human T cell. In some aspects, the modified polynucleotides result in in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell. Such polynucleotides can be utilized in constructs for generation of engineered cells that express the encoded BCMA-binding cell surface protein. Thus, also provided are cells expressing the recombinant receptors encoded by the polynucleotides provided herein and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with BCMA expression.

Among the provided polynucleotides are those that encode recombinant receptors, such as antigen receptors, that specifically recognize, such as specifically bind, BCMA. In some aspects, the encoded receptors, such as those containing BCMA-binding polypeptides, and compositions and articles of manufacture and uses of the same, also are provided.

Among the BCMA-binding polypeptides are antibodies, such as single-chain antibodies (e.g., antigen binding antibody fragments), or portions thereof. In some examples, the recombinant receptors are chimeric antigen receptors, such as those containing anti-BCMA antibodies or antigen-binding fragments thereof. In any of the embodiments, an antibody or antigen binding fragment, in the provided CARs, that specifically recognizes an antigen, e.g. BCMA, specifically binds to the antigen. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the encoded recombinant BCMA-binding receptors.

In some cases, the polynucleotide encoding the BCMA-binding receptor contains a signal sequence that encodes a signal peptide, in some cases encoded upstream of the nucleic acid sequences encoding the BCMA-binding receptor, or joined at the 5′ terminus of the nucleic acid sequences encoding the antigen-binding domain. In some cases, the polynucleotide containing nucleic acid sequences encoding the BCMA-binding receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptide include a signal peptide of the IgG kappa chain set forth in SEQ ID NO: 620, or encoded by the nucleotide sequence set forth in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain set forth in SEQ ID NO:851 and encoded by the nucleotide sequence set forth in SEQ ID NO:850; a CD8 alpha signal peptide set forth in SEQ ID NO:852; or a CD33 signal peptide set forth in SEQ ID NO:853. In some cases, the polynucleotide encoding the BCMA-binding receptor can contain nucleic acid sequence encoding additional molecules, such as a surrogate marker or other markers, or can contain additional components, such as promoters, regulatory elements and/or multicistronic elements. In some embodiments, the nucleic acid sequence encoding the BCMA-binding receptor can be operably linked to any of the additional components.

A. Components of Encoded Recombinant BCMA-Binding Receptors

The provided BCMA-binding receptors generally contain an extracellular binding molecule and an intracellular signaling domain. Among the provided binding molecules are polypeptides containing antibodies, including single chain cell surface proteins, e.g., recombinant receptors such as chimeric antigen receptors, containing such antibodies.

Among the provided binding molecules (e.g., BCMA-binding molecules) are single chain cell surface proteins, such as recombinant receptors (e.g., antigen receptors), that include one of the provided antibodies or fragment thereof (e.g., BCMA-binding fragment). The recombinant receptors include antigen receptors that specifically bind to or specifically recognize BCMA, such as antigen receptors containing the provided anti-BCMA antibodies, e.g., antigen-binding fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with BCMA expression.

Exemplary antigen receptors, including CARs, and methods for engineering and introducing such antigen receptors into cells, include those described, for example, in international patent application publication Nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013166321, WO2013071154, WO2013123061 U.S. patent application publication Nos. US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application No. EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No. WO2014055668. Exemplary CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282, and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody or an antigen-binding fragment thereof, as provided herein.

In some embodiments, the provided CAR has an amino acid sequence selected from among SEQ ID NOs: 757-762, or exhibits at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs 757-762. In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in any of SEQ ID NOs 751-756, or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 751-756.

In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in any of SEQ ID NOs:755 and 756, or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 755 and 756.

In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in SEQ ID NOs:755 or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in SEQ ID NOs:755.

In some embodiments, the nucleic acid encoding the antigen-binding domain comprises (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 648, 330-352, 647, 716, or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 648, 330-352, 647, 716, or 718; or (c) a degenerate sequence of (a) or (b).

1. Antigen-Binding Domain

Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or is comprised within or comprises, one of the provided anti-BCMA antibodies. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more BCMA-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable regions, and/or antibody molecules, such as those described herein.

The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′) 2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V H ) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, Nanobody®) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof also referred to herein as “antigen-binding fragments.” The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); A1-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.

Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.

Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given V H or V L region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g. CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.

Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (V H and V L , respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a V H or V L domain from an antibody that binds the antigen to screen a library of complementary V L or V H domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Among the antibodies included in the provided CARs are antibody fragments. An “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; heavy chain variable (V H ) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the V H region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding domain in the provided CARs is or comprises an antibody fragment comprising a variable heavy chain (V H ) and a variable light chain (V L ) region. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable (V H ) region and/or a light chain variable (V L ) region, such as scFvs.

Single-domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Among the anti-BCMA antibodies included in the provided CARs are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.

Among the antibodies included in the provided CARs are those that are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.

In some embodiments, the CAR includes a BCMA-binding portion or portions of the antibody molecule, such as a heavy chain variable (V H ) region and/or light chain variable (V L ) region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the provided BCMA-binding CARs contain an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment thereof that confers the BCMA-binding properties of the provided CAR. In some embodiments, the antibody or antigen-binding domain can be any anti-BCMA antibody described or derived from any anti-BCMA antibody described. See, e.g., Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060, WO 2016090320, WO2016090327, WO2010104949 and WO2017173256. Any of such anti-BCMA antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the anti-BCMA CAR contains an antigen-binding domain that is an scFv containing a variable heavy (V H ) and/or a variable light (V L ) region derived from an antibody described in WO 2016090320 or WO2016090327.

In some embodiments, the antibody, e.g., the anti-BCMA antibody or antigen-binding fragment, contains a heavy and/or light chain variable (V H or V L ) region sequence as described, or a sufficient antigen-binding portion thereof. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a V H region sequence or sufficient antigen-binding portion thereof that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a V L region sequence or sufficient antigen-binding portion that contains a CDR-L1, CDR-L2 and/or CDR-L3 as described. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a V H region sequence that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described and contains a V L region sequence that contains a CDR-L1, CDR-L2 and/or CDR-L3 as described. Also among the antibodies are those having sequences at least at or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to such a sequence.

In some embodiments, the antibody, e.g., antigen-binding fragment thereof, in the provided CAR, has a heavy chain variable (V H ) region having the amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, and 772-774, and 814-832, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V H region amino acid selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832, or contains a CDR-H1, CDR-H2, and/or CDR-H3 present in such a V H sequence. In some embodiments, the antibody or antibody fragment, in the provided CAR, has a V H region of any of the antibodies or antibody binding fragments described in WO 2016090327, WO 2016090320, or WO 2017173256.

In some embodiments, the V H region of the anti-BCMA antibody is one that includes a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 (SEQ ID NO:355), wherein X 1 is A, D, E, G, L, V or W; X 2 is A, D, G, L, P, Q or S; X 3 is A, D, G, L or Y; X 4 is D, G, P, R, S, V, Y or null; X 5 is D, I, P, S, T, Y or null; X 6 is A, G, I, S, T, V, Y or null; X 7 is A, D, E, F, L, P, S, Y or null; X 8 is P, Q, T, Y or null; X 9 is D, G, R, Y or null; X 10 is A, F, Y or null; X 11 is D, F or null; X 12 is F or null; X 13 is D, T or Y; and X 14 is I, L, N, V or Y. In some such embodiments, in said CDR-H3, X 1 is V; X 2 is D; X 3 is G; X 4 is D; X 5 is Y; X 6 is V; X 7 is D; X 8 is null; X 9 is null; X 10 is null; X 11 is null; X 12 is null; X 13 is D; and X 14 is Y.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, according to Kabat numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering or AbM numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence comprising the amino acid sequence selected from SEQ ID NOs: 606 and 613. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence of SEQ ID NO: 517, 595, 606, or 613. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V H region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H3 sequence contained therein (e.g. corresponding to amino acid residues H95 to H102 by Kabat numbering) is replaced by the CDR-H3 sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Kabat numbering, any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering or AbM numbering, or any one of SEQ ID NOs: 606 and 613.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof comprises a CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

In some embodiments, the V H region of the antibody or antigen-binding fragment thereof is one that includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of X 1 X 2 X 3 MX 4 (SEQ ID NO:353) X 1 is D or S; X 2 is Y or S; X 3 is A, G, W, or Y; and X 4 is H, Q, or S. In some embodiments, in said CDR-H1, X 1 is D; X 2 is Y; X 3 is Y; and X 4 is S.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:19-22, 165-169, 298, 299, 509, 577, and 598 according to AbM numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs 604, and 611. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence of SEQ ID NO:507, 532, 577, 593, 596, 598, 604, and 611. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V H region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H1 sequence contained therein (e.g. corresponding to amino acid residues H31 to H35 by Kabat numbering) is replaced by the CDR-H1 sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering, any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering, any one of SEQ ID NOs:19-22, 165-169, 509, 298, 299, 509, 577, and 598 according to AbM numbering, or any one of SEQ ID NOs: 604 and 611.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H1 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof is one that includes a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of X 1 IX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 YX 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO:354), wherein X 1 is F, G, H, V, W or Y; X 2 is N, R, S or V; X 3 is P, Q, S, V, W or Y; X 4 is K or null; X 5 is A or null; X 6 is D, G, N, S, or Y; X 7 is G or S; X 8 is G or S; X 9 is E, G, N, T or S; X 10 is I, K, or T; X 11 is E, G, N or Y; X 12 is A or V; X 13 is A, D or Q; X 14 is K or S; X 15 is F or V; X 16 is K or Q; and X 17 is E or G. In some embodiments in said CDR-H2, X 1 is Y; X 2 is S, X 3 is S; X 4 is null; X 5 is null; X 6 is S; X 7 is G; X 8 is S; X 9 is T; X 10 is I; X 11 is Y; X 12 is A; X 13 is D; X 14 is S; X 15 is V; X 16 is K; and X 17 is G.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs:16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 23-25, 170-173, 300, 301, 510-512, 587, and 599 according to AbM numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 605 and 612. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 having the amino acid sequence of any of SEQ ID NOs: 513, 551, 587, 594, 597, 599, 605, or 612. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V H region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H2 sequence contained therein (e.g. corresponding to amino acid residues H50 to H65 by Kabat numbering) is replaced by the CDR-H2 sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering, any one of SEQ ID NOs: 16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering, any one of SEQ ID NOs: 23-25, 170-173, 300, 301, 510-512, 587, and 599 according to AbM numbering, or any one of SEQ ID NOs 605 or 612.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof contains a CDR-H2 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Kabat numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NO:19-22, 165-169, 509, 298, 299, 509, 577, and 598 according to AbM numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:23-25, 170-173, 300, 201, 510-512, 587, and 599 according to AbM numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, and 613 according to AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NO:604 and 611; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:605 and 612; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:606 and 613.

In some embodiments, the V H region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to Kabat numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to Chothia numbering. In some embodiments, the V H region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to AbM numbering.

In some embodiments, the antibody or antigen-binding fragment thereof comprises an V H region comprising a CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of: a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 374, and 9; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, 293; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively, according to Kabat numbering.

For example, the antibody or antigen-binding fragment thereof provided herein comprises a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence selected from among: SEQ ID NOs:1, 4, and 7; SEQ ID NOs:2, 5, and 8; SEQ ID NOs:2, 5, and 9; SEQ ID NOs:2, 5, and 10; SEQ ID NOs:3, 6, and 11; SEQ ID NOs:140, 145, and 149; SEQ ID NOs:141, 145, and 149; SEQ ID NOs:141, 145, and 150; SEQ ID NOs:142, 146, and 151; SEQ ID NOs:2, 5, and 152; SEQ ID NOs:143, 147, and 153; SEQ ID NOs:144, 148, and 154; SEQ ID NOs:3, 6, and 155; SEQ ID NOs:2, 5, and 156; SEQ ID NOs:2, 5, and 157; SEQ ID NOs:2, 6, and 376; SEQ ID NOs:3, 372, and 376; SEQ ID NOs:3, 6, and 376; SEQ ID NOs:3, 6, and 377; SEQ ID NOs:2, 373, and 152; SEQ ID NOs:2, 5, and 378; SEQ ID NOs:2, 374, and 9, SEQ ID NOs:288, 290, and 292; SEQ ID NOs:289, 291, 293; SEQ ID NOs:507, 513, and 517; and SEQ ID NOs:593, 594, and 595, respectively, according to Kabat numbering.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequence of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequence of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the V H region amino acid sequence of SEQ ID NO:609 or SEQ ID NO: 617. In some embodiments, the antibody or antigen-binding fragment thereof comprises a V H region that comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively; SEQ ID NOS: 596, 597, and 595, respectively; SEQ ID NOS: 598, 599, and 595, respectively; or SEQ ID NOS: 611, 612, and 613, respectively.

In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the V H region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. For example, the anti-BCMA antibody or antigen-binding fragment thereof can comprise a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832, and a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the V H region comprises a FR1, a FR2, a FR3 and/or a FR4 selected from a FR1 comprising the amino acid sequence selected from any one of SEQ ID NOs:59-63, 195-203, 308, 309, and 434-439; a FR2 comprising the amino acid sequence selected from any one of SEQ ID NOs:64-66, 204-209, 310, and 311; a FR3 comprising the amino acid sequence selected from any one of SEQ ID NOs:67-69, 210-216, 312, 313, 441 and 443; and/or a FR4 comprising the amino acid sequence selected from any one of SEQ ID NOs:70-71, 217-220, 314, 315, 444 and 445. In some embodiments, the V H region comprises a FR1 comprising the amino acid sequence of SEQ ID NO:61, a FR2 comprising the amino acid sequence of SEQ ID NO:65, a FR3 comprising the amino acid sequence of SEQ ID NO:69, and/or a FR4 comprising the amino acid of SEQ ID NO:70.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a V H region comprising the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

Also provided are antibodies and antigen-binding fragments thereof having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. For example, provided herein is an antibody or antigen-binding fragment comprising a V H region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

In some embodiments, the antibody is a single domain antibody (sdAb) comprising only a V H region sequence or a sufficient antigen-binding portion thereof, such as any of the above described V H sequences (e.g., a CDR-H1, a CDR-H2, a CDR-H3 and/or a CDR-H4).

In some embodiments, an antibody provided herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof comprising a V H region further comprises a light chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody or antigen-binding fragment thereof contains a V H region and a V L region, or a sufficient antigen-binding portion of a V H and V L region. In such embodiments, a V H region sequence can be any of the above described V H sequence. In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or an scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a light chain variable (V L ) region having the amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody or antigen-binding fragment has a V L region described in any of WO 2016090327, WO 2016090320, or WO 2017173256

In some embodiments, the V L region of the antibody described herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 , (SEQ ID NO:358), wherein X 1 is A, C, G, H, I, Q or S; X 2 is A, Q, S or V; X 3 is S, W or Y; X 4 is D, F, G, H or Y; X 5 is D, G, M, R, S or T; X 6 is A, G, H, L, R, S, T or Y; X 7 is L, P, R, S or null; X 8 is D, G, N, R, S, T or null; X 9 is A, G, H, L, P or null; X 10 is F, S or null; X 11 is L, P, W or Y; and X 12 is S, T or V. In some embodiments, in said CDR-L3, X 1 is H; X 2 is V; X 3 is W; X 4 is D; X 5 is R; X 6 is S; X 7 is R; X 8 is D; X 9 is H; X 10 is null; X 11 is Y; and X 12 is V.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L3 comprising the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L3 having the amino acid sequence of SEQ ID NO:591 or 603 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V L region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L3 sequence contained therein (e.g. corresponding to amino acid residues L89 to L97 by Kabat numbering) is replaced by the CDR-L3 sequence selected from any one of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering.

In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L3 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L3 contained within the V L region amino acid sequence of SEQ ID NO:610 or SEQ ID NO: 618.

In some embodiments, the V L region of the antibody described herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 1 (CDR-L1) that contains the amino acid sequence: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO:356), wherein X 1 is G, K, R, S or T; X 2 is A, G or S; X 3 is G, N, S or T; X 4 is G, K, N, Q, R or S; X 5 is S or null; X 6 is D, N, V or null; X 7 is L, V or null; X 8 is H, S, Y or null; X 9 is S, T or null; X 10 is S or null; X 11 is D, G, I, N, S or null; X 12 is D, E, G, K, I, N or null; X 13 is F, G, K, N, R, S, Y or null; X 14 is D, K, N, T or null; X 15 is A, D, G, L, N, S, T or Y; X 16 is L or V; X 17 is A, H, N, Q or S. In some embodiments, X 1 is G; X 2 is A; X 3 is N; X 4 is N; X 5 is null; X 6 is null; X 7 is null; X 8 is null; X 9 is null; X 10 is null; X 11 is I; X 12 is G; X 13 is S; X 14 is K; X 15 is S; X 16 is V; X 17 is H.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 comprising the amino acid sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 comprising the amino acid sequence selected from any one of SEQ ID NOs: 607 and 614. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 having the amino acid sequence of SEQ ID NO:589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V L region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L1 sequence contained therein (e.g. corresponding to amino acid residues L24 to L34 by Kabat numbering) is replaced by the CDR-L1 sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering.

In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L1 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L1 contained within the V L region amino acid sequence of SEQ ID NO:589, 601, 607 or 614.

In some embodiments, the V L region of the antibody provided herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 2 (CDR-L2) that contains the amino acid sequence of X 1 X 2 X 3 X 4 X 5 X 6 X 7 (SEQ ID NO:357), wherein X 1 is A, D, E, N, S, V or W; X 2 is A, D, N, S or V; X 3 is A, D, H, I, N or S; X 4 is D, K, N, Q, R or T; X 5 is L, R or V; X 6 is A, E, P or Q; and X 7 is A, D, S or T. In some embodiments, X 1 is D; X 2 is D; X 3 is D; X 4 is D; X 5 is R; X 6 is P; and X 7 is S.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 comprising the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 608 and 615. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 having the amino acid sequence of SEQ ID NO:590 or SEQ ID NO: 602 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a V L region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L2 sequence contained therein (e.g. corresponding to amino acid residues L50 to L56 by Kabat numbering) is replaced by the CDR-L2 sequence selected from any one of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering, or with any of SEQ ID NOs: 608 and 615.

In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L2 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L2 contained within the V L region amino acid sequence of SEQ ID NO: 589, 601, 607 or 614.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering; a CDR-L2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering; and a CDR-L3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering.

In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to Kabat numbering. In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to Chothia numbering. In some embodiments, the V L region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to AbM numbering.

In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the V L region comprises a CDR-L1, a CDR-L2, and a CDR-L3 selected from among: a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively. In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs:589, 590, and 591, respectively; SEQ ID NOs:607, 608, and 591, respectively; SEQ ID NOs: 601, 602, and 603, respectively; or SEQ ID NOs:614, 615, and 603, respectively.

For example, the antibody or antigen-binding fragment thereof provided herein comprises an V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence selected from among: SEQ ID NOs:26, 37, and 47; SEQ ID NOs:27, 38, and 48; SEQ ID NOs:28, 39, and 49; SEQ ID NOs:29, 40, and 50; SEQ ID NOs:30, 39, and 51; SEQ ID NOs:31, 41, and 52; SEQ ID NOs:32, 42, and 53; SEQ ID NOs:30, 39, and 54; SEQ ID NOs:33, 43, and 55; SEQ ID NOs:34, 44, and 56; SEQ ID NOs:35, 45, and 57; SEQ ID NOs:36, 46, and 58; SEQ ID NOs:174, 179, and 184; SEQ ID NOs:174, 179, and 185; SEQ ID NOs:174, 179, and 186; SEQ ID NOs:174, 179, and 187; SEQ ID NOs:175, 180, and 188; SEQ ID NOs:174, 179, and 189; SEQ ID NOs:176, 181, and 190; SEQ ID NOs:177, 182, and 191; SEQ ID NOs:174, 179, and 192; SEQ ID NOs:178, 183, and 193; SEQ ID NOs:178, 183, and 194; SEQ ID NOs:30, 399, and 415; SEQ ID NOs:380, 400, and 416; SEQ ID NOs:33, 43, and 421; SEQ ID NOs:381, 401, and 417; SEQ ID NOs:382, 402, and 418; SEQ ID NOs:383, 403, and 419; SEQ ID NOs:384, 39, and 54; SEQ ID NOs:385, 180, and 58; SEQ ID NOs:175, 180, and 188; SEQ ID NOs:386, 404, and 420; SEQ ID NOs:387, 405, and 422; SEQ ID NOs:388, 406, and 423; SEQ ID NOs:388, 407, and 424; SEQ ID NOs:389, 408, and 425; SEQ ID NOs:390, 183, and 193; SEQ ID NOs:391, 409, and 426; SEQ ID NOs:392, 40, and 427; SEQ ID NOs:394, 39, and 429; SEQ ID NOs:395, 411, and 430; SEQ ID NOs:396, 412, and 431; SEQ ID NOs:396, 412, and 58; SEQ ID NOs:397, 413, and 432; SEQ ID NOs:398, 414, and 433; SEQ ID NOs:589, 590, and 591; SEQ ID NOs:607, 608, and 591; SEQ ID NOs: 601, 602, and 603; or SEQ ID NOs:614, 615, and 603, respectively.

In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the V L region amino acid sequence selected of SEQ ID NO: 610 or SEQ ID NO: 618.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a V L region that comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or SEQ ID NOS: 614, 615, and 603, respectively.

In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the V L region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. For example, the anti-BCMA antibody or antigen-binding fragment thereof can comprise a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849, and a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the V L region comprises a FR1, a FR2, a FR3 and/or a FR4 selected from a FR1 comprising the amino acid sequence selected from any one of SEQ ID NOs:72-82, 221-227, 316, 317, 446-459 and 461-466; a FR2 comprising the amino acid sequence selected from any one of SEQ ID NOs:83-92, 228-232, 318, 319, 467-477 and 479-482; a FR3 comprising the amino acid sequence selected from any one of SEQ ID NOs:93-101, 233-242, 320, 321, 483-495 and 497-501; and/or a FR4 comprising the amino acid sequence selected from any one of SEQ ID NOs:102-109, 243-246, 322, 323, 502-506 and 508. In some embodiments, the V L region comprises a FR1 comprising the amino acid sequence of SEQ ID NO:79, a FR2 comprising the amino acid sequence of SEQ ID NO:89, a FR3 comprising the amino acid sequence of SEQ ID NO:98, and/or a FR4 comprising the amino acid sequence of SEQ ID NO:108.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a V L region comprising an amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody or antigen-binding fragment thereof contains a V L region comprises the amino acid sequence of SEQ ID NO: 610 or SEQ ID NO: 618.

Also provided are antibodies having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the V H region of the antibody or fragment comprises the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 and the V L region of the antibody or fragment comprises the amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849.

Also provided are antibodies and antigen-binding fragments thereof having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. For example, provided herein is an antibody or antigen-binding fragment containing a V L region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a V L region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 and/or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a V H region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the antibody or antigen-binding fragment contains a V L region comprising the amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 and a V H region the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.

In some embodiments, the V H region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V H region sequence of any of SEQ ID NOs:617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the V L region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.

In some embodiments, the V H region and the V L regions comprise the sequence of SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids having at least 90% identity thereto; the V H region and the V L regions comprise the sequence of SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids having at least 90% identity thereto; or the V H region and the V L regions comprise the sequence of SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids having at least 90% identity thereto.

In some embodiments, the V H region of the antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, a CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the V H region amino acid sequence selected from any one of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, and 814-832; and comprises a CDR-L1, a CDR-L2, a CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, respectively contained within the V L region amino acid sequence selected from any one of SEQ ID NOs: 618,116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, and 833-849.

In some of any embodiments, the V H is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H sequence of SEQ ID NO: 617; and the V L is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L sequence of SEQ ID NO: 618; the V H is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H sequence of SEQ ID NO: 256; and the V L is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L sequence of SEQ ID NO: 267; the V H is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H sequence of SEQ ID NO: 519; and the V L is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L sequence of SEQ ID NO: 535; the V H is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H sequence of SEQ ID NO: 115; and the V L is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L sequence of SEQ ID NO: 536; or the V H is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H sequence of SEQ ID NO: 609; and the V L is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L sequence of SEQ ID NO: 610. In some of any embodiments, the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 617; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 618; the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 256; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 267; the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 519; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 535; the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO:115; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 536; or the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 609; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 610.

In some embodiments, the V H region is or comprises (a) a CDR-H1 comprising the sequence selected from any one of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289, 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 comprising the sequence selected from any one of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 comprising the sequence selected from any one of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517, or 606; and the V L region is or comprises (a) a CDR-L1 comprising the sequence selected from any one of SEQ ID NOs: 601, 614, 26-36, 174-178, 302, 303, 380-392, 394-398, 589, or 607; (b) a CDR-L2 comprising the sequence selected from any one of SEQ ID NOs: 602, 615, 37-46, 179-183, 304, 305, 399-409, 411-414, 590, or 608; and (c) a CDR-L3 comprising the sequence selected from any one of SEQ ID NOs: 603, 47-58, 184-194, 306, 307, 415-427, 429-433, or 591.

In some embodiments, the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:26, 37, and 47, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 8, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:27, 38, and 48, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:28, 39, and 49, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS: 1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:29, 40, and 50, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 39, and 51, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:31, 41, and 52, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VLregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:32, 42, and 53, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 39, and 54, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 55, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:34, 44, and 56, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 11, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:35, 45, and 57, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:36, 46, and 58, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:140, 145, and 149, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 184, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:141, 145, and 149, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 185, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:141, 145, and 150, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 186, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:142, 146, and 151, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 187, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 152, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:175, 180, and 188, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:143, 147, and 153, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 189, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:144, 148, and 154, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:176, 181, and 190, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 155, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:177, 182, and 191, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 156, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 192, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:178, 183, and 193, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:178, 183, and 194, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 6, and 376, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 399, and 415, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS: 1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:380, 400, and 416, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 421, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 155, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:177, 182, and 191, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 372, and 376, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:381, 401, and 417, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 376, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:382, 402, and 418, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 377, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:383, 403, and 419, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:384, 39, and 54, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:385, 180, and 58, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 373, and 152, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:175, 180, and 188, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 11, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:386, 404, and 420, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 378, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 421, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:387, 405, and 422, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:388, 406, and 423, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:388, 407, and 424, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 376, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:389, 408, and 425, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:390, 183, and 193, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 374, and 9, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:391, 409, and 426, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:392, 40, and 427, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VLregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:394, 39, and 429, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:395, 411, and 430, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS: 1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:28, 39, and 49, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:396, 412, and 431, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:396, 412, and 58, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:397, 413, and 432, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:398, 414, and 433, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:288, 290, and 292, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:302, 304, and 306, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:288, 290, and 292, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:302, 304, and 306, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:289, 291, and 293, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:303, 305, and 307, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:289, 291, and 293, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:303, 305, and 307, respectively; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:507, 513, and 517, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:589, 590, and 591, respectively.

In some embodiments, the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.

In some embodiments, the V H region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the V L region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.

In some embodiments, the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 116, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 111 and 117, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 118, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 119, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 120, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 121, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 122, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 123, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:112 and 124, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:113 and 125, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:114 and 126, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 127, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:247 and 257, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:248 and 258, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:249 and 259, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:250 and 260, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:251 and 261, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:252 and 262, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:253 and 263, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:254 and 264, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:255 and 265, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 266, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 267, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:518 and 534, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 535, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 536, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:520 and 264, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:521 and 537, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:522 and 538, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:523 and 539, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 540, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:524 and 541, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:525 and 261, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:526 and 542, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:527 and 543, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:528 and 544, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:529 and 545, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:528 and 546, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:522 and 547, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 548, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:530 and 549, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:531 and 550, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 552, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 553, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 118, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:533 and 554, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 555, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:524 and 556, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 557, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:324 and 326, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:325 and 327, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:609 and 610, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:617 and 618, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:772 and 775, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:773 and 776, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:774 and 777, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:815 and 833, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:816 and 834, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:817 and 835, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:818 and 836, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:819 and 837, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:820 and 838, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:821 and 839, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:822 and 840, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:823 and 841, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:824 and 842, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:825 and 843, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:826 and 844, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:827 and 845, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:828 and 846, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:829 and 847, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:830 and 847, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:831 and 848, respectively; the V H and V L regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:832 and 849, respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above V H and V L , such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

For example, the V H and V L regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:110 and 116; SEQ ID NOs:111 and 117; SEQ ID NOs:110 and 118; SEQ ID NOs:110 and 119; SEQ ID NOs:110 and 120; SEQ ID NOs:110 and 121; SEQ ID NOs:110 and 122; SEQ ID NOs:110 and 123; SEQ ID NOs:112 and 124; SEQ ID NOs:113 and 125; SEQ ID NOs:114 and 126; SEQ ID NOs:115 and 127; SEQ ID NOs:247 and 257; SEQ ID NOs:248 and 258; SEQ ID NOs:249 and 259; SEQ ID NOs:250 and 260; SEQ ID NOs:251 and 261; SEQ ID NOs:252 and 262; SEQ ID NOs:253 and 263; SEQ ID NOs:254 and 264; SEQ ID NOs:255 and 265; SEQ ID NOs:256 and 266; SEQ ID NOs:256 and 267; SEQ ID NOs:518 and 534; SEQ ID NOs:519 and 535; SEQ ID NOs:115 and 536; SEQ ID NOs:520 and 264; SEQ ID NOs:521 and 537; SEQ ID NOs:522 and 538; SEQ ID NOs:523 and 539; SEQ ID NOs:519 and 540; SEQ ID NOs:524 and 541; SEQ ID NOs:525 and 261; SEQ ID NOs:526 and 542; SEQ ID NOs:527 and 543; SEQ ID NOs:528 and 544; SEQ ID NOs:529 and 545; SEQ ID NOs:528 and 546; SEQ ID NOs:522 and 547; SEQ ID NOs:256 and 548; SEQ ID NOs:530 and 549; SEQ ID NOs:531 and 550; SEQ ID NOs:519 and 552; SEQ ID NOs:110 and 553; SEQ ID NOs:110 and 118; SEQ ID NOs:533 and 554; SEQ ID NOs:115 and 555; SEQ ID NOs:524 and 556; SEQ ID NOs:519 and 557, SEQ ID NOs:324 and 326, SEQ ID NOs:325 and 327, SEQ ID NOs:609 and 610; SEQ ID NOs:617 and 618; SEQ ID NOs:772 and 775; SEQ ID NOs:773 and 776; SEQ ID NOs:774 and 777; SEQ ID NOs:815 and 833; SEQ ID NOs:816 and 834; SEQ ID NO:817 and 835; SEQ ID NO:818 and 836; SEQ ID NO:819 and 837; SEQ ID NO:820 and 838; SEQ ID NO:821 and 839; NO:822 and 840; SEQ ID NO:823 and 841; SEQ ID NO:824 and 842; SEQ ID NO:825 and 843; SEQ ID NO:826 and 844; SEQ ID NO:827 and 845; SEQ ID NO:828 and 846; SEQ ID NO:829 and 847; SEQ ID NO:830 and 847; SEQ ID NO:831 and 848; and SEQ ID NO:832 and 849, respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above V H and V L , such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region and a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region of any of the above V H and V L .

In some embodiments, the V H and V L regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:617 and 618; SEQ ID NOs:256 and 267; SEQ ID NOs:519 and 535; SEQ ID NOs:115 and 536; or SEQ ID NOs:609 and 610; respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above V H and V L , such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region and a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region of any of the above V H and V L .

In some embodiments, the V H and V L regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:617 and 618, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above V H and V L , such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region and a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region of any of the above V H and V L .

In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody fragment, such as a single chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the V H region. In some embodiments, the antibody or antigen binding fragment is an scFv comprising a heavy chain variable (V H ) region and a light chain variable (V L ) region. In some embodiments, the single-chain antibody fragment (e.g. scFv) includes one or more linkers joining two antibody domains or regions, such as a heavy chain variable (V H ) region and a light chain variable (V L ) region. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker. Among the linkers are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.

Accordingly, the provided anti-BCMA antibodies include single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain antibody fragments, typically comprising linker(s) joining two antibody domains or regions, such V H and V L regions. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker, such as one rich in glycine and serine.

In some aspects, the linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO:359) or GGGS (3GS; SEQ ID NO:360), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of an sequence set forth in SEQ ID NO:361 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:362 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:778 (SRGGGGSGGGGSGGGGSLEMA).

Accordingly, in some embodiments, the provided embodiments include single-chain antibody fragments, e.g., scFvs, comprising one or more of the aforementioned linkers, such as glycine/serine rich linkers, including linkers having repeats of GGGS (SEQ ID NO: 360) or GGGGS (SEQ ID NO: 359), such as the linker set forth in SEQ ID NO:361.

In some embodiments, the linker has an amino acid sequence containing the sequence set forth in SEQ ID NO:361. The fragment, e.g., scFv, may include a V H region or portion thereof, followed by the linker, followed by a V L region or portion thereof. The fragment, e.g., the scFv, may include the V L region or portion thereof, followed by the linker, followed by the V H region or portion thereof.

In some embodiments, the antigen-binding domain comprises the sequence selected from any one of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence selected from any one of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585, or 769-771.

In some aspects, an scFv provided herein comprises the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 328, 329, 442, 478, 558-576, 578-583, 585, 586, and 769-771, or has an amino acid sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 328, 329, 442, 478, 558-576, 578-583, 585, 586, and 769-771.

For example, the scFv provided herein comprises the amino acid sequence selected from any of SEQ ID NOS:128, 129, 130, 132, 133, 136, 137, 269, 273, 274, 275, 276, 277, 278, 328, 329, 442, 478, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583 585, 586, 769, 770, 771, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, or 813 or has an amino acid sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOS: 128, 129, 130, 132, 133, 136, 137, 269, 273, 274, 275, 276, 277, 278, 328, 329, 442, 478, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583 585, 586, 769, 770, 771, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, or 813.

Table 2 provides the SEQ ID NOS: of exemplary antigen-binding domains, such as antibodies or antigen-binding fragments, that can be comprised in the provided BCMA-binding receptors, such as anti-BCMA chimeric antigen receptors (CARs). In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a V H region that comprises the CDR-H1, CDR-H2, and CDR-H3 sequence and a V L region that comprises the CDR-L1, CDR-L2 and CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below (by Kabat numbering). In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a V H region sequence and a V L region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below, or an antibody comprising a V H and V L region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V H region sequence and the V L region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a V H region sequence and a V L region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising an scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below, or an antibody comprising an scFv amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising an scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below.

Among the antibodies, e.g. antigen-binding fragments, in the provided CARs, are human antibodies. In some embodiments of a provided human anti-BCMA antibody, e.g., antigen-binding fragments, the human antibody contains a V H region that comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 10000 sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or contains a V L region that comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 1000% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the portion of the V H region corresponds to the CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the V H region corresponds to the framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the V L region corresponds to the CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the V L region corresponds to the FR1, FR2, FR2 and/or FR4.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment. For example, the human antibody in some embodiments contains a CDR-H3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment. For example, the human antibody in some embodiments contains a CDR-L3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment.

In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the human antibody contains a V H region in which the framework region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. In some embodiments, the human antibody contains a V L region in which the framework region e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. For example, in some such embodiments, the framework region sequence contained within the V H region and/or V L region differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region sequence encoded by a human germline antibody segment.

In some embodiments, the reference antibody can be a mouse anti-BCMA scFv described in International Patent App. Pub. No. WO 2010/104949.

The antibody, e.g., antigen-binding fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CHI). In some embodiments, the antibody includes a C H 2 and/or C H 3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.

2. Spacer

In some embodiments, the recombinant receptor such as a CAR comprising an antibody (e.g., antigen-binding fragment) provided herein, further includes a spacer or spacer region. The spacer typically is a polypeptide spacer and in general is located within the CAR between the antigen binding domain and the transmembrane domain of the CAR. In some aspects, the spacer may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region of an immunoglobulin, such as an IgG hinge region, e.g., an IgG4 or IgG4-derived hinge region, and/or a C H 1/CL and/or Fc region. In some embodiments, the constant region or one or more of the portion(s) thereof is of a human IgG, such as of a human IgG4 or IgG1 or IgG2. In general, the spacer, such as the portion of the constant region, serves as a spacer region between the antigen-recognition component (e.g., scFv) and transmembrane domain. In some embodiments, the length and/or composition of the spacer is designed to optimize or promote certain features of the interaction between the CAR and its target; in some aspects, it is designed to optimize the biophysical synapse distance between the CAR-expressing cell and the cell expressing the target of the CAR during or upon or following binding of the CAR to its target on the target-expressing cell; in some aspects, the target expressing cell is a BCMA-expressing tumor cell. In some embodiments, The CAR is expressed by a T-cell, and the length of the spacer is of a length that is compatible for T-cell activation or to optimize CAR T-cell performance. In some embodiments, the spacer is a spacer region, located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor, e.g., CAR. In some embodiments, the spacer region is a region located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor, e.g., CAR.

In some embodiments, the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer and/or in the presence of a different spacer, such as one different only in length. In some embodiments, the spacer is at least 100 amino acids in length, such as at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 300 amino acids, about 10 to 200 amino acids, about 50 to 175 amino acids, about 50 to 150 amino acids, about 10 to 125 amino acids, about 50 to 100 amino acids, about 100 to 300 amino acids, about 100 to 250 amino acids, about 125 to 250 amino acids, or about 200 to 250 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer or spacer region is at least about 12 amino acids, at least about 119 amino acids or less, at least about 125 amino acids, at least about 200 amino acids, or at least about 220 amino acids, or at least about 225 amino acids in length.

In some embodiments, the spacer has a length of 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the foregoing.

Exemplary spacers include those containing portion(s) of an immunoglobulin constant region such as those containing an Ig hinge, such as an IgG hinge domain. In some aspects, the spacer includes an IgG hinge alone, an IgG hinge linked to one or more of a C H 2 and C H 3 domain, or IgG hinge linked to the C H 3 domain. In some embodiments, the IgG hinge, C H 2 and/or C H 3 can be derived all or in part from IgG4 or IgG2. In some embodiments, the spacer can be a chimeric polypeptide containing one or more of a hinge, C H 2 and/or C H 3 sequence(s) derived from IgG4, IgG2, and/or IgG2 and IgG4. In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region and/or of an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; the C H 2 region comprises all or a portion of an IgG4 C H 2 region and/or of an IgG2 C H 2 region, wherein the IgG4 C H 2 region is optionally a human IgG4 C H 2 region and the IgG2 C H 2 region is optionally a human IgG2 C H 2 region; and/or the C H 3 region comprises all or a portion of an IgG4 C H 3 region and/or of an IgG2 C H 3 region, wherein the IgG4 C H 3 region is optionally a human IgG4 C H 3 region and the IgG2 C H 3 region is optionally a human IgG2 C H 3 region. In some embodiments, the hinge, C H 2 and C H 3 comprises all or a portion of each of a hinge region, C H 2 and C H 3 from IgG4. In some embodiments, the hinge region is chimeric and comprises a hinge region from human IgG4 and human IgG2; the C H 2 region is chimeric and comprises a C H 2 region from human IgG4 and human IgG2; and/or the C H 3 region is chimeric and comprises a C H 3 region from human IgG4 and human IgG2. In some embodiments, the spacer comprises an IgG4/2 chimeric hinge or a modified IgG4 hinge comprising at least one amino acid replacement compared to human IgG4 hinge region; an human IgG2/4 chimeric C H 2 region; and a human IgG4 C H 3 region.

In some embodiments, the spacer can be derived all or in part from IgG4 and/or IgG2 and can contain mutations, such as one or more single amino acid mutations in one or more domains. In some examples, the amino acid modification is a substitution of a proline (P) for a seine (S) in the hinge region of an IgG4. In some embodiments, the amino acid modification is a substitution of a glutamine (Q) for an asparagine (N) to reduce glycosylation heterogeneity, such as an N177Q mutation at position 177, in the C H 2 region, of the full-length IgG4 Fc sequence set forth in SEQ ID NO: 750 or an N176Q. at position 176, in the C H 2 region, of the full-length IgG2 Fc sequence set forth in SEQ ID NO: 749. In some embodiments, the spacer is or comprises an IgG4/2 chimeric hinge or a modified IgG4 hinge; an IgG2/4 chimeric C H 2 region; and an IgG4 C H 3 region and optionally is about 228 amino acids in length; or a spacer set forth in SEQ ID NO: 649. In some embodiments, the spacer comprises the amino acid sequence

(SEQ ID NO: 649) ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK

encoded by a polynucleotide that has been optimized for codon expression and/or to eliminate splice sites such as cryptic splice sites. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 622. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 855 or 856.

Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135, or international patent application publication number WO2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity following expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or reduce the likelihood of a splice event at a splice site.

In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:363, and is encoded by the polynucleotide sequence set forth in SEQ ID NO:364. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 630, and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 629. In some embodiments, the spacer has an amino acid sequence set forth in SEQ ID NO: 649, encoded by the polynucleotide sequence set forth in SEQ ID NO: 621,622, 855 or 856 or a polynucleotide that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 621, 622, 855 or 856. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 649, encoded by a polynucleotide that has been optionally optimized for codon usage and/or to reduce RNA heterogeneity.

In some embodiments, the spacer is or comprises an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:622.

3. Transmembrane Domain and Intracellular Signaling Components

The antigen-recognition component generally is linked to one or more intracellular signaling regions containing signaling components, such as signaling components that mimic stimulation and/or activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the BCMA-binding molecule (e.g., antibody or antigen binding fragment thereof) is linked to one or more transmembrane domains such as those described herein and intracellular signaling regions or domains comprising one or more intracellular components such as those described herein. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane domains include those derived from (i.e. comprise at least the transmembrane domain(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and/or CD154. For example, the transmembrane domain can be a CD28 transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 624, encoded by the nucleic acid sequence set forth in SEQ ID NO: 623 or SEQ ID NO:688. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling regions or domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the intracellular signaling domain of the CAR.

The receptor, e.g., the CAR, generally includes an intracellular signaling region comprising at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the BCMA-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon or following ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR stimulates and/or activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.

In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such classes of cytoplasmic signaling sequences.

In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary stimulation and/or activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, the intracellular signaling region or domain in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments the CD3 zeta comprises the sequence of amino acids set forth in SEQ ID NO: 628, encoded by the nucleic acid sequence set forth in SEQ ID NO: 627 or SEQ ID NO: 652.

In some embodiments, the CAR includes a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and/or transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule. Exemplary costimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS. For example, a costimulatory molecule can be derived from 4-1BB and can comprise the amino acid sequence set forth in SEQ ID NO: 626, encoded by the nucleotide sequence set forth in SEQ ID NO: 625 or SEQ ID NO: 681. In some aspects, the same CAR includes both the stimulatory or activating components (e.g., cytoplasmic signaling sequence) and costimulatory components.

In some embodiments, the stimulatory or activating components are included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, and costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the BCMA-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than BCMA, whereby a stimulatory or an activating signal delivered through the BCMA-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and a stimulatory or activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the provided chimeric antigen receptor comprises: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA), such as any antigen-binding domain described herein; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region and a V L region comprising the amino acid sequence of SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence of SEQ ID NO: 617; and a V L region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence of SEQ ID NO: 618. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS:593, 594, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS:596, 597, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS: 598, 599, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS: 611, 612, and 613, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS: 614, 615, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a V H region that is or comprises the amino acid sequence of SEQ ID NO: 617; and a V L region that is or comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, the antigen-binding domain of such receptor, comprising the amino acid sequence of SEQ ID NO: 478. In some embodiments, the intracellular signaling region includes an stimulating cytoplasmic signaling domain. In some embodiments, the stimulating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the stimulating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a CD3-zeta (CD3) chain or a functional variant or signaling portion thereof. In some embodiments, the stimulating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the stimulating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628. In some embodiments, the nucleic acid encoding the stimulating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:627 or is a codon-optimized sequence and/or degenerate sequence thereof. In other embodiments, the nucleic acid encoding the stimulating cytoplasmic signaling domain is or includes the sequence set forth in SEQ ID NO:652. In some embodiments, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In other embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626. In some embodiments, the nucleic acid encoding the costimulatory region is or includes the sequence set forth in SEQ ID NO:625 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the costimulatory signaling region includes the sequence set forth in SEQ ID NO:681. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In other embodiments, the transmembrane domain is or includes the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.

Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (V H ) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V H region sequence of SEQ ID NO: 617; and (ii) a variable light chain (V L ) region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region sequence of any of SEQ ID NO: 618; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3) chain and an intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such a chimeric antigen receptor.

In some embodiments, the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.

Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: a variable heavy (V H ) region comprising a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and a variable light (V L ) region comprising a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region sequence of SEQ ID NO: 617; and the V L region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region sequence of SEQ ID NO: 618; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the V H region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the V L region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a human CD3-zeta (CD3) chain and an intracellular signaling domain of a T cell costimulatory molecule, optionally from a human 4-1BB or a human CD28. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some embodiments, the extracellular antigen-binding domain comprises the V H region sequence of SEQ ID NO:617 and the V L region sequence of SEQ ID NO:618. In some embodiments, the antigen-binding domain of such receptor, comprising the amino acid sequence of SEQ ID NO: 478. In some embodiments, other domains, regions, or components of the chimeric antigen receptor includes any domains, regions, or components described herein.

4. Surrogate Marker

In some embodiments, the CAR further includes a surrogate marker, such as a cell surface marker (e.g., a truncated cell surface marker), which may be used to confirm transduction or engineering of the cell to express the receptor. For example, in some aspects, extrinsic marker genes are utilized in connection with engineered cell therapies to permit detection or selection of cells and, in some cases, also to promote cell suicide by ADCC. Exemplary marker genes include truncated epidermal growth factor receptor (EGFRt), which can be co-expressed with a transgene of interest (e.g., a CAR or TCR) in transduced cells (see, e.g., U.S. Pat. No. 8,802,374). EGFRt contains an epitope recognized by the antibody cetuximab (Erbitux®). For this reason, Erbitux® can be used to identify or select cells that have been engineered with the EGFRt construct, including in cells also co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR). Additionally, EGFRt is commonly used as a suicide mechanism in connection with cell therapies. In some aspects, when EGFRt is co-expressed in cells with a transgene of interest (e.g. CAR or TCR), it can be targeted by the cetuximab monoclonal antibody to reduce or deplete the transferred gene-modified cells via ADCC (see U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). Importantly, the suicide killing approach using tEGFR requires availability of the antibody epitope. Another example of such a marker gene is prostate-specific membrane antigen (PSMA) or a modified form thereof. PSMA or modified forms thereof may comprise a sequence of amino acids bound by or recognized by a PSMA-targeting molecule, such as an antibody or an antigen-binding fragment thereof. PSMA-targeting molecules can be used to identify or select cells that have been engineered with a PSMA or modified construct, including in cells also co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR) provided herein. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a nerve growth factor receptor (NGFR), epidermal growth factor receptor (e.g., EGFR), or PSMA.

Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and to not transduce or are not capable of transducing a signal or a signal ordinarily transduced by the full-length form of the cell surface polypeptide, and/or do not or are not capable of internalizing. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO: 11 or 76) or a prostate-specific membrane antigen (PSMA) or modified form thereof tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and an encoded exogenous protein, and/or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19 or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and/or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS) or variants thereof.

In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the CAR and surrogate marker, separated by a T2A ribosome switch, can express two proteins from the same construct, such that the surrogate marker can be used as a marker to detect cells expressing such construct. In some embodiments, the surrogate marker, and optionally a linker sequence, can be any as disclosed in international publication no. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) or PSMA that is, optionally, linked to a linker sequence, such as a 2A cleavable linker sequence (e.g., a T2A, P2A, E2A or F2A cleavable linker, described elsewhere herein). An exemplary polypeptide for a truncated EGFR surrogate marker comprises the sequence of amino acids set forth in SEQ ID NO: 634 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 634. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells following adoptive transfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon or in response to antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv or a single-domain antibody comprising only the V H region and the intracellular signaling domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a co-stimulatory molecule (e.g., T cell costimulatory molecule), such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1). In some embodiments, the intracellular signaling domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB or functional variant thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1). In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190.

For example, in some embodiments, the CAR includes a BCMA antibody or fragment, such as any of the human BCMA antibodies, including sdAbs and scFvs, described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes the BCMA antibody or fragment, such as any of the human BCMA antibodies, including sdAbs and scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR.

In certain embodiments, multispecific binding molecules, e.g., multispecific chimeric receptors, such as multispecific CARs, can contain any of the multispecific antibodies, including, e.g. bispecific antibodies, multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs, such as any described above in Section I.A.

B. Exemplary Features

In some aspects, the antibodies or antigen-binding fragments thereof, in the provided CARs, have one or more specified functional features, such as binding properties, including recognizing or binding to particular epitopes, such as to epitopes that are similar to or overlap with those specifically bound by other antibodies such as reference antibodies, or epitopes that are different from those specifically bound by other antibodies such as reference antibodies, the ability to compete for binding with other antibodies such as reference antibodies, and/or particular binding affinities. In other embodiments, the antibodies or antigen-binding fragments thereof, in the provided CARs, recognize, such as specifically recognize, or bind, e.g., specifically bind, to epitopes that are different from, or do not overlap with those specifically bound by other antibodies such as reference antibodies. For example, the epitopes specifically bound by the antibodies, in the provided CARs, are different from those specifically bound by other antibodies such as reference antibodies. In some embodiments, the antibodies and antigen binding fragments thereof do not directly compete for, or compete to a lower degree, with binding with other antibodies such as reference antibodies.

In some embodiments, the antibodies or antigen-binding fragments thereof specifically recognize or specifically bind to BCMA protein. In any of the embodiments, an antibody or antigen binding fragment, in the provided CARs, that specifically recognize BCMA, specifically binds BCMA. In some embodiments provided herein, BCMA protein refers to human BCMA, a mouse BCMA protein, or a non-human primate (e.g., cynomolgus monkey) BCMA protein. In some embodiments of any of the embodiments herein, BCMA protein refers to human BCMA protein. The observation that an antibody or other binding molecule binds to BCMA protein or specifically binds to BCMA protein does not necessarily mean that it binds to a BCMA protein of every species. For example, in some embodiments, features of binding to BCMA protein, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human BCMA protein and the antibody may not have this feature with respect to a BCMA protein of another species, such as mouse.

In some embodiments, the antibody or antigen-binding fragment binds to a mammalian BCMA protein, including to naturally occurring variants of BCMA, such as certain splice variants or allelic variants.

In some embodiments, the antibodies specifically bind to human BCMA protein, such as to an epitope or region of human BCMA protein, such as the human BCMA protein comprising the amino acid sequence of SEQ ID NO:367 (GenBank® No. BAB60895.1), or SEQ ID NO:368 (NCBI No. NP_001183.2) or an allelic variant or splice variant thereof. In one embodiment, the human BCMA protein is encoded by a transcript variant or is an isoform that has the sequence of amino acids forth in SEQ ID NO:369. In some embodiments, the antibodies bind to cynomolgus monkey BCMA protein, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank® No. EHH60172.1). In some embodiments, the antibodies bind to human BCMA but do not bind to or bind in a lower level or degree or affinity to cynomolgus monkey BCMA protein, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank® No. EHH60172.1). In some embodiments, the antibodies do not bind to or bind in a lower level or degree or affinity to mouse BCMA protein, such as the mouse BCMA protein set forth in SEQ ID NO:370 (NCBI No. NP_035738.1). In some embodiments, the antibodies bind to mouse BCMA protein, such as the mouse BCMA protein set forth in SEQ ID NO:370 (NCBI No. NP_035738.1). In some embodiments, the antibodies bind to mouse BCMA protein, with lower affinity than its binding to a human BCMA protein and/or a cynomolgus monkey BCMA protein. In some embodiments, the antibodies bind to mouse BCMA protein and/or a cynomolgus monkey BCMA protein with lower affinity than its binding to a human BCMA protein. In some embodiments, the antibodies bind to mouse BCMA protein and/or a cynomolgus monkey BCMA protein with similar binding affinity compared to its binding to a human BCMA protein.

In some embodiments, the provided antigen-binding domain or CAR exhibits preferential binding to membrane-bound BCMA as compared to soluble BCMA. In some embodiments, the provided antigen-binding domain or CAR exhibits greater binding affinity for, membrane-bound BCMA compared to soluble BCMA.

In one embodiment, the extent of binding of an anti-BCMA antibody or antigen-binding domain or CAR to an unrelated, non-BCMA protein, such as a non-human BCMA protein or other non-BCMA protein, is less than at or about 10% of the binding of the antibody or antigen-binding domain or CAR to human BCMA protein or human membrane-bound BCMA as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies or antigen-binding domains or CARs in which binding to mouse BCMA protein is less than or at or about 10% of the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies in which binding to cynomolgus monkey BCMA protein is less than or at or about 10% of the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies in which binding to cynomolgus monkey BCMA protein and/or a mouse BCMA protein is similar to or about the same as the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies or antigen-binding domains or CARs in which binding to soluble BCMA protein is less than or at or about 10% of the binding of the antibody to membrane-bound BCMA protein.

In some embodiments, the antibody specifically binds to, and/or competes for binding thereto with a reference antibody, and/or binds with a particular affinity or competes to a particular degree, to a BCMA protein, e.g., human BCMA, a mouse BCMA protein, or a non-human primate (e.g., cynomolgus monkey) BCMA protein.

In some embodiments, the antibodies, in the provided CARs, are capable of binding BCMA protein, such as human BCMA protein, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by an equilibrium dissociation constant (K D ); in some embodiments, the affinity is represented by EC 50 .

A variety of assays are known for assessing binding affinity and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen, such as a BCMA protein). It is within the level of a skilled artisan to determine the binding affinity of a binding molecule, e.g., an antibody, for an antigen, e.g., BCMA, such as human BCMA or cynomolgus BCMA or mouse BCMA, such as by using any of a number of binding assays that are well known in the art. For example, in some embodiments, a BIAcore® (surface plasmon resonance (SPR)) instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or fragment thereof, and an antigen, such as a BCMA protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N. Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or the equivalent).

SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detection of expressed polynucleotides or binding of proteins.

In some embodiments, the binding molecule, e.g., antibody or fragment thereof or antigen-binding domain of a CAR, binds, such as specifically binds, to an antigen, e.g., a BCMA protein or an epitope therein, with an affinity or K A (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M; equal to the ratio of the on-rate [k on or k a ] to the off-rate [k off or k a ] for this association reaction, assuming bimolecular interaction) equal to or greater than 10 5 M −1 . In some embodiments, the antibody or fragment thereof or antigen-binding domain of a CAR exhibits a binding affinity for the peptide epitope with a K D (i.e., an equilibrium dissociation constant of a particular binding interaction with units of M; equal to the ratio of the off-rate [k off or k a ] to the on-rate [k on or k a ] for this association reaction, assuming bimolecular interaction) of equal to or less than 10 −5 M. For example, the equilibrium dissociation constant K D ranges from 10 −5 M to 10 −13 M, such as 10 −7 M to 10 −11 M, 10 −8 M to 10 −10 M, or 10 −9 M to 10 −10 M. The on-rate (association rate constant; k on or k a ; units of 1/Ms) and the off-rate (dissociation rate constant; k off or k a ; units of 1/s) can be determined using any of the assay methods known in the art, for example, surface plasmon resonance (SPR).

In some embodiments, the binding affinity (EC 50 ) and/or the dissociation constant of the antibody (e.g. antigen-binding fragment) or antigen-binding domain of a CAR to about BCMA protein, such as human BCMA protein, is from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 50 nM, from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM. In certain embodiments, the binding affinity (EC 50 ) and/or the equilibrium dissociation constant, K D , of the antibody to a BCMA protein, such as human BCMA protein, is at or less than or about 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies bind to a BCMA protein, such as human BCMA protein, with a sub-nanomolar binding affinity, for example, with a binding affinity less than about 1 nM, such as less than about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM or about 0.1 nM or less.

In some embodiments, the binding affinity may be classified as high affinity or as low affinity. In some cases, the binding molecule (e.g. antibody or fragment thereof) or antigen-binding domain of a CAR that exhibits low to moderate affinity binding exhibits a K A of up to 10 7 M −1 , up to 10 6 M −1 , up to 10 5 M −1 . In some cases, a binding molecule (e.g. antibody or fragment thereof) that exhibits high affinity binding to a particular epitope interacts with such epitope with a K A of at least 10 7 M −1 , at least 10 8 M −1 , at least 10 9 M −1 , at least 10 10 M −1 , at least 10 11 M −1 , at least 10 12 M −1 , or at least 10 13 M −1 . In some embodiments, the binding affinity (EC 50 ) and/or the equilibrium dissociation constant, K D , of the binding molecule, e.g., anti-BCMA antibody or fragment thereof or antigen-binding domain of a CAR, to a BCMA protein, is from or from about 0.01 nM to about 1 μM, 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC 50 ) and/or the dissociation constant of the equilibrium dissociation constant, K D , of the binding molecule, e.g., anti-BCMA antibody or fragment thereof or antigen-binding domain of a CAR, to a BCMA protein, is at or about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. The degree of affinity of a particular antibody can be compared with the affinity of a known antibody, such as a reference antibody.

In some embodiments, the binding affinity of a binding molecule, such as an anti-BCMA antibody or antigen-binding domain of a CAR, for different antigens, e.g., BCMA proteins from different species can be compared to determine the species cross-reactivity. For example, species cross-reactivity can be classified as high cross reactivity or low cross reactivity. In some embodiments, the equilibrium dissociation constant, K D , for different antigens, e.g., BCMA proteins from different species such as human, cynomolgus monkey or mouse, can be compared to determine species cross-reactivity. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be high, e.g., the anti-BCMA antibody binds to human BCMA and a species variant BCMA to a similar degree, e.g., the ratio of K D for human BCMA and K D for the species variant BCMA is or is about 1. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be low, e.g., the anti-BCMA antibody has a high affinity for human BCMA but a low affinity for a species variant BCMA, or vice versa. For example, the ratio of K D for the species variant BCMA and K D for the human BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the anti-BCMA antibody has low species cross-reactivity. The degree of species cross-reactivity can be compared with the species cross-reactivity of a known antibody, such as a reference antibody.

In some embodiments, the binding affinity of the anti-BCMA antibody or antigen-binding domain of a CAR, for different form or topological type of antigens, e.g., soluble BCMA protein compared to the binding affinity to a membrane-bound BCMA, to determine the preferential binding or relative affinity for a particular form or topological type. For example, in some aspects, the provided anti-BCMA antibodies or antigen-binding domains can exhibit preferential binding to membrane-bound BCMA as compared to soluble BCMA and/or exhibit greater binding affinity for, membrane-bound BCMA compared to soluble BCMA. In some embodiments, the equilibrium dissociation constant, K D , for different form or topological type of BCMA proteins, can be compared to determine preferential binding or relative binding affinity. In some embodiments, the preferential binding or relative affinity to a membrane-bound BCMA compared to soluble BCMA can be high. For example, in some cases, the ratio of K D for soluble BCMA and the K D for membrane-bound BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more and the antibody or antigen-binding domain preferentially binds or has higher binding affinity for membrane-bound BCMA. In some cases, the ratio of K A for membrane-bound BCMA and the K A for soluble BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more and the antibody or antigen-binding domain preferentially binds or has higher binding affinity for membrane-bound BCMA. In some cases, the antibody or antigen-binding domain of CAR binds soluble BCMA and membrane-bound BCMA to a similar degree, e.g., the ratio of K D for soluble BCMA and K D for membrane-bound BCMA is or is about 1. In some cases, the antibody or antigen-binding domain of CAR binds soluble BCMA and membrane-bound BCMA to a similar degree, e.g., the ratio of K A for soluble BCMA and K A for membrane-bound BCMA is or is about 1. The degree of preferential binding or relative affinity for membrane-bound BCMA or soluble BCMA can be compared with that of a known antibody, such as a reference antibody.

In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a similar degree to a human BCMA protein and a non-human BCMA protein or other non-BCMA proteins. For example, in some embodiments, the antibodies or antigen binding fragments thereof or antigen-binding domain of a CAR bind to a human BCMA protein, such as the human BCMA protein comprising the amino acid sequence of SEQ ID NO:367 (GenBank® No. BAB60895.1), or SEQ ID NO:368 (NCBI No. NP_001183.2) or an allelic variant or splice variant thereof, with an equilibrium dissociation constant (K D ), and to a non-human BCMA, such as a cynomolgus monkey BCMA, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank® No. EHH60172.1), with a K D that is similar, or about the same, or less than 2-fold different, or less than 5-fold different.

In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a similar degree to a soluble BCMA protein and a membrane-bound BCMA protein, with an equilibrium dissociation constant (K D ) that is similar, or about the same, or less than 2-fold different, or less than 5-fold different.

For example, in some embodiments, the antibodies, in the provided CARs, or antigen binding fragments thereof bind to a human BCMA with a K D of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less, and binds to a cynomolgus monkey BCMA with a K D of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies or antigen binding fragments thereof bind to a mouse BCMA protein with a K D of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a human BCMA, a cynomolgus monkey BCMA and a mouse BCMA with high affinity. In some embodiments, the antibodies or antigen binding fragments thereof bind to a human BCMA and cynomolgus monkey BCMA with a high affinity, and to a mouse BCMA with low affinity. In some embodiments, the antibodies or antigen binding fragments thereof bind to a human BCMA and BCMA from other species, or other variants of the BCMA protein, with high affinity.

In some embodiments, the total binding capacity (R max ), as measured using particular surface plasmon resonance (SPR) conditions, is used to determine the ability or capacity of binding of the antibody or antigen binding fragment thereof, to the antigen, e.g., a BCMA protein, such as a human BCMA protein. For SPR analysis, the “ligand” is the immobilized target molecule on the surface of the sensor, for example, a BCMA protein, and the “analyte” is the tested molecule, e.g., antibody, for binding to the “ligand”. For example, the “analyte” can be any of the antibodies, or antigen binding fragments thereof, that binds to a BCMA protein. For a particular ligand and analyte pair in SPR, the R max can be determined assuming a 1:1 binding stoichiometry model, for a particular condition. Binding capacity (R max ) was determined using the following formula: R max (RU)=(analyte molecular weight)/(ligand molecular weight)×immobilized ligand level (RU). For example, in a particular SPR conditions, the R max of binding between any of the antibody or antigen binding fragment thereof and a BCMA protein, such as a human BCMA or a cynomolgus BCMA, is at least or at least about 50 resonance units (RU), such as about 25 RU, 20 RU, 15 RU, 10 RU, 5 RU or 1 RU.

In some embodiments, the antibodies, such as the human antibodies, in the provided CAR, specifically bind to a particular epitope or region of BCMA protein, such as generally an extracellular epitope or region. BCMA protein is a type III membrane 184 amino acid protein that contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. With reference to a human BCMA amino acid sequence set forth in SEQ ID NO:367, the extracellular domain corresponds to amino acids 1-54, amino acids 55-77 correspond to the transmembrane domain, and amino acids 78-184 correspond to the cytoplasmic domain.

Among the provided CARs are CARs that exhibit antigen-dependent activity or signaling, i.e. signaling activity that is measurably absent or at background levels in the absence of antigen, e.g. BCMA. Thus, in some aspects, provided CARs do not exhibit, or exhibit no more than background or a tolerable or low level of, tonic signaling or antigen-independent activity or signaling in the absence of antigen, e.g. BCMA, being present. In some embodiments, the provided anti-BCMA CAR-expressing cells exhibit biological activity or function, including cytotoxic activity, cytokine production, and ability to proliferate.

In some embodiments, biological activity or functional activity of a chimeric receptor, such as cytotoxic activity, can be measured using any of a number of known methods. The activity can be assessed or determined either in vitro or in vivo. In some embodiments, activity can be assessed once the cells are administered to the subject (e.g., human). Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as interlekukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and/or TGF-beta (TGFβ). Assays to measure cytokines are well known in the art, and include but are not limited to, ELISA, intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT, flow cytometry and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g. proliferation) in the presence of a test sample. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

In some aspects, a reporter cell line can be employed to monitor antigen-independent activity and/or tonic signaling through anti-BCMA CAR-expressing cells. In some embodiments, a T cell line, such as a Jurkat cell line, contains a reporter molecule, such as a fluorescent protein or other detectable molecule, such as a red fluorescent protein, expressed under the control of the endogenous Nur77 transcriptional regulatory elements. In some embodiments, the Nur77 reporter expression is cell intrinsic and dependent upon signaling through a recombinant reporter containing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), such as a CD3 chain. Nur77 expression is generally not affected by other signaling pathways such as cytokine signaling or toll-like receptor (TLR) signaling, which may act in a cell extrinsic manner and may not depend on signaling through the recombinant receptor. Thus, only cells that express the exogenous recombinant receptor, e.g. anti-BCMA CAR, containing the appropriate signaling regions is capable of expressing Nur77 upon stimulation (e.g., binding of the specific antigen). In some cases, Nur77 expression also can show a dose-dependent response to the amount of stimulation (e.g., antigen).

In some embodiments, the provided anti-BCMA CARs exhibit improved expression on the surface of cells, such as compared to an alternative CAR that has an identical amino acid sequence but that is encoded by non-splice site eliminated and/or a codon-optimized nucleotide sequence. In some embodiments, the expression of the recombinant receptor on the surface of the cell can be assessed. Approaches for determining expression of the recombinant receptor on the surface of the cell may include use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 March; 5(177): 177 ra38), Protein L (Zheng et al., J. Transl. Med. 2012 February; 10:29), epitope tags, and monoclonal antibodies that specifically bind to a CAR polypeptide (see international patent application Pub. No. WO2014190273). In some embodiments, the expression of the recombinant receptor on the surface of the cell, e.g., primary T cell, can be assessed, for example, by flow cytometry, using binding molecules that can bind to the recombinant receptor or a portion thereof that can be detected. In some embodiments, the binding molecules used for detecting expression of the recombinant receptor an anti-idiotypic antibody, e.g., an anti-idiotypic agonist antibody specific for a binding domain, e.g., scFv, or a portion thereof. In some embodiments, the binding molecule is or comprises an isolated or purified antigen, e.g., recombinantly expressed antigen.

C. Multispecific Antibodies

In certain embodiments, the BCMA-binding molecules, e.g., antibodies or polypeptides, such as chimeric receptors containing the same, are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific antibodies. Multispecific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for BCMA and the other is for another antigen. In some embodiments, additional binding molecules bind to and/or recognize a third, or more antigens. In certain embodiments, bispecific antibodies may bind to two different epitopes of BCMA. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express BCMA. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the multispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also provided are multispecific chimeric receptors, such as multispecific CARs, containing the antibodies (e.g., antigen-binding fragments). Also provided are multispecific cells containing the antibodies or polypeptides including the same, such as cells containing a cell surface protein including the anti-BCMA antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope on BCMA.

Exemplary antigens include B cell specific antigens, other tumor-specific antigens, such as antigens expressed specifically on or associated with a leukemia (e.g., B cell leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), or a myeloma, e.g., a multiple myeloma (MM), a plasma cell malignancy (e.g., plasmacytoma). For example, antigens include those expressed specifically on or associated with B cell chronic lymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkitt's lymphoma (e.g., endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma), mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), chronic myeloid (or myelogenous) leukemia (CML), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Marginal zone lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), refractory follicular lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma such as multiple myeloma (e.g., non-secretory multiple myeloma, smoldering multiple myeloma), stomach cancer, esophageal cancer, brain cancer, lung cancer (e.g., small-cell lung cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer (e.g., hepatic carcinoma, hepatoma, etc.), bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, spleen cancer (e.g., splenic lymphoma), adrenal cancer and/or head and neck cancer, and antigens expressed on T cells.

In some embodiments, among the second or additional antigens for multi-targeting strategies includes those in which at least one of the antigens is a universal tumor antigen, or a family member thereof. In some embodiments, the second or additional antigen is an antigen expressed on a tumor. In some embodiments, the BCMA-binding molecules provided herein target an antigen on the same tumor type as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a tumor type.

Exemplary second or additional antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, ROR1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, erbB dimers, EGFR viii, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor class C group 5 member D (GPRC5D), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, dual antigen, an antigen associated with a universal tag, a cancer-testes antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, oncofetal antigen, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, livin, AFP, p53, cyclin (D1), CS-1, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGE A3, a cyclin, such as cyclin A1 (CCNA1) and/or a pathogen-specific antigen, biotinylated molecules, molecules expressed by HIV, HCV, HBV and/or other pathogens, and/or in some aspects, neoepitopes or neoantigens thereof. In some embodiments, the antigen is associated with or is a universal tag.

In some aspects, the antigen, e.g., the second or additional antigen, such as the disease-specific antigen and/or related antigen, is expressed on multiple myeloma, such as G protein-coupled receptor class C group 5 member D (GPRC5D), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACI and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol. (2012) 30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058; Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med. (2014) 17(91):37-46. In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Pat. Nos. 8,153,765; 8,603,477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748; and/or International PCT Publication Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) are contained in multispecific antibodies, multispecific chimeric receptors, such as multispecific CARs, and/or multispecific cells.

II. METHODS OF OPTIMIZING AND PRODUCING POLYNUCLEOTIDES, E.G., POLYNUCLEOTIDES ENCODING BCMA CARS, AND OPTIMIZED POLYNUCLEOTIDES

Provided herein are methods for optimizing polynucleotides for expression and/or therapeutic use, and polynucleotides optimized, e.g., according to the methods. In some embodiments, the provided methods or optimizations reduce heterogeneity and/or increase homogeneity of transcribed RNA, such as messenger RNA (mRNA), for example, when the polynucleotide is expressed in a cell, such as in a particular cell type, such as in a mammalian, e.g., human cell type such as a human T cell such as a primary human T cell or T cell line. In some embodiments, the methods for optimizing polynucleotides include methods to identify and remove or alter the sequence of one or more cryptic splice site, such as one or both of a donor splice site or an acceptor splice site. In some embodiments, the methods can additionally or further include codon optimization. In some embodiments, codon optimization can be performed prior to and/or after methods of reducing heterogeneity of transcribed RNA (e.g., mRNA), such as by removal or elimination of predicted splice sites. In some embodiments, codon optimization is integrated in any one or more steps of the method of reducing heterogeneity of transcribed RNAs. In some embodiments, methods of reducing heterogeneity, such as by removal or elimination of predicted splice sites, can be performed after codon optimization. In some embodiments, provided are methods in which a polynucleotide encoding a transgene, including a polynucleotide encoding any of the provided anti-BCMA CAR polypeptides, can be optimized for expression and/or for therapeutic use. In some embodiments, the polynucleotides are modified to optimize codon usage. In some embodiments, the polynucleotides are codon optimized for expression in a human cell such as a human T cell such as a primary human T cell. In some embodiments, the polynucleotides, such as those encoding any of the antibodies, receptors (such as antigen receptors such as chimeric antigen receptors) and/or BCMA-specific binding proteins provided herein, are or have been modified to reduce heterogeneity or contain one or more nucleic acid sequences observed herein (such as by the optimization methods) to result in improved features of the polypeptides, such as the CARs, as compared to those containing distinct, reference, sequences or that have not been optimized. Among such features include improvements in RNA heterogeneity, such as that resulting from the presence of one or more splice sites, such as one or more cryptic splice sites, and/or improved expression and/or surface expression of the encoded protein, such as increased levels, uniformity, or consistency of expression among cells or different therapeutic cell compositions engineered to express the polypeptides. In some embodiments, the polynucleotides can be codon optimized for expression in human cells.

Genomic nucleic acid sequences generally, in nature, in a mammalian cell, undergo processing co-transcriptionally or immediately following transcription, wherein a nascent precursor messenger ribonucleic acid (pre-mRNA), transcribed from a genomic deoxyribonucleic acid (DNA) sequence, is in some cases edited by way of splicing, to remove introns, followed by ligation of the exons in eukaryotic cells. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products following expression in eukaryotic cells.

Polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences, such as complementary DNA (cDNA), or portions thereof, that do not contain introns. Thus, splicing of such sequences is not expected to occur. However, the presence of cryptic splice sites within the cDNA sequence can lead to unintended or undesired splicing reactions and heterogeneity in the transcribed mRNA. Such heterogeneity results in translation of unintended protein products, such as truncated protein products with variable amino acid sequences that exhibit modified expression and/or activity.

Also provided are methods and approaches for determining the heterogeneity of a transcribed nucleic acid such as one encoding or containing a transgene or encoding a recombinant protein. In some embodiments, the methods include determining the heterogeneity of a transcribed nucleic acid sequence that includes all or a portion of the 5′ untranslated region (5′ UTR), and/or all or a portion of the 3′ untranslated region (3′ UTR), of the transcribed nucleic acid. Also provided herein are methods of identifying the presence of splice sites, such as cryptic splice sites, based on the heterogeneity of the transcribed nucleic acid. Also provided are methods of identifying a transgene candidate for the removal of splice sites, such as cryptic splice sites, using the provided methods of determining the heterogeneity of the transcribed nucleic acid of the transgene. Also provided are methods of reducing the heterogeneity of an expressed transgene transcript.

Also provided herein are methods of identifying a transgene or recombinant protein or nucleic acid candidate for the removal or modification of one or more splice sites, such as cryptic splice sites, such as based on the determined heterogeneity of the transcribed nucleic acid, e.g., of the transgene.

Also provided are methods and approaches for reducing the heterogeneity of a transcribed nucleic acid (e.g., transcript) of a transgene (e.g., an expressed transgene transcript) or other nucleic acid. Such methods and approaches can include identifying a transgene candidate for the removal of splice sites (such as cryptic splice sites) according to the provided methods and identifying one or more potential splice donor and/or splice acceptor sites within the transgene. In embodiments of the provided methods the splice donor and/or splice acceptor sites can be in the translated and/or untranslated regions of the transcribed nucleic acid (e.g., transcript).

In some embodiments, eliminating splice sites, such as cryptic splice sites, can improve or optimize expression of a transgene product, such as a polypeptide translated from the transgene, such as an anti-BCMA CAR polypeptide. Splicing at cryptic splice sites of an encoded transgene, such as an encoded BMCA CAR molecule, can lead to reduced protein expression, e.g., expression on cell surfaces, and/or reduced function, e.g., reduced intracellular signaling. Provided herein are polynucleotides, encoding anti-BMCA CAR proteins that have been optimized to reduce or eliminate cryptic splice sites. Also provided herein are polynucleotides encoding anti-BCMA CAR proteins that have been optimized for codon expression and/or in which one or more sequence, such as one identified by the methods or observations herein regarding splice sites, is present, and/or in which an identified splice site, such as any of the identified splice sites herein, is not present. Among the provided polynucleotides are those exhibiting below a certain degree of RNA heterogeneity or splice forms when expressed under certain conditions and/or introduced into a specified cell type, such as a human T cell, such as a primary human T cell, and cells and compositions and articles of manufacture containing such polypeptides and/or exhibiting such properties.

In some embodiments, reducing RNA heterogeneity or removing potential splice site comprises modifying a polynucleotide. In some embodiments, the modification includes one or more nucleotide modifications, such as a replacement or substitution, compared to a reference polynucleotide such as an unmodified polynucleotide that encodes the same polypeptide. In some embodiments, the reference polynucleotide is one in which the transcribed RNA (e.g. mRNA), when expressed in a cell, exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some embodiments, the provided methods can result in polynucleotides in which RNA heterogeneity of transcribed RNA is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some embodiments, the provided methods produce polynucleotides in which RNA homogeneity of transcribed RNA is at least 70%, 75%, 80%, 85%, 90%, or 95% or greater.

A. Methods of Measuring and Reducing RNA Heterogeneity

Provided herein are methods, approaches, and strategies for measuring, evaluating and/or reducing RNA heterogeneity of a nucleic acid, such as of a transcribed RNA, e.g., when expressed in a particular cell type or context, as well as polynucleotides exhibiting reduction in such heterogeneity and/or risk thereof, as compared to a reference polynucleotide. In some embodiments, a reference polynucleotide can be assessed for RNA heterogeneity, such as by methods as described in this Section. In some embodiments, the provided approaches involve identifying RNA (e.g., mRNA) heterogeneity or likelihood thereof, such as in a particular cell or context, such as due to cryptic splice sites. In some aspects, such heterogeneity is identified by amplifying RNA transcripts using a first primer specific to the 5′ untranslated region (5′ UTR), corresponding to a portion of an element located upstream of the transgene in the transcribed RNA, such as a promoter, and a second primer specific to a 3′ untranslated region (3′ UTR), located downstream of the expressed transgene in the transcribed RNA sequence or specific to a sequence within the transgene. In some embodiments, the methods involve amplifying a transcribed nucleic acid using at least one 5′ and 3′ primer pair, wherein at least one pair comprises a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR) of the transcribed nucleic acid to generate one or more amplified products. In some embodiments, the methods involve detecting the amplified products, wherein the presence of two or more amplified products from at least one 5′ and 3′ primer pair indicates heterogeneity in the amplified products. In some embodiments, the detected difference in transcripts are different lengths of the amplified transcript. In some embodiments, the detected difference in transcripts are differences in chromatographic profiles. Exemplary methods for identifying a polynucleotide with RNA heterogeneity are described below. In some embodiments, the methods comprise evaluating RNA heterogeneity for the need of being modified to reduce heterogeneity. In some embodiments, polynucleotides that exhibit RNA heterogeneity greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more are selected for nucleotide modification to remove one or more splice sites, such as one or more cryptic splice sites.

1. Measuring RNA Heterogeneity

RNA heterogeneity can be determined by any of a number of methods provided herein or described or known. In some embodiments, RNA heterogeneity of a transcribed nucleic acid is determined by amplifying the transcribed nucleic acid, such as by reverse transcriptase polymerase chain reaction (RT-PCR) followed by detecting one or more differences, such as differences in size, in the one or more amplified products. In some embodiments, the RNA heterogeneity is determined based on the number of differently sized amplified products, or the proportion of various differently sized amplified products. For example, in some embodiments, RNA heterogeneity is quantified by determining the number, amount or proportion of differently sized amplified product compared to the number or amount of total amplified products. In some cases, all or substantially all of a particular transcript is determined to be equal in size, and in this case, the RNA heterogeneity is low. In some cases, a variety of differently sized transcripts are present, or a large proportion of a particular transcript is of a different size compared to the predicted size of the amplified product without cryptic or undesired splicing events. In some embodiments, RNA heterogeneity can be calculated by dividing the total number or amount of all of amplified products that are of a different size compared to the predicted size of the amplified product by the total number or amount of all amplified products. In some embodiments, the predicted size of the transcript or amplified product is from an RNA that does not contain or is not predicted to contain a cryptic splice site. In some embodiments, the predicted size of the transcript or amplified product takes into account one or more splice sites that are desired or intentionally placed.

In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is harvested from cells, expressing the transgene to be optimized, and amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using a primer specific to the 5′ untranslated region (5′ UTR), in some cases corresponding to a portion of the promoter sequence in the expression vector, located upstream of the transgene in the transcribed RNA, and a primer specific to the 3′ untranslated region (3′ UTR), located downstream of the expressed transgene in the transcribed RNA sequence or a primer specific to a sequence within the transgene. In particular embodiments, at least one primer complementary to a sequence in the 5′ untranslated region (UTR) and at least one primer complementary to a sequence in the 3′ untranslated region (UTR) are employed to amplify the transgene. An exemplary depiction of the amplification of a transcript and resulting product using a forward primer specific to the 5′ UTR and a primer specific to a nucleotide sequence in the 3′ UTR and a predicted amplified product, where no splice events have occurred, is provided in FIG. 21 A . An exemplary depiction of exemplary multiple amplified products (i.e., heterogeneity) resulting from amplification of a transcript that has a 5′ UTR, with a transcribed promoter sequence that contains a known splice donor site (P-SD) and a known splice acceptor site (P-SD), a transcribed transgene containing an unknown (cryptic) splice donor site (T-SD) and two unknown (cryptic) splice acceptor sites (T-SA) and a 3′ UTR, using primers specific to regions of the 5′ UTR and 3′ UTR, is shown in FIG. 21 B .

Exemplary primers specific for the 5′ untranslated region (UTR) include primers directed to sequences within the promoter of the transgene. In some examples, a primer specific to an EF1a/HTLV promoter. An exemplary forward primer, specific to an EF1a-HTLV promoter is set forth in SEQ ID NO: 763.

Exemplary primers specific for the 3′ untranslated region (UTR) include primers directed to 3′ posttranscriptional regulatory elements located downstream of the transgene. Exemplary 3′ posttranscriptional regulatory elements include the woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE), set forth in SEQ ID NO: 636. An exemplary forward primer, specific to a WPRE is set forth in SEQ ID NO: 764.

In some embodiments, multiple primer pairs can be used to amplify the transgene, such as for long transgenes. In some embodiments, sequential or nested pairs of forward and reverse primers, to crease a sliding window of amplified products, can be used to gain full and overlapping coverage of the sequence. Typically, the primers are designed to amplify a length of transgene that is approximately 1.5-6 kb, 2-6 kb, or 3-6 kb. An exemplary depiction of the amplification of a transcript using nested primer pairs is provided in FIG. 21 C .

The amplified nucleic acid sequence is then analyzed for heterogeneity in terms of amplified transcript lengths. In some examples, heterogeneity is determined by the number and intensity of the bands for the expressed sequence. In some embodiments, RNA sequences having splice events upon expression generate multiple bands with different mobilities. In some embodiments, a major band is detected at the predicted mobility for a sequence not having any unpredicted splice events, and 1 or more additional bands of varying intensities and mobilities indicate the occurrence of one or more cryptic splice events within the transgene sequence.

The skilled artisan can resolve RNA, such as messenger RNA, and analyze the heterogeneity thereof by several methods. Non-limiting, exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field flow fractionation, and chromatography, such as size exclusion chromatography or liquid chromatography.

One or more steps of the above techniques can be performed under denaturing conditions, partially denaturing conditions, or non-denaturing conditions. The denaturing conditions can include conditions that cause denaturing of the nucleic acid transcript (e.g., mRNA) due to temperature, chaotropic agents (including salts), organic agents, among other mechanisms for denaturing. With thermal denaturing conditions, an elevated temperature can be applied. The elevated temperature can be one that is sufficient to denature intramolecular hydrogen bonds, to cause a change in or loss of secondary or tertiary structure, and so forth. For example, the temperature or thermal denaturing conditions can include a temperature of 25 degrees Celsius to 95 degrees Celsius, 35 to 85 degrees Celsius, 55 to 75 degrees Celsius, or of another range within those ranges. Similarly, higher or lower temperatures can be used as appropriate to cause the desired level of denaturing. The temperature or thermal denaturing conditions can also be dependent on the identity of the nucleic acid transcript, such that different temperatures are used for different nucleic acid transcripts or types of nucleic acid transcripts. The denaturing conditions can also include using chaotropic agents, such as lithium perchlorate and other perchlorate salts, guanidinium chloride and other guanidinium salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or others. The denaturing conditions can further include organic denaturing agents, such as dimethyl sulfoxide (DMSO), acetonitrile, and glyoxal. In addition, the denaturing conditions can include a combination of two or more of these types of denaturing conditions. Any one or more of the steps of the RNA heterogeneity determining techniques can be performed at an elevated temperature or at ambient temperature, with or without chaotropic or organic agents.

a) Gel Electrophoresis

In some embodiments, RNA transcript topology and apparent (hydrodynamic) size can be analyzed by gele electrophoresis, such as agarose gel electrophoresis. In some examples, RNA transcript can be resolved on a 0.05% to 2% agarose gel, such as a 1.2% agarose gel, and visualized by staining or using probes that are specific to a particular sequence. In some embodiments, RNA transcripts can be directly assessed by gel electrophoresis, or can be assessed after amplification, such as quantitative amplification methods. Nucleic acid stains for visualizing nucleic acid on agarose gel are well known. Exemplary stains include BlueView™ Nucleic Acid Stain (Millipore Sigma), SYBR® Gold Nucleic Acid Stain (ThermoFisher), SYBR® Green Nucleic Acid Stain (Millipore Sigma), SYBR® Green II (ThermoFisher), PicoGreen® nucleic acid stain (Invitrogen), and ethidium bromide: 0.5 μg/mL prepared in distilled water, or incorporated into the gel. In some examples, the nucleic acid is stained using Quant-iT™ PicoGreen® binding followed by fluorescence detection and quantitation of the amplified products. The agarose gel method gives a more quantitative, but less resolving, measure of size distribution. In some embodiments, the nucleic acid fragments, resolved by agarose gel electrophoresis can be visualized by Northern blot for RNA or Southern blot for amplified reverse transcriptase-polymerase chain reaction (RT-PCR) products.

b) Chip-Based Capillary Electrophoresis

Chip-based capillary electrophoresis (e.g., with the AGILENT 2100 BIOANALYZER™) can be used a rapid and routine method for monitoring RNA transcript integrity and its size distribution. The separation is based on hydrodynamic size and charge, and is affected by the nucleotide length and folded structure of the RNA transcript. In one embodiment, the method includes delivering the sample into a channel of a chip with an electrolyte medium and applying an electric field to the chip that causes the RNA transcript and the impurities migrate through the channel. The RNA transcript has a different electrophoretic mobility than the impurities such that the RNA transcript migrates through the channel at rate that is different from a rate at which the impurities migrate through the channel. The electrophoretic mobility of the RNA transcript is proportional to an ionic charge the RNA transcript and inversely proportional to frictional forces in the electrolyte medium. The method also includes collecting from the chip the sample comprising the RNA transcript and one or more separate portions of the sample comprising the impurities. In addition, the method includes characterizing an aspect of at least one of the portion of the sample comprising the RNA transcript and the one or more separate portions of the sample comprising the impurities. The characterizing can include, for example, quantifying charge variants.

c) Analytical Ultracentrifugation (AUC)

Analytical ultracentrifugation (AUC) is a solution phase method for measuring molecular weight distribution, without the potential artifacts that could be introduced by matrix (resin or gel) interaction in the SEC, agarose, or other methods. Both equilibrium AUC and sedimentation ultracentrifugation are used, and the latter provides sedimentation coefficients that are related to both size and shape of the RNA transcript. A BECKMAN™ analytical ultracentrifuge equipped with a scanning UV/visible optics is used for analysis of the RNA transcript.

d) Field Flow Fractionation (FFF)

Another solution phase method for assessing hydrodynamic size distribution is field flow fractionation (FFF). FFF is a separation technique where a field is applied to a fluid suspension or solution pumped through a long and narrow channel, perpendicular to the direction of flow, to cause separation of the polynucleotides (RNA transcripts) present in the fluid, under the force exerted by the field. The field can be asymmetrical flow through a semi-permeable membrane, gravitational, centrifugal, thermal-gradient, electrical, magnetic etc.

e) Chromatography

Chromatography also can be used to detect heterogeneity of RNA transcript lengths. Methods of size exclusion chromatography and liquid chromatography for determining mRNA heterogeneity are described in WO2014144711 which is incorporated herein by reference. B. Methods of Optimizing Polynucleotides, e.g., Polynucleotides Encoding BCMA CARs

In some embodiments, the provided methods include optimizing and/or modifying the polynucleotide, for example, to reduce RNA heterogeneity and/or removing or eliminating cryptic or undesired splice sites. In some aspects, provided are methods of reducing the heterogeneity of an expressed transgene transcript that involves identifying a transgene candidate for the removal of splice sites, such as by the methods described above in Section I.A.; identifying one or more potential splice donor and/or splice acceptor sites; and modifying the nucleic acid sequence at or near the one or more identified splice donor sites that were identified, thereby generating a modified polynucleotide. In some aspects, the methods also involve assessing the transgene candidacy for the removal of splice sites. In some embodiments, the methods also include repeating one or more steps above until the heterogeneity of the transcript is reduced compared to the initial heterogeneity of the transcript as determined (such as before modification).

In some embodiments, methods of reducing heterogeneity, such as by removal or elimination of predicted splice sites, can be performed after codon optimization, or on non codon-optimized RNA. In some aspects, the methods involve identifying splice sites, such as one or more potential splice donor and/or acceptor sites, and modifying or change the RNA sequence (e.g., by replacing or substituting one or more nucleotides at or near the splice site. In some embodiments, codon optimization can be performed prior to and/or after methods of reducing heterogeneity of transcribed RNA (e.g., mRNA), such as by removal or elimination of predicted splice sites. In some embodiments, whether a transcript is a candidate for reducing RNA heterogeneity is determined based on the method of measuring RNA heterogeneity, e.g., as described in Section II.A herein. In some aspects, a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more splice site. In some embodiments, a transgene sequence can be a candidate for reducing heterogeneity when the transcribed nucleic acid of the transgene candidate exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity following expression in a cell. In some embodiments, following transcription and processing of the polynucleotide in a human cell, optionally a human T cell, the messenger RNA (mRNA) from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity.

1. Methods of Reducing RNA Heterogeneity

Provided are methods of reducing heterogeneity of an expressed transgene transcript. In some embodiments, the methods involve identifying one or more potential splice donor and/or splice acceptor sites and modifying the nucleic acid sequence at or near the one or more of the identified splice donor sites. In some embodiments, the methods also involve assessing the transgene candidacy for removal of splice sites. In some aspects, one or more steps described herein can be repeated, for example, until the potential RNA heterogeneity is reduced compared to the starting or unmodified transcript.

a) Splice Site Identification

In some aspects, the presence of potential cryptic splice sites (splice donor and/or acceptor sites that are present in a transcript, such as a transgene transcript, can result in RNA heterogeneity of the transcript following expression in a cell. In some embodiments, the methods involve identifying one or more potential splice sites that can be present in the transgene transcript, that are not desired and/or that may be created in a transgene transcript from various underlying sequences, following codon optimization of a transcript and/or by mutation or mistake or error in transcription. In some aspects of the provided embodiments, the splice donor sites and splice acceptor sites are identified independently. In some embodiments, the splice acceptor and/or donor site(s) is/are canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s).

In some embodiments, the provided methods include identifying one or more potential splice site (e.g., canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s) or branch sites) in a polynucleotide, such as a polynucleotide encoding a transgene, such as a recombinant receptor, that may exhibit RNA heterogeneity or contain undesired. Also provided are polypeptides having reduced numbers of such splice sites as compared to such reference polynucleotides.

In some aspects, identification of the one or more splice sites in a nucleic acid sequence is an iterative process. In some embodiments, splice sites can be identified using a splice site and/or codon optimization prediction tool, such as by submitting the starting or reference sequence encoding the transgene, such as a BCMA-binding receptor, e.g., anti-BCMA CAR, to a database, a gene synthesis vendor or other source able to computationally or algorithmically compare the starting or reference sequence to identify or predict splice sites and/or for codon optimization and/or splice site removal. In some embodiments, after modifying the sequence for codon optimization and/or splice site removal, one or more further assessment of a sequence, such as a revised or modified nucleic acid sequence, is carried out to further evaluate for splice site removal, such as cryptic splice sites, using one or more other or additional splice site prediction tool(s).

In some aspects, RNA heterogeneity can be a result of the activity of the spliceosome present in a eukaryotic cell. In some aspects, splicing is typically carried out in a series of reactions catalyzed by the spliceosome. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products following expression in eukaryotic cells. In some cases, within spliceosomal introns, a donor site (usually at the 5′ end of the intron), a branch site (near the 3′ end of the intron) and an acceptor site (3′ end of the intron) are required for a splicing event. The splice donor site can include a GU sequence at the 5′ end of the intron, with a large less highly conserved region. The splice acceptor site at the 3′ end of the intron can terminate with an AG sequence.

In some embodiments, splice sites, including potential cryptic splice sites can be identified by comparing sequences to known splice site sequences, such as those in a sequence database. In some embodiments, splice sites can be identified by computationally by submitting nucleotide sequences for analysis by splice site prediction tools, such as Human Splice Finder (Desmet et al., Nucl. Acids Res. 37(9):e67 (2009)), a neural network splice site prediction tool, NNSplice (Reese et al., J. Comput. Biol., 4(4):311 (1997)), GeneSplicer (Pertea et al., Nucleic Acids Res. 2001 29(5): 1185-1190) or NetUTR (Eden and Brunak, Nucleic Acids Res. 32(3):1131 (2004)), which identify potential splice sites and the probability of a splicing event at such sites. Additional splice prediction tools include RegRNA, ESEfinder, and MIT splice predictor. Splice site prediction tools such as GeneSplicer has been trained and/or tested successfully on databases for different species, such as human, Drosophila melanogaster, Plasmodium falciparum, Arabidopsis thaliana, and rice. In some embodiments, different prediction tools may be adapted for different extents on different database and/or for different species. In some embodiments, the one or more prediction tools are selected based upon their utility in certain database and/or for certain species. See, e.g., Saxonov et al., (2000) Nucleic Acids Res., 28, 185-190.

In some embodiments, one or more splice site prediction tools are selected for use in the determination of potential splice donor and/or acceptor sites. In some embodiments, splice site prediction tools that can be run locally; that can be retrained with a set of data at the user site; that can use databases for particular species (such as human), that can be compiled for multiple platforms, that allow real-time predictions for sequence selections, and/or that is an OSI certified open source software such that particular tools or plugins can be modified, can be employed. Exemplary tools that can be employed include NNSplice, GeneSplicer or both.

In some aspects, the splice site prediction tools be used to identify a list of potential splice donor and/or splice acceptor sites in a sequence such as a polynucleotide sequence containing transgene sequences. In some aspects, the prediction tools also can generate one or more prediction scores for one or more sequences in the polynucleotide, that can indicate the likelihoods of the one or more sequences being a splice donor or acceptor site sequence.

In some embodiments, the method involves comparing the prediction score for a particular splice site with a threshold score or reference score to determine or identify a particular splice sites that are candidate for elimination or removal. For example, in some embodiments, the predicted splice site is identified as a potential splice site when the prediction score is greater or no less than the threshold score or reference score. In some aspects, considerations for eliminating or removing a particular splice site include the prediction score as compared to a reference score or a threshold score; and whether a particular splice site is desired or intentional (for example, when the splicing event is more advantageous or is required for regulation of transcription and/or translation). In some aspects, the likelihood that the resulting splice variant loses the desired function or has compromised function can also be considered when determining particular donor and/or acceptor sites for elimination or removal. In some aspects, the one or more potential splice donor and/or splice acceptor sites exhibit a score about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 (e.g., on a scale with a maximum of 1.0) of a splice event or probability of a splice event, and the site can be a candidate for splice site elimination or removal. In some aspects, the score, e.g., used by GeneSplicer, at the one or more potential splice donor and/or splice site is based on the difference between the log-odds score returned for that sequence by the true Markov model and the score is computed by the false Markov model. In particular embodiments, the splice donor sites and splice acceptor sites are evaluated independently, or individually. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor/acceptor pair.

b) Splice Site Elimination

In some embodiments, the provided methods involve eliminating or eliminating one or more splice donor and/or splice acceptor site(s), such as the potential splice donor and/or acceptor sites that may be involved in a cryptic splicing event that is not desired or that results in undesired RNA heterogeneity. In some embodiments, eliminating one or more splice sites comprises modifying one or more nucleotides (e.g., by substitution or replacement) in at, containing or near the splice donor and/or acceptor sites that are candidates for removal. In some aspects, a particular nucleotide within a codon that is at, contains or is near the splice site is modified (e.g., substituted or replaced). In some aspects, the modification (such as substitution or replacement) retains or preserves the amino acid encoded by the particular codon at the site, at the same time removing the potential splice donor and/or acceptor sites.

In some embodiments, the codon at or near the splice site for modification comprises one or more codons that involve one or both of the two nucleotides at the potential splice site (in some cases referred to as “splice site codon”). When the potential splicing is predicted to occur between two nucleotides in a codon, the codon is the only splice site codon for this splice site. If the potential splicing is predicted to occur between two adjacent codons, for example, between the last nucleotide of the first codon and the first nucleotide of the next codon, the two codons are splice site codons. For example, for splice sites that are predicted to be at boundaries of two codons, the two adjacent codons can be candidates for nucleotide modification. In some embodiments, the one or more codons comprise one splice site codon. In some embodiments, the one or more codons comprise both splice site codons. In some embodiments, the method involves eliminating potential potential splice donor site by modifying one or both splice site codons. In some embodiments, the method involves eliminating a potential splice acceptor donor site by modifying one or both splice site codons. In some embodiments, the one or both codons at the splice site is not modified, for example, when there are no synonymous codon for the splice site codon. In some embodiments, if there are no synonymous codons available for the particular splice site codon, one or more nucleotides in a nearby codon can be modified. In some embodiments, one or more codons that are modified include a splice site codon, wherein the modification comprises changing one or both nucleotides at the splice site to a different nucleotide or different nucleotides. In some embodiments, In some embodiments, the method involves eliminating the splice donor site by modifying one or both splice site codons, wherein the modification does not change one or two of the nucleotides of the at the splice site to a different nucleotide, but a nearby nucleotide, e.g., a part of a codon adjacent to the splice site, is modified. In some embodiments, the nearby or adjacent nucleotides that can be modified include modification of a nucleotide that is a part of a nearby or adjacent codon, such as a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon.

In some cases, manual modification of the polynucleotides can be employed, while preserving the encoded amino acid sequence, to reduce the probability of a predicted splice site. In some embodiments, one or more of the predicted splice sites having at least 80%, 85%, 90%, or 95% probability of a splice site are manually modified to reduce the probability of the splicing event. In some embodiments, the one or more modification(s) is/are by nucleotide replacement or substitution of 1, 2, 3, 4, 5, 6 or 7 nucleotides. In some embodiments, the modification(s) is/are at the junction of the splice donor site or are at the junction of the splice acceptor site. In some embodiments, at least one of the one or more nucleotide modifications is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the splice acceptor and/or splice donor site. In some embodiments, libraries of modified nucleic acid sequences can be generated with reduced probability of cryptic splice sites. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor/acceptor pair. In particular embodiments, the splice donor sites and splice acceptor sites are evaluated independently, or individually, and not part as a splice donor/acceptor pair. In some embodiments, one or more predicted splice sites are not eliminated. In some embodiments, splice sites, such as known or predicted splice sites, within the promoter region of the transcript are not eliminated.

In some embodiments, the method involves eliminating one or more potential donor splice site by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, the method involves eliminating one or more potential acceptor splice site by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, the nearby or adjacent codon that is subject to modification include a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon, such as a codon that is within one, two or three codons from the splice site. In some embodiments, the methods can include removal or elimination of a potential branch site for splicing. In some aspects, a nucleotide within the codon at or near the branch site can be modified, e.g., substituted or replaced, to eliminate cryptic splicing and/or reduce RNA heterogeneity. In some embodiments, the modification of the one or more nucleotides can involve a substitution or replacement of one of the nucleotides that may be involved in splicing (such as at the splice donor site, splice acceptor site or splice branch site), such that the amino acid encoded by the codon is preserved, and the nucleotide substitution or replacement does not change the polypeptide sequence that is encoded by the polynucleotide. In some cases, the third position in the codon is more degenerate than the other two positions. Thus, various synonymous codons can encode a particular amino acid (see, e.g., Section II.B.2 below). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the species of the cell into which the polynucleotide is introduced (e.g., human). In some embodiments, the species is human. In some embodiments, the one or more codon is replaced with a corresponding synonymous codons that the most frequently used in the species or synonymous codons that have a similar frequency of usage (e.g., most closest frequency of usage) as the corresponding codon (see, e.g., Section II.B.2 below).

In some embodiments, the methods also involve assessing the transgene candidacy for the removal of splice sites, after initial proposed modification. In some aspects, the proposed modification can be evaluated again, to assess the proposed modification and identify any further potential splice sites after modification and/or codon optimization. In some aspects, after modifying the sequence for codon optimization and/or splice site removal, one or more further assessment of a sequence, such as a revised or modified nucleic acid sequence, is carried out to further evaluate for splice site removal, such as cryptic splice sites, using the same or one or more other or additional splice site prediction tool(s). In some aspects, proposed modifications are considered for subsequent steps, and iterative optimization can be used. In some aspects, the methods also include repeating any of the identification and/or modification step, for example, until heterogeneity of the transcript is reduced compared to the heterogeneity of the transcript as initially determined. In some embodiments, a further or a different modification, such as with a different nucleotide replacement at the same codon or a modification at a different position or codon, can be done after an interative evaluation and assessment. In some embodiments, corresponding different synonymous codon can be used, such as the second most frequently used in the particular species or a codon that has a similar frequency of usage (e.g., the next closest frequency of usage) as the corresponding codon (see, e.g., Section II.B.2 below).

In some aspects, a proposed modification can be further evaluated, for example, to assess whether the modification generates an undesired or additional restriction site in the polynucleotide. In some aspects, an additional restriction site may not be desired, and a further or a different modification (e.g., with a different nucleotide replacement at the same codon or a modification at a different position or codon) can be considered. In some aspects, particular restriction site, such as a designated restriction site, is avoided. In some aspects, if the modification does not substantially reduce or, the splice site prediction score, an additional or alternative modification can be proposed. In some embodiments, the splice site prediction score can be is reduced or lowered by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%, after one or more iteration of the methods.

In some embodiments of any of the methods provided herein, a computer system can be used to execute one or more steps, tools, functions, processes or scripts. In certain embodiments, methods provided herein are computer implemented methods and/or are performed with the aid of a computer. In some embodiments, the splice site prediction, evaluation and modification for elimination or removal of a splice site can be performed by computer implemented methods and/or by methods which include steps that are computer implemented steps. In some embodiments, comparison of the sequences to a known database, calculating a splice site prediction score, determining potential nucleotide modifications, codon optimization and/or any one of the iterative steps can be implemented by a computer or using a computer-implemented steps, tools, functions, processes or scripts. In particular embodiments, a computer system comprising a processor and memory is provided, wherein the memory contains instructions operable to cause the processor to carry out any one or more of steps of the methods provided herein. In some embodiments, the methods include steps, functions, processes or scripts that are performed computationally, e.g., performed using one or more computer programs and/or via the use of computational algorithms.

Exemplary steps, functions, processes or scripts of the provided methods for identifying and/or removing possible splice sites include one or more steps of: selecting sequence, writing FASTA format sequences, loading codon table (e.g., from www.kazusa.or.jp/codon, running GeneSplicer, loading predictions, parsing codons, determining overlaps in prediction, identifying next highest usage synonymous codon, reviewing for restriction site, creating annotations or assessing other codons. Particular steps can assess both forward and reverse strands. In some aspects, previously annotated splice site modifications can also be considered, to allow for iterative optimization. In some embodiments, any one or more of the steps, functions, processes or scripts can be repeated.

In certain embodiments, methods provided herein may be practiced, at least in part, with computer system configurations, including single-processor or multi-processor computer systems, minicomputers, mainframe computers, as well as personal computers, hand-held computing devices, microprocessor-based and/or programmable consumer electronics and the like, each of which may operatively communicate with one or more associated devices. In particular embodiments, the methods provided herein may be practiced, at least in part, in distributed computing environments such that certain tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in local and/or remote memory storage devices. In particular embodiments, some or all steps of the methods provided herein may be practiced on stand-alone computers.

In particular embodiments, some or all of the steps of the methods provided herein can operate in the general context of computer-executable instructions, such as program modules, plugins and/or scripts executed by one or more components. Generally, program modules include routines, programs, objects, data structures and/or scripts, that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired. In certain embodiments, instructions operable to cause the processor to carry out any one or more steps of the methods provided herein can be embodied on a computer-readable medium having computer-executable instructions and transmitted as signals manufactured to transmit such instructions as well as the results of performing the instructions, for instance, on a network. In some embodiments, also provided are computer systems, computer readable instructions, software, systems, networks and/or devices for carrying out or performing one or more steps of the methods provided herein.

2. Codon Optimization

In some embodiments the polynucleotides are modified by optimization of the codons for expression in humans. In some aspects, codon optimization can be considered before and/or after the steps for splice site identification and/or splice site elimination, and/or at each of the iterative steps for reducing RNA heterogeneity. Codon optimization generally involves balancing the percentages of codons selected with the abundance, e.g., published abundance, of human transfer RNAs, for example, so that none is overloaded or limiting. In some cases, such balancing is necessary or useful because most amino acids are encoded by more than one codon, and codon usage generally varies from organism to organism. Differences in codon usage between transfected or transduced genes or nucleic acids and host cells can have effects on protein expression from the nucleic acid molecule. Table 3 below sets forth an exemplary human codon usage frequency table. In some embodiments, to generate codon-optimized nucleic acid sequences, codons are chosen to select for those codons that are in balance with human usage frequency. The redundancy of the codons for amino acids is such that different codons code for one amino acid, such as depicted in Table 3. In selecting a codon for replacement, it is desired that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon (e.g., at the third position) can remain unchanged without affecting the amino acid sequence.

For example, the codons TCT, TCC, TCA, TCG, AGT and AGC all code for Serine (note that T in the DNA equivalent to the U in RNA). From a human codon usage frequency, such as set forth in Table 3 above, the corresponding usage frequencies for these codons are 15.2, 17.7, 12.2, 4.4, 12.1, and 19.5, respectively. Since TCG corresponds to 4.4%, if this codon were commonly used in a gene synthesis, the tRNA for this codon would be limiting. In codon optimization, the goal is to balance the usage of each codon with the normal frequency of usage in the species of animal in which the transgene is intended to be expressed.

C. Optimized Anti-BCMA CAR

In some embodiments, a starting or reference sequence encoding a transgene, such as a BCMA-binding receptor, e.g., anti-BCMA CAR, is assessed for codon optimization and/or splice site removal.

In some embodiments, the methods are carried out on an anti-BCMA CAR, such as a CAR containing an scFv antigen-binding domain specific to BCMA, a spacer, such as a spacer set forth in SEQ ID NO:649, a costimulatory signaling region, such as a costimulatory signaling domain from 4-1BB and a CD3 zeta signaling region. Exemplary identified splice donor sites and splice acceptor sites, and their corresponding scores, are listed in Tables 3 and 4 below for exemplary anti-BCMA CARs.

In some embodiments, the resulting modified nucleic acid sequence(s) is/are then synthesized and used to transduce cells to test for splicing as indicated by RNA heterogeneity. Exemplary methods are as follows and described in the Examples. Briefly, RNA is harvested from the expressing cells, amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and resolved by agarose gel electrophoresis to determine the heterogeneity of the RNA, compared to the starting sequence. In some cases, improved sequences can be resubmitted to the gene synthesis vendor for further codon optimization and splice site removal, followed by further cryptic splice site evaluation, modification, synthesis and testing, until the RNA on the agarose gel exhibits minimal RNA heterogeneity.

In some embodiments, the provided methods for optimizing a coding nucleic acid sequence encoding a transgene, such as an anti-BCMA CAR provided herein, or a construct provided herein, is to both reduce or eliminate cryptic splice sites (see, e.g., SEQ ID NO: 622 for an exemplary codon optimized and splice site eliminated spacer sequence) and optimize human codon usage (see, e.g., SEQ ID NO: 855 for an exemplary codon optimized and spacer sequence). An exemplary optimization strategy is described in the Examples.

In some embodiments, provided are polynucleotides encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes BCMA, including any of the antigen-binding domains described below; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments the antigen-binding domain comprises a V H region and a V L region comprising the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain comprises a V H region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from SEQ ID NO: 617; and a V L region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from SEQ ID NO: 618. In some embodiments, In some embodiments, the antigen-binding domain comprises a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:596, 597, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612, and 613, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615, and 603, respectively; or a V H region that is or comprises the amino acid sequence set forth in SEQ ID NO: 617; and a V L region that is or comprises the amino acid sequence set forth in SEQ ID NO: 618. In some embodiments, exemplary antigen-binding domain in the chimeric antigen receptor encoded by the polynucleotide include those described in each row of Table 2 herein. In any of such embodiments, the transmembrane domain of the CAR is or comprises a transmembrane domain derived from a CD28; the intracellular signaling region comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof and a costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.

In some embodiments, provided are polynucleotides encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes BCMA, including any of the antigen-binding domains described below; (b) (b) a spacer, wherein the encoding nucleic acid is or comprises, or consists or consists essentially of, the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments the antigen-binding domain comprises a V H region and a V L region comprising the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain comprises a V H region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the V H region amino acid sequence selected from SEQ ID NO: 617; and a V L region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the V L region amino acid sequence selected from SEQ ID NO: 618. In some embodiments, In some embodiments, the antigen-binding domain comprises a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:596, 597, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599, and 595, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a V H region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612, and 613, respectively, and a V L region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615, and 603, respectively; or a V H region that is or comprises the amino acid sequence set forth in SEQ ID NO: 617; and a V L region that is or comprises the amino acid sequence set forth in SEQ ID NO: 618. In some embodiments, exemplary antigen-binding domain in the chimeric antigen receptor encoded by the polynucleotide include those described in each row of Table 2 herein. In any of such embodiments, the transmembrane domain of the CAR is or comprises a transmembrane domain derived from a CD28; the intracellular signaling region comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof and a costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.

Also provided herein are exemplary modified polynucleotides, including polynucleotides that were modified for codon optimization (O) and/or splice site elimination (SSE). Examples of such polynucleotides are set forth in Table 5, wherein exemplary nucleotide (nt) sequences for the components of the exemplary CAR constructs prior to splice site elimination and codon optimization (non-opt), nucleic acid (nt) sequences for the components of the CAR constructs following splice site elimination and optimization (O/SSE), and the corresponding amino acid (aa) sequences encoded by the nucleic acid sequences are provided. The components include the IgG-kappa signaling sequence (ss), the anti-BCMA scFv, spacer region, transmembrane (tin) domain, co-signaling sequence (4-1BB co-sig or CD28 co-sig), CD3-ζ signaling domain (CD3-ζ), T2A ribosomal skip element (T2A) and truncated EGF receptor (EGFRt) sequence. Polynucleotide sequences of exemplary CAR constructs are set forth in SEQ ID NOs: 751-756, encoding the amino acid sequences set forth in SEQ TD NOs: 757-762.

Also provided are cells such as engineered cells that contain a recombinant receptor (e.g., a chimeric antigen receptor) such as one that contains an extracellular domain including an anti-BCMA antibody or fragment as described herein. Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the BCMA-binding molecule make up at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

Thus also provided are genetically engineered cells expressing the recombinant receptors containing the antibodies, e.g., cells containing the CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (T N ) cells, effector T cells (T EFF ), memory T cells and sub-types thereof, such as stem cell memory T (T SCM ), central memory T (T CM ), effector memory T (T EM ), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

In some embodiments, the cells include one or more polynucleotides introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such polynucleotides. In some embodiments, the polynucleotides are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the polynucleotides are not naturally occurring, such as a polynucleotide not found in nature, including one comprising chimeric combinations of polynucleotides encoding various domains from multiple different cell types. In some embodiments, the cells (e.g., engineered cells) comprise a vector (e.g., a viral vector, expression vector, etc.) as described herein such as a vector comprising a nucleic acid encoding a recombinant receptor described herein.

A. Vectors and Methods for Genetic Engineering

Also provided are methods, polynucleotides, compositions, and kits, for expressing the binding molecules (e.g., anti-BCMA binding molecules), including recombinant receptors (e.g., CARs) comprising the binding molecules, and for producing the genetically engineered cells expressing such binding molecules. In some embodiments, one or more binding molecules, including recombinant receptors (e.g., CARs) can be genetically engineered into cells or plurality of cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.

Also provided are polynucleotides encoding the chimeric antigen receptors and/or portions, e.g., chains, thereof. Among the provided polynucleotides are those encoding the anti-BCMA chimeric antigen receptors (e.g., antigen-binding fragment) described herein. Also provided are polynucleotides encoding one or more antibodies and/or portions thereof, e.g., those encoding one or more of the anti-BCMA antibodies (e.g., antigen-binding fragment) described herein and/or other antibodies and/or portions thereof, e.g., antibodies and/or portions thereof that binds other target antigens. The polynucleotides may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

Also provided are polynucleotides that have been optimized for codon usage and/or to eliminate splice sites, such as cryptic splice sites. Also provided are methods of optimizing and producing the coding sequences of chimeric antigen receptors, such as any of the chimeric antigen receptors described herein. Such methods are described in Section II herein.

Also provided are vectors containing the polynucleotides, such as any of the polynucleotides described herein, and host cells containing the vectors, e.g., for producing the antibodies or antigen-binding fragments thereof. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, or a lentiviral vector. Also provided are methods for producing the antibodies or antigen-binding fragments thereof. The nucleic acid may encode an amino acid sequence comprising the V L region and/or an amino acid sequence comprising the V H region of the antibody (e.g., the light and/or heavy chains of the antibody). The nucleic acid may encode one or more amino acid sequence comprising the V L region and/or an amino acid sequence comprising the V H region of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such polynucleotides are provided. In a further embodiment, a host cell comprising such polynucleotides is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the V H region of the antibody. In another such embodiment, a host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the V L region of the antibody and an amino acid sequence comprising the V H region of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the V L region of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the V H region of the antibody. In some embodiments, a host cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and/or portions thereof, e.g., antigen-binding fragments thereof. In some embodiments, one or more such host cells are provided. In some embodiments, a composition containing one or more such host cells are provided. In some embodiments, the one or more host cells can express different antibodies, or the same antibody. In some embodiments, each of the host cells can express more than one antibody.

Also provided are methods of making the anti-BCMA chimeric antigen receptors. For recombinant production of the chimeric receptors, a nucleic acid sequence encoding a chimeric receptor antibody, e.g., as described herein, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid sequences may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). In some embodiments, a method of making the anti-BCMA chimeric antigen receptor is provided, wherein the method comprises culturing a host cell comprising a nucleic acid sequence encoding the antibody, as provided above, under conditions suitable for expression of the receptor.

In some aspects, for production of isolated or secreted polypeptides, in addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Exemplary eukaryotic cells that may be used to express polypeptides, including isolated or secreted polypeptides, include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, the antibody heavy chains and/or light chains (e.g., V H region and/or V L region) may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains (e.g., V H region and/or V L region). For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

In particular examples immune cells, such as human immune cells are used to express the provided polypeptides encoding chimeric antigen receptors. In some examples, the immune cells are T cells, such as CD4+ and/or CD8+ immune cells, including primary cells, such as primary CD4+ and CD8+ cells.

In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.

In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as following administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 2:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of polynucleotides encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

In some embodiments, recombinant polynucleotides are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant polynucleotides are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or human immunodeficiency virus type 1 (HIV-1). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

In some embodiments, recombinant polynucleotides are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant polynucleotides are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the polynucleotides encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.

Among additional polynucleotides, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

In some embodiments, one or more binding molecules, including antibodies and/or recombinant receptors (e.g., CARs), can be genetically engineered to be expressed in cells or plurality of cells. In some embodiments, a first recombinant receptor and a second binding molecule, e.g., recombinant receptor, are encoded by the same or separate nucleic acid molecules. In some embodiments, additional binding molecules are engineered to be expressed in cells or a plurality of cells.

In some cases, the polynucleotide containing nucleic acid sequences encoding the BCMA-binding receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptide include a signal peptide of the IgG kappa chain set forth in SEQ ID NO: 620, or encoded by the nucleotide sequence set forth in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain set forth in SEQ ID NO:851 and encoded by the nucleotide sequence set forth in SEQ ID NO:850; a CD8 alpha signal peptide set forth in SEQ ID NO:852; or a CD33 signal peptide set forth in SEQ ID NO:853.

In some embodiments the vector or construct can contain promoter and/or enhancer or regulatory elements to regulate expression of the encoded recombinant receptor. In some examples the promoter and/or enhancer or regulatory elements can be condition-dependent promoters, enhancers, and/or regulatory elements. In some examples these elements drive expression of the transgene. In some examples, the CAR transgene can be operatively linked to a promoter, such as an EF1alpha promoter with an HTLV1 enhancer (SEQ ID NO: 635). In some examples, the CAR transgene is operatively linked to a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE; SEQ ID NO: 636), located downstream of the transgene.

In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g. encoding a first and second chimeric receptor) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding a first and second binding molecules, e.g., antibody recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A cleavage sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 659 or 660), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 657 or 658), Thosea asigna virus (T2A, e.g., SEQ ID NO: 631, 653, or 654), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 655 or 656) as described in U.S. Patent Publication No. 20070116690. In some embodiments, the one or more different or separate promoters drive the expression of one or more nucleic acid molecules encoding the one or more binding molecules, e.g., recombinant receptors.

Any of the binding molecules, e.g., antibodies and/or recombinant receptors provided herein, e.g., BCMA-binding molecules and/or the additional recombinant receptors, can be encoded by polynucleotides containing one or more nucleic acid molecules encoding the receptors, in any combinations or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different receptors or domains. In some embodiments, one vector or construct contains nucleic acid molecules encoding one or more binding molecules, e.g., antibody and/or recombinant receptor, and a separate vector or construct contains nucleic acid molecules encoding an additional binding molecule, e.g., antibody and/or recombinant receptor. Each of the nucleic acid molecules can also encode one or more marker(s), such as a surface marker, e.g., truncated EGFR (tEGFR).

Also provided are compositions containing one or more of the nucleic acid molecules, vectors or constructs, such as any described above. In some embodiments, the nucleic acid molecules, vectors, constructs or compositions can be used to engineer cells, such as T cells, to express any of the binding molecules, e.g., antibody or recombinant receptor, and/or the additional binding molecules.

B. Preparation of Cells for Engineering

In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the recombinant receptor (e.g., CAR) may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.

In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contain cells other than red blood cells and platelets.

In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ /Mg ++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.

For example, CD3+, CD28 + T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander, MACSiBeads™, etc.).

In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker + ) at a relatively higher level (marker high ) on the positively or negatively selected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, CD4+ and/or CD8+ selection steps are used to separate CD4+ helper and CD8+ cytotoxic T cells from a composition, such as from a PBMC composition such as one obtained via leukapheresis. Such CD4+ and CD8+ populations, in some aspects, can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In some embodiments, CD4+ and CD8+ cells are mixed at a desired ratio

In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (T CM ) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining T CM -enriched CD8+ T cells and CD4 + T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (T CM ) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for T CM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (T CM ) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

CD4+T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4 + T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.

In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, NJ).

In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads® or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084, are other examples.

The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody—or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS®) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS®) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS® operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out using CliniMACS® system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

The CliniMACS® system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy® system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy® system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy® system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.

Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.

In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

C. Engineered Cells, Vectors and Compositions for Multi-Targeting

Also provided are cells such as engineered cells that can bind to and/or target multiple antigens. In some embodiments, improved selectivity and specificity is achieved through strategies targeting multiple antigens. Such strategies generally involve multiple antigen-binding domains, which typically are present on distinct genetically engineered antigen receptors and specifically bind to distinct antigens. In some embodiments, the cells are engineered with the ability to bind more than one antigen. For example, in some embodiments, the cells are engineered to express multispecific binding molecules. In some embodiments, the cells express multiple binding molecules, e.g., recombinant receptors, each of which can target one antigen or multiple antigens, e.g., one receptor that targets BCMA, such as any described herein, and another receptor that targets another antigen, e.g., tumor antigen. In some aspects, a plurality of genetically engineered antigen receptors are introduced into the cell, which specifically bind to different antigens, each expressed in or on the disease or condition to be targeted with the cells or tissues or cells thereof. Such features can in some aspects address or reduce the likelihood of off-target effects or increase efficacy. For example, where a single antigen expressed in a disease or condition is also expressed on or in non-diseased or normal cells, such multi-targeting approaches can provide selectivity for desired cell types by requiring binding via multiple antigen receptors in order to activate the cell or induce a particular effector function. In some embodiments, a plurality of cells can be engineered to express one or more different binding molecules, e.g., recombinant receptors, each of which can target one antigen or multiple antigens.

Also provided are multispecific cells containing any of the binding molecules described herein, such as cells containing a cell surface protein including the anti-BCMA antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope on BCMA. In some embodiments, provided are compositions of cells that express recombinant receptors, wherein one or more of the binding molecules, multispecific binding molecules and/or recombinant receptors bind and/or target BCMA. In some embodiments, the multispecific binding molecules and/or recombinant receptors target one or more different epitopes on BCMA.

In some embodiments, provided are composition of cells, wherein each type of cell expresses one or more binding molecules, e.g., recombinant receptors. In some embodiments, the cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and/or portions thereof, e.g., antigen-binding fragments thereof. In some embodiments, one or more such cells are provided. In some embodiments, a composition containing one or more such cells is provided. In some embodiments, the one or more cells can express different antibodies, or the same antibody. In some embodiments, each of the cells expresses one or more antibodies, such as more than one antibody. In some embodiments, each of the cells expresses a multispecific binding molecule, e.g., a multispecific receptor, e.g., CAR.

In some embodiments, the cells include multi-targeting strategies that target BCMA and a second or additional antigen associated with a particular disease or condition. In some embodiments, the second or additional antigen is targeted by a multispecific binding molecule and/or multiple binding molecules and/or a plurality of cells, e.g., one or more cells, each engineered to express one or more recombinant receptors. In some embodiments, a recombinant receptor targeting a second or additional antigen is expressed on the same cell as a BCMA binding molecule, or on a different cell.

In some embodiments, among the second or additional antigens for multi-targeting strategies includes those in which at least one of the antigens is a universal tumor antigen, or a family member thereof. In some embodiments, the second or additional antigen is an antigen expressed on a tumor. In some embodiments, the BCMA-binding molecules provided herein target an antigen on the same tumor type as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a tumor type. In some embodiments, the cell further comprises an additional genetically engineered antigen receptor that recognizes a second or additional antigen expressed on a disease or condition to be treated and induces a stimulatory or activating signal.

Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, ROR1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), B cell maturation antigen (BCMA), tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, erbB dimers, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor class C group 5 member D (GPRCSD), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, dual antigen, an antigen associated with a universal tag, a cancer-testes antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, oncofetal antigen, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, β-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, livin, AFP, p53, cyclin (D1), CS-1, BCMA, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGE A3, a cyclin, such as cyclin A1 (CCNA1) and/or a pathogen-specific antigen, biotinylated molecules, molecules expressed by HIV, HCV, HBV and/or other pathogens, and/or in some aspects, neoepitopes or neoantigens thereof. In some embodiments, the antigen is associated with or is a universal tag.

In some embodiments, the plurality of antigens, e.g., the first antigen, e.g., BCMA, and the second or additional antigens, are expressed on the cell, tissue, or disease or condition being targeted, such as on the cancer cell. In some aspects, the cell, tissue, disease or condition is multiple myeloma or a multiple myeloma cell. One or more of the plurality of antigens generally also is expressed on a cell which it is not desired to target with the cell therapy, such as a normal or non-diseased cell or tissue, and/or the engineered cells themselves. In such embodiments, by requiring ligation of multiple receptors to achieve a response of the cell, specificity and/or efficacy is achieved.

In some aspects, the antigen, e.g., the second or additional antigen, such as the disease-specific antigen and/or related antigen, is expressed on multiple myeloma, such as G protein-coupled receptor class C group 5 member D (GPRCSD), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACI and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol . (2012) 30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058; Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med . (2014) 17(91):37-46. In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Pat. Nos. 8,153,765; 8,603,477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748; and/or International PCT Publication Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) are contained in multispecific antibodies, multispecific chimeric receptors, such as multispecific CARs, and/or multispecific cells.

In some embodiments, the cells and methods include multi-targeting strategies, such as expression of two or more genetically engineered receptors on the cell, each recognizing a different antigen and typically each including a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application, Publication No.: WO 2014055668 A1 (describing combinations of a stimulatory or activating and costimulatory CARs, e.g., targeting two different antigens present individually on off-target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013) (describing cells expressing a stimulatory or an activating and an inhibitory CAR, such as those in which the stimulatory or activating CAR binds to one antigen expressed on both normal or non-diseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on the normal cells or cells which it is not desired to treat).

In some embodiments, a plurality of cells, each engineered to express one or more recombinant receptors, are provided. For example, in some embodiments, one cell is engineered to express a binding molecule that binds and/or targets BCMA, and another cell is engineered to express a binding molecule that binds and/or targets an additional or second antigen. In some embodiments, the cells can each express a multispecific binding molecule, e.g., a multispecific recombinant receptor, where one or more of the target antigen is BCMA. In some of such embodiments, the plurality of cells can be administered together or separately. In some embodiments, the plurality of cells are administered simultaneously or concurrently with the cells, e.g., administered on the same day, and/or sequentially with or intermittently with, in any order, another engineered cell in the plurality. For example, in some embodiments, an engineered cell expressing a BCMA-binding molecule, e.g., CAR, is administered simultaneously with or sequentially with, in any order, another engineered cell expressing a binding molecule that binds a different target antigen or a different epitope on BCMA. In some embodiments, the plurality of cells can be in the same composition. Exemplary compositions of the cells include compositions described in Section II below.

Also provided are compositions including the BCMA-binding molecules, immunoconjugates, recombinant receptors, and engineered cells, including pharmaceutical compositions and formulations. Among such compositions are those that include engineered cells, such as a plurality of engineered cells, expressing the provided anti-BCMA recombinant receptors (e.g CARs).

Provided are pharmaceutical formulations comprising a BCMA-binding recombinant chimeric antigen receptors or engineered cells expressing said receptors, a plurality of engineered cells expressing said receptors and/or additional agents for combination treatment or therapy. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier(s) or excipient(s). In some embodiments, the composition includes at least one additional therapeutic agent.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

Formulations of the antibodies described herein can include lyophilized formulations and aqueous solutions.

The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.

Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

In certain embodiments, in the context of genetically engineered cells containing the binding molecules, e.g., CAR, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject. In some aspects, in the context of genetically engineered cells expressing the binding molecules, e.g., CAR, a composition can contain at least the number of cells for administration for a dose of cell therapy, such as about or at least a number of cells described herein for administration, e.g., in Section V.A.

The may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

Also provided are pharmaceutical compositions for combination therapy. Any of the additional agents for combination therapy described herein, such as agents described in Section III.B, can be prepared and administered as one or more pharmaceutical compositions, with the BCMA-binding molecule (e.g., antibody), immunoconjugate, recombinant receptor (e.g., chimeric antigen receptor) and/or engineered cells expressing said molecules (e.g., recombinant receptor) described herein. The combination therapy can be administered in one or more pharmaceutical compositions, e.g., where the binding molecules, recombinant receptors and/or cells are in the same pharmaceutical composition as the additional agent, or in separate pharmaceutical compositions. For example, in some embodiments, the additional agent is an additional engineered cell, e.g., cell engineered to express a different recombinant receptor, and is administered in the same composition or in a separate composition. In some embodiments, each of the pharmaceutical composition is formulated in a suitable formulation according to the particular binding molecule, recombinant receptor, cell, e.g., engineered cell, and/or additional agent, and the particular dosage regimen and/or method of delivery.

Also provided methods of using and uses of the BCMA-binding molecules, immunoconjugates, recombinant receptors, engineered cells, and pharmaceutical compositions and formulations thereof, such as in the treatment of diseases, conditions, and disorders in which BCMA is expressed, and/or detection, diagnostic, and prognostic methods. Among such methods, such as methods of treatment, and uses are those that involve administering to a subject engineered cells, such as a plurality of engineered cells, expressing the provided anti-BCMA recombinant receptors (e.g CARs). Also provided are methods of combination therapy and/or treatment.

A. Therapeutic and Prophylactic Methods and Uses

Also provided are methods of administ