BCMA Chimeric Antigen Receptor and its Use

Abstract

To provide an isolated nucleic acid molecule encoding a chimeric antigen receptor.SOLUTION: In one aspect, the chimeric antigen receptor is an isolated nucleic acid molecule comprising an anti-BCMA binding domain, a transmembrane domain and an intracellular signaling domain, the anti-BCMA binding domain comprising a heavy chain variable region comprising heavy chain complementarity determining region 1, heavy chain complementarity determining region 2 and heavy chain complementarity determining region 3, and a light chain variable region comprising light chain complementarity determining region 1, light chain complementarity determining region 2 and light chain complementarity determining region 3, and each of which comprising specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Patent Application No. 62 / 684,628, filed on 13 June 2018, and U.S. Patent Application No. 62 / 832,991, filed on 12 April 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, created on June 11, 2019, is named N2067-7155WO_SL.txt and has a size of 228,604 bytes.

[0003] The present invention generally relates to the use of immunoeffector cells (e.g., T cells, NK cells) engineered to express chimeric antigen receptors (CARs) for treating diseases associated with the expression of B cell maturation antigen proteins (BCMAs). [Background technology]

[0004] B-cell maturation antigens (BCMAs) are B-cell lineage cells that express tumor necrosis family receptor (TNFR) members. BCMA expression is highest in terminally differentiated B cells. BCMAs are involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has recently been associated with many cancers, autoimmune diseases, and infectious diseases. Cancers with increased BCMA expression include multiple myeloma, Hodgkin lymphoma and non-Hodgkin lymphoma, various leukemias, and several hematological malignancies such as glioblastoma. [Overview of the project] [Means for solving the problem]

[0005] In one embodiment, the present invention is characterized by an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain, e.g., the human anti-BCMA binding domain described herein), a transmembrane domain, and an intracellular signaling domain.

[0006] In another embodiment, the present invention provides an isolated CAR comprising an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain, e.g., the human anti-BCMA binding domain described herein), a transmembrane domain, and an intracellular signaling domain.

[0007] In some embodiments, the anti-BCMA binding domain (e.g., human anti-BCMA binding domain) comprises one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the anti-BCMA binding domain described herein, and / or one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the anti-BCMA binding domain described herein. In some embodiments, the anti-BCMA binding domain comprises the heavy chain variable region and / or the light chain variable region described herein (e.g., Table 2, 6, or 10). In some embodiments, the anti-BCMA binding domain comprises an scFv containing the light and heavy chains of the amino acid sequences in Table 2, 6, or 10. In some embodiments, the anti-BCMA binding domain comprises an scFv as described herein (e.g., Tables 2, 6, or 10). In some embodiments, the CAR comprises a CAR sequence as disclosed herein (e.g., Tables 2, 6, or 10).

[0008] In some embodiments, the anti-BCMA binding domain includes HC CDR1, HC CDR2, and HC CDR3 of any anti-BMCA heavy chain binding domain amino acid sequence listed in Tables 2-13 (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 7, 6, or 5 modifications). In some embodiments, the anti-BCMA binding domain includes LC CDR1, LC CDR2, and LC CDR3 of any anti-BMCA light chain binding domain amino acid sequence listed in Tables 2-13 (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 7, 6, or 5 modifications).

[0009] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 3-5 (e.g., a single column in Tables 3-5) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 3-5 (e.g., a single column in Tables 3-5) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, and 84, respectively (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions)).In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are (i) amino acid sequences of SEQ ID NOs. 44, 45, and 46, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) amino acid sequences of SEQ ID NOs. 44, 45, and 68, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 44, 45, and 76, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0010] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NOs. 54, 55, and 56, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0011] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 46, 54, 55, and 56, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 68, 54, 55, and 56, respectively, or sequences having at least 85%, 90%, 95%, or 99% sequence identity with them, or fewer (iii) A sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications; or (iii) a sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 76, 54, 55, and 56, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications.

[0012] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 52, 70, or 78, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0013] In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NOs. 53, 71, or 79, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0014] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions).

[0015] In some embodiments, the anti-BCMA binding domain comprises VL, the nucleic acid molecule comprises a nucleic acid sequence encoding VL, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 62 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0016] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 52 and 61, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 70 and 61, respectively. (iii) an amino acid sequence having sequence identity or at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications, or (iii) a sequence having sequence identity of at least approximately 85%, 90%, 95%, or 99% with sequence numbers 78 and 61, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications.

[0017] In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 64, 72, or 80, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv), the nucleic acid molecule comprises a nucleic acid sequence encoding the scFv, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 65, 73, or 81, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0018] In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 66, 74, or 82, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 67, 75, or 83, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0019] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 7-9 (e.g., a single column in Tables 7-9) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 7-9 (e.g., a single column in Tables 7-9) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications).

[0020] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 each contain the amino acid sequences of SEQ ID NOs. 86, 130, and 88 (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions)). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 is (i) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 87, and 88, respectively, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 109, and 88, respectively, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 109, and 88, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0021] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 95, 131, and 132, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. CDR3 is (i) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 114, and 115, respectively, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 114, and 97, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0022] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 87, 88, 95, 96, and 97, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 109, 88, 95, 114, and 115, respectively, or sequences having at least 85%, 90%, 95%, or 99% sequence identity with them, or fewer (iii) A sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications; or (iii) a sequence having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 109, 88, 95, 114, and 97, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications.

[0023] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 93 or 112 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 260, 94, or 113 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0024] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 102, 118, or 124 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises the VL, the nucleic acid molecule comprises a nucleic acid sequence encoding the VL, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 261, 103, 119, or 125 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0025] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 93 and 102, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 112 and 118, respectively. (iii) sequences having sequence identity or at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (iii) sequences having sequence numbers 112 and 124, or at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0026] In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 105, 120, or 126, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv), the nucleic acid molecule comprises a nucleic acid sequence encoding the scFv, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 253, 106, 121, or 127, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0027] In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 107, 122, or 128, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 259, 108, 123, or 129, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 258, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0028] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 11-13 (e.g., a single column in Tables 11-13) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 11-13 (e.g., a single column in Tables 11-13) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications).

[0029] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 include the amino acid sequences of SEQ ID NOs. 179, 180, and 181, respectively, or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 include (i) amino acid sequences of SEQ ID NOs. 137, 138, and 139, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); or (ii) amino acid sequences of SEQ ID NOs. 160, 161, and 162, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0030] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NOs. 147, 182, and 183, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 include (i) amino acid sequences of SEQ ID NOs. 147, 148, and 149, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); or (ii) amino acid sequences of SEQ ID NOs. 147, 170, and 171, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0031] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 include (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 137, 138, 139, 147, 148, and 149, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 160, 161, 162, 147, 170, and 171, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications.

[0032] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 145 or 168 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 146 or 169 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0033] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 154 or 173 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises the VL, the nucleic acid molecule comprises a nucleic acid sequence encoding the VL, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 155 or 174 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0034] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL comprise (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 145 and 154, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 168 and 173, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0035] In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 156 or 175 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises a single-chain variable fragment (scFv), the nucleic acid molecule comprises a nucleic acid sequence encoding the scFv, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 157 or 176 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0036] In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 158 or 177, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 159 or 178, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0037] In some embodiments, the anti-BCMA binding domain comprises VH and VL, which are linked by a linker, for example, a linker described herein, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0038] In some embodiments, the transmembrane domain includes the transmembrane domain of a protein selected from the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conserved substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conserved substitutions). In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0039] In some embodiments, the anti-BCMA binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region includes the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the hinge region, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0040] In some embodiments, the transmembrane domain and hinge region include the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the transmembrane domain and hinge region are encoded by the nucleic acid sequence of SEQ ID NO: 254 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0041] In some embodiments, the intracellular signaling domain includes a primary signaling domain, for example, the primary signaling domain described herein, which optionally includes a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the primary signaling domain includes the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 256 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0042] In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain, such as the co-stimulatory signaling domain described herein, which optionally includes MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, and CD7. CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, C D49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, C D18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly It contains functional signaling domains derived from ligands that specifically bind to 9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83.In some embodiments, the co-stimulus signaling domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the co-stimulus signaling domain, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the co-stimulus signaling domain, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 255 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0043] In some embodiments, the intracellular signaling domain includes a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3 zeta, and optionally, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conserved substitutions) but not exceeding 30, 20, or 10 modifications) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conserved substitutions) but not exceeding 30, 20, or 10 modifications), and optionally, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

[0044] In some embodiments, the CAR further comprises a leader sequence containing the amino acid sequence of SEQ ID NO: 1.

[0045] In some embodiments, CAR has one or more (e.g., one, two, or all) of the following properties: (i) CAR activates NFAT signaling in cells in the presence of BCMA-expressing cells when expressed in cells (e.g., T cells), measured by, for example, the JNL screening reporter assay described in Example 1 and evaluated using, for example, the method described in Example 1 with respect to Figure 1A or 1C; (ii) CAR induces cytotoxicity in BCMA-expressing cells when expressed in cells (e.g., T cells), evaluated using, for example, the method described in Example 1 with respect to Figure 3A; and (iii) CAR induces the expression of cytokines (e.g., IFN-γ) in cells in the presence of BCMA-expressing cells when expressed in cells (e.g., T cells), evaluated using, for example, the method described in Example 1 with respect to Figure 3C.

[0046] In another embodiment, the present invention provides an anti-BCMA binding domain comprising a heavy chain variable region (VH) including heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3), and a light chain variable region (VL) including light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the CDR amino acid sequences disclosed herein.

[0047] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 3-5 (e.g., a single column in Tables 3-5) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 3-5 (e.g., a single column in Tables 3-5) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, and 84, respectively (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions)).In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are (i) amino acid sequences of SEQ ID NOs. 44, 45, and 46, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) amino acid sequences of SEQ ID NOs. 44, 45, and 68, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 44, 45, and 76, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0048] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NOs. 54, 55, and 56, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0049] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 46, 54, 55, and 56, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 68, 54, 55, and 56, respectively, or sequences having at least 85%, 90%, 95%, or 99% sequence identity with them, or fewer (iii) A sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications; or (iii) a sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with sequence numbers 44, 45, 76, 54, 55, and 56, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications.

[0050] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 52, 70, or 78, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0051] In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NOs. 53, 71, or 79, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0052] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions).

[0053] In some embodiments, the anti-BCMA binding domain comprises VL, the nucleic acid molecule comprises a nucleic acid sequence encoding VL, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 62 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0054] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 52 and 61, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 70 and 61, respectively. (iii) an amino acid sequence having sequence identity or at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications, or (iii) a sequence having sequence identity of at least approximately 85%, 90%, 95%, or 99% with sequence numbers 78 and 61, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications.

[0055] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 7-9 (e.g., a single column in Tables 7-9) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 7-9 (e.g., a single column in Tables 7-9) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications).

[0056] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 each contain the amino acid sequences of SEQ ID NOs. 86, 130, and 88 (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions)). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 is (i) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 87, and 88, respectively, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 109, and 88, respectively, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 86, 109, and 88, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0057] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 95, 131, and 132, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. CDR3 is (i) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 114, and 115, respectively, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. (iii) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications; or (iii) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the amino acid sequences of SEQ ID NOs. 95, 114, and 97, respectively, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding seven, six, or five modifications.

[0058] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 87, 88, 95, 96, and 97, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 109, 88, 95, 114, and 115, respectively, or sequences having at least 85%, 90%, 95%, or 99% sequence identity with them, or fewer (iii) A sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications; or (iii) a sequence having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 86, 109, 88, 95, 114, and 97, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications.

[0059] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 93 or 112 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 260, 94, or 113 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0060] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 102, 118, or 124 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises the VL, the nucleic acid molecule comprises a nucleic acid sequence encoding the VL, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 261, 103, 119, or 125 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0061] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 93 and 102, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 112 and 118, respectively. (iii) sequences having sequence identity or at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (iii) sequences having sequence numbers 112 and 124, or at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0062] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are HC CDR sequences listed in Tables 11-13 (e.g., a single column in Tables 11-13) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are LC CDR sequences listed in Tables 11-13 (e.g., a single column in Tables 11-13) (or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but with no more than 7, 6, or 5 modifications).

[0063] In some embodiments, HC CDR1, HC CDR2, and HC CDR3 include the amino acid sequences of SEQ ID NOs. 179, 180, and 181, respectively, or sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or sequences having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding seven, six, or five modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, HC CDR1, HC CDR2, and HC CDR3 include (i) amino acid sequences of SEQ ID NOs. 137, 138, and 139, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); or (ii) amino acid sequences of SEQ ID NOs. 160, 161, and 162, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0064] In some embodiments, LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NOs. 147, 182, and 183, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, LC CDR1, LC CDR2, and LC CDR3 include (i) amino acid sequences of SEQ ID NOs. 147, 148, and 149, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions); or (ii) amino acid sequences of SEQ ID NOs. 147, 170, and 171, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications (e.g., substitutions, e.g., conservative substitutions).

[0065] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 include (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 137, 138, 139, 147, 148, and 149, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with sequence numbers 160, 161, 162, 147, 170, and 171, respectively, or sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but not exceeding 7, 6, or 5 modifications.

[0066] In some embodiments, the anti-BCMA binding domain includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 145 or 168 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises VH, the nucleic acid molecule comprises a nucleic acid sequence encoding VH, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 146 or 169 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0067] In some embodiments, the anti-BCMA binding domain includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 154 or 173 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or an amino acid sequence having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions) but not exceeding 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the anti-BCMA binding domain comprises the VL, the nucleic acid molecule comprises a nucleic acid sequence encoding the VL, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 155 or 174 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0068] In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL comprise (i) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 145 and 154, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10; or (ii) sequences having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 168 and 173, respectively, or amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions, e.g., conservative substitutions), but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10.

[0069] In one embodiment, the present invention provides an isolated polypeptide molecule encoded by a nucleic acid molecule described herein. In one embodiment, the present invention provides a vector comprising a nucleic acid molecule described herein or a nucleic acid molecule encoding a CAR described herein. In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenovirus vector, or a retroviral vector. In some embodiments, the vector comprises an EF-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the present invention provides a cell (e.g., a T cell or an NK cell) comprising a nucleic acid molecule described herein, a CAR described herein, a polypeptide molecule described herein, or a vector described herein. In some embodiments, the cell further expresses an inhibitor comprising a first polypeptide comprising at least a portion of an inhibitory molecule related to a second polypeptide comprising a positive signal from an intracellular signaling domain, optionally the inhibitor comprising a first polypeptide comprising at least a portion of PD-1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

[0070] In one embodiment, the present invention provides a method for producing cells, comprising transducing cells (e.g., T cells or NK cells) with a vector described herein. In one embodiment, the present invention provides a method for producing RNA-modified cells, comprising introducing in vitro transcribed RNA or synthetic RNA into cells (e.g., T cells or NK cells), wherein the RNA includes nucleic acid molecules described herein or nucleic acid molecules encoding CARs described herein.

[0071] In one embodiment, the present invention provides a method for providing antitumor immunity in a subject, comprising administering to the subject an effective amount of the cells described herein. In one embodiment, the present invention provides a method for treating a subject having a disease associated with BCMA expression, comprising administering to the subject an effective amount of the cells described herein. In some embodiments, the cells are autologous T cells or allogeneic T cells. In some embodiments, the disease associated with BCMA expression is (i) cancer or malignant tumor or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or preleukemia, or (ii) a non-cancer-related indication associated with BCMA expression. In some embodiments, the disease is hematological cancer or solid tumor. In some embodiments, the disease is acute leukemia, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, WALK The treatment is selected from Denström macroglobulinemia, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferation disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), monoclonal hypergammaglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell hyperplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain amyloidosis or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome)) or combinations thereof. In some embodiments, the disease is multiple myeloma. In some embodiments, the method further comprises administering a second therapeutic agent to the subject.In some embodiments, the second therapeutic agent is a PD-1 inhibitor, which is optionally selected from the group consisting of PDR001, nivolumab, pembrolizumab, pizilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the second therapeutic agent is a PD-L1 inhibitor, which is optionally selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In some embodiments, the second therapeutic agent is a LAG-3 inhibitor, which is optionally selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767. In some embodiments, the second therapeutic agent is a TIM-3 inhibitor, which is optionally selected from the group consisting of MBG453, TSR-022, and LY3321367. In some embodiments, the second therapeutic agent is a CTLA-4 inhibitor, which is optionally ipilimumab or tremelimumab. In some embodiments, the second therapeutic agent is an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both IL-15 polypeptide and IL-15Ra polypeptide, e.g., hetIL-15. In some embodiments, the second therapeutic agent is an interleukin-12 (IL-12) polypeptide. In some embodiments, the second therapeutic agent is an mTOR inhibitor, which is optionally RAD001 or rapamycin.

[0072] The anti-BCMA binding domains disclosed herein and CARs containing such anti-BCMA binding domains have improved properties compared to previous anti-BCMA binding domains and CARs containing them, such as increased binding affinity to BCMA, increased CAR expression levels in cells (e.g., T cells or NK cells), and / or enhanced cytotoxicity and / or the ability to mediate cytokine production in cells (e.g., T cells or NK cells).

[0073] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference as a whole. In addition, materials, methods, and examples are illustrative and not intended to limit the scope. Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are provided simply for readability. The use of headings or numbered or lettered elements herein does not require that the steps or elements be performed in alphabetical order, nor do the steps or elements necessarily have to be separate from one another. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings and the claims. [Brief explanation of the drawing]

[0074] [Figure 1-1]The function of BCMA CAR was tested using a Jurkat NFAT luciferase (JNL) reporter assay with an automated system. CAR clones were evaluated for antigen-dependent activity using the JNL reporter assay. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTDs) were co-cultured in medium alone (Figures 1G and 1H) or in different ratios with target cell lines (KMS11 as a BCMA-positive cell line (Figures 1A and 1C) and NALM6 as a BCMA-negative cell line (Figures 1E and 1F)), and luciferase activity was measured as luminescence intensity. If the luminescence intensity in the presence of antigen-expressing cells was more than twice the level of UTD cells, the clone was considered active. The luminescence reading is a direct measurement of CAR stimulation. Figures 1B and 1D are graphs showing the expression levels of BCMA CAR on JNL cells as detected by flow cytometry using human recombinant(r) BCMA_Fc-AF647. The 1x or 2x platform showed 40,000 or 80,000 H293 cells seeded for virus production. [Figure 1-2] The function of BCMA CAR was tested using a Jurkat NFAT luciferase (JNL) reporter assay with an automated system. CAR clones were evaluated for antigen-dependent activity using the JNL reporter assay. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTDs) were co-cultured in medium alone (Figures 1G and 1H) or in different ratios with target cell lines (KMS11 as a BCMA-positive cell line (Figures 1A and 1C) and NALM6 as a BCMA-negative cell line (Figures 1E and 1F)), and luciferase activity was measured as luminescence intensity. If the luminescence intensity in the presence of antigen-expressing cells was more than twice the level of UTD cells, the clone was considered active. The luminescence reading is a direct measurement of CAR stimulation. Figures 1B and 1D are graphs showing the expression levels of BCMA CAR on JNL cells as detected by flow cytometry using human recombinant(r) BCMA_Fc-AF647. The 1x or 2x platform showed 40,000 or 80,000 H293 cells seeded for virus production. [Figure 1-3] The function of BCMA CAR was tested using a Jurkat NFAT luciferase (JNL) reporter assay with an automated system. CAR clones were evaluated for antigen-dependent activity using the JNL reporter assay. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTDs) were co-cultured in medium alone (Figures 1G and 1H) or in different ratios with target cell lines (KMS11 as a BCMA-positive cell line (Figures 1A and 1C) and NALM6 as a BCMA-negative cell line (Figures 1E and 1F)), and luciferase activity was measured as luminescence intensity. If the luminescence intensity in the presence of antigen-expressing cells was more than twice the level of UTD cells, the clone was considered active. The luminescence reading is a direct measurement of CAR stimulation. Figures 1B and 1D are graphs showing the expression levels of BCMA CAR on JNL cells as detected by flow cytometry using human recombinant(r) BCMA_Fc-AF647. The 1x or 2x platform showed 40,000 or 80,000 H293 cells seeded for virus production. [Figure 1-4]The function of BCMA CAR was tested using a Jurkat NFAT luciferase (JNL) reporter assay with an automated system. CAR clones were evaluated for antigen-dependent activity using the JNL reporter assay. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTDs) were co-cultured in medium alone (Figures 1G and 1H) or in different ratios with target cell lines (KMS11 as a BCMA-positive cell line (Figures 1A and 1C) and NALM6 as a BCMA-negative cell line (Figures 1E and 1F)), and luciferase activity was measured as luminescence intensity. If the luminescence intensity in the presence of antigen-expressing cells was more than twice the level of UTD cells, the clone was considered active. The luminescence reading is a direct measurement of CAR stimulation. Figures 1B and 1D are graphs showing the expression levels of BCMA CAR on JNL cells as detected by flow cytometry using human recombinant(r) BCMA_Fc-AF647. The 1x or 2x platform showed 40,000 or 80,000 H293 cells seeded for virus production. [Figure 2] Expression levels of BCMA CARs on primary human T cells. Cells were stained with human rBCMA_Fc-AF647 reagent and assayed by flow cytometry. The percentages of CAR+ cells and MFIs are shown graphically for days 5 and 9 of cell culture. The data are summarized in Table 17, which includes the viral titers achieved for each CAR. [Figure 3-1]The ability of T cells expressing the indicated CAR to mediate cell lysis and cytokine production was evaluated against KMS11 target cell lines expressing firefly luciferase (KMS11-luc). Figure 3A: CART cells were co-cultured with KMS11-luc target cells at the indicated E:T ratio. % cell death was determined by the difference in luciferase signaling between target cells without effector T cells (control) and target cells with effector T cells (examples), and is expressed as a percentage of the control. UTD represents untransduced T cells. Figure 3B: Background death was observed for the BCMA-negative NALM6 line. Figure 3C: IFNγ was measured by MSD of the supernatant collected 24 hours after these co-culture systems at an E:T ratio of 2.5. All data are expressed as mean + / - standard deviation. [Figure 3-2] The ability of T cells expressing the indicated CAR to mediate cell lysis and cytokine production was evaluated against KMS11 target cell lines expressing firefly luciferase (KMS11-luc). Figure 3A: CART cells were co-cultured with KMS11-luc target cells at the indicated E:T ratio. % cell death was determined by the difference in luciferase signaling between target cells without effector T cells (control) and target cells with effector T cells (examples), and is expressed as a percentage of the control. UTD represents untransduced T cells. Figure 3B: Background death was observed for the BCMA-negative NALM6 line. Figure 3C: IFNγ was measured by MSD of the supernatant collected 24 hours after these co-culture systems at an E:T ratio of 2.5. All data are expressed as mean + / - standard deviation. [Figure 3-3]The ability of T cells expressing the indicated CAR to mediate cell lysis and cytokine production was evaluated against KMS11 target cell lines expressing firefly luciferase (KMS11-luc). Figure 3A: CART cells were co-cultured with KMS11-luc target cells at the indicated E:T ratio. % cell death was determined by the difference in luciferase signaling between target cells without effector T cells (control) and target cells with effector T cells (examples), and is expressed as a percentage of the control. UTD represents untransduced T cells. Figure 3B: Background death was observed for the BCMA-negative NALM6 line. Figure 3C: IFNγ was measured by MSD of the supernatant collected 24 hours after these co-culture systems at an E:T ratio of 2.5. All data are expressed as mean + / - standard deviation. [Modes for carrying out the invention]

[0075] definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in the field relating to this invention.

[0076] The terms "a" and "an" refer to one or more (i.e., at least one) of the grammatical referents of the articles. For example, "element" means one or more elements.

[0077] The term “approximately” means that, when referring to a measurable value such as quantity or duration of time, a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value is included as appropriate for carrying out the method of this disclosure.

[0078] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a specific sequence or a sequence substantially identical or similar thereto, for example, a sequence that is at least 85%, 90%, or 95% identical or more than identical to a specific sequence. In relation to amino acid sequences, the term “substantially identical” is used herein to mean an amino acid sequence having a common structural domain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a first amino acid sequence, for example, a reference sequence, for example, a sequence provided herein, such that the first and second amino acid sequences may have a common structural domain and / or common functional activity.

[0079] In relation to nucleotide sequences, the term “substantially identical” is used herein to mean a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a first nucleic acid sequence, e.g., a reference sequence, e.g., a sequence provided herein, such as a first and second nucleotide sequence, which contain a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides of the second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity.

[0080] The term "mutant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the mutant is a functional mutant.

[0081] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and can possess one or more activities of the reference amino acid sequence.

[0082] As used herein, the term "BCMA" refers to the B cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is primarily expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called the B cell activator (BAFF) and proliferation-inducing ligand (APRIL) of the TNF family. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16 and produces a 994-nucleotide primary mRNA transcript (NCBI accession number NM_001192.2) encoding a 184-amino acid protein (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described, which may play a role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22:1147-1154). Further transcriptional variants have been described as having no significance (Smirnova AS et al. Mol Immunol., 2008, 45(4):1179-1183). A second isoform, also known as TV4, has been identified (Uniprot identifier Q02223-2). As used herein, "BCMA" includes the protein, including mutations in the wild-type full-length BCMA, such as point mutations, fragments, insertions, deletions, and splice variants.

[0083] The term “chimeric antigen receptor” or, instead, “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) comprising a functional signaling domain derived from a stimulating molecule as defined below. In some embodiments, the domains of the CAR polypeptide construct are on the same polypeptide chain, e.g., including a chimeric fusion protein. In some embodiments, the domains of the CAR polypeptide construct are not contiguous with each other, but are on different polypeptide chains, e.g., as provided in the RCAR described herein.

[0084] In one embodiment, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one embodiment, the co-stimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, the leader sequence being optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and cell membrane localization of the CAR.

[0085] A CAR containing an antigen-binding domain (e.g., scFv, single-domain antibody, or TCR (e.g., TCR alpha-binding domain or TCR beta-binding domain)) that targets a specific tumor marker X (where X may be a tumor marker as described herein) is also referred to as an XCAR. For example, a CAR containing an antigen-binding domain that targets BCMA is referred to as a BCMA CAR. CARs can be expressed in any cell, such as immune effector cells as described herein (e.g., T cells or NK cells).

[0086] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate cellular activity via a defined signaling pathway, either by generating secondary messengers or by acting as an effector in response to such messengers.

[0087] As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0088] The term "antibody fragment" refers to at least a portion of an intact antibody or its recombinant variant, and specifically to the antigen-binding domain of the intact antibody, such as the antigen-determining variable region, which is sufficient to result in the recognition and specific binding of the antibody fragment to a target such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), multispecific molecules formed from antibody fragments such as a bivalent fragment containing two Fab fragments linked by disulfide crosslinks at the camelid VHH domain and hinge region, and two or more bound antibodies, such as two isolated CDRs or other epitope-binding fragments. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0089] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked by a short, mobile polypeptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in either order relative to, for example, the N-terminal and C-terminal ends of the polypeptide, and the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0090] The term “complementarity-determining region” or “CDR” as used herein refers to the sequence of amino acids within the antibody variable region that confers antigen specificity and binding affinity. For example, generally there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof. In combinations of Kabat and Chothia numbering schemes, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both.

[0091] A portion of the CAR composition of the present invention, comprising an antibody or an antibody fragment thereof, may exist in various forms, for example, and the antigen-binding domain may be expressed as, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or as part of a polypeptide chain comprising, for example, a human or humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0092] As used herein, the terms “binding domain” or “antibody molecule” (also referred to herein as “antitarget binding domain”) refer to a protein, such as an immunoglobulin chain or a fragment thereof, that contains at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, which comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity to a first epitope, and a second immunoglobulin variable domain sequence has binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to two or fewer antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope.

[0093] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule that exhibits a naturally occurring conformation, and this usually determines the class to which the antibody belongs.

[0094] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules that conform to their natural state. Kappa (κ) and lambda (λ) light chains are the two main antibody light chain isotypes.

[0095] The term "recombinant antibody" refers to antibodies created using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. This term should also be interpreted as meaning an antibody created by the synthesis of a DNA molecule that encodes an antibody and expresses an antibody protein, or an amino acid sequence that specifies the antibody, and the DNA or amino acid sequence is obtained using well-known recombinant DNA or amino acid sequence technologies available in the art.

[0096] The terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immune-qualified cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can be an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will encode an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be sequenced in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded by a “gene” at all. It is readily apparent that antigens may be synthesized, obtained from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids along with other biological components.

[0097] The term "antitumor effect" refers to biological effects that can be manifested by various means, including, but are not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell survival, or an improvement in various physiological symptoms associated with cancerous conditions. The "antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumor development in the first place.

[0098] The term "anti-cancer effect" refers to a biological effect that can be demonstrated by various means, but is not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with cancerous conditions. "Anti-cancer effect" can also be demonstrated by the ability of peptides, polynucleotides, cells, and antibodies to prevent cancer development in the first place. The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, but is not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. The term "self" refers to any material originating from the same individual that will later be reintroduced into that individual.

[0099] The term "homogeneous" refers to any material originating from different animals of the same species as the individual into which it is introduced. Two or more individuals are said to be homogeneous if they do not have identical genes at one or more loci. In some embodiments, homogeneous material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0100] The term "heterogeneous" refers to a graft derived from an animal of a different species.

[0101] The term "apheresis," as used herein, refers to an extracorporeal process recognized in the art, in which blood from a donor or patient is taken from the donor or patient, passed through an apparatus to remove one or more selected specific components, and the remainder is returned to the donor or patient's circulation, for example, by a blood reinfusion method. Accordingly, in relation to "apheresis sample," it refers to a sample obtained using apheresis.

[0102] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Various examples of cancers described herein, but not limited to, include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. Preferred cancers treated by the methods described herein include multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.

[0103] The terms “tumor” and “cancer” are used synonymously herein, and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. When used herein, the terms “cancer” or “tumor” include precancerous and malignant cancers and tumors.

[0104] "Derived from," as used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include limiting methods or sources relating to the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains a sufficient CD3 zeta structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include limiting to a specific method of constructing an intracellular signaling domain, and does not mean, for example, that one must start with a CD3 zeta sequence and delete unwanted sequences or confer mutations to arrive at the intracellular signaling domain.

[0105] The phrase “diseases associated with BCMA expression” includes, but is not limited to, diseases or conditions associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), or non-cancer-related signs associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), including, for example, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplastic syndromes or preleukemia. To avoid misunderstanding, diseases associated with BCMA expression may include conditions associated with cells that previously expressed BCMA but do not currently express it because BCMA expression is downregulated by, for example, treatment with BCMA-targeting molecules such as BCMA inhibitors as described herein. In one embodiment, cancers associated with BCMA (e.g., wild-type or mutant BCMA) expression are hematological cancers. In one embodiment, hematological cancers are leukemia or lymphoma. In one embodiment, cancers associated with the expression of BCMA (e.g., wild-type or mutant BCMA) are malignancies of differentiated plasma cell B cells. In one embodiment, cancers associated with the expression of BCMA (e.g., wild-type or mutant BCMA) include, but are not limited to, one or more acute leukemias, including, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL), and one or more chronic leukemias, including, for example, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).Further cancers or hematological conditions associated with the expression of BMCA (e.g., wild-type or mutant BCMA) include, but are not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndromes and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemia,” a group of hematological conditions encompassed by ineffective production (or dysplasia) of myeloid hematopoiesis. In some embodiments, the cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, or glioblastoma. In embodiments, diseases associated with the expression of BCMA include plasma cell proliferation disorders, e.g., asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), monoclonal immunoglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell hyperplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Further diseases associated with the expression of BCMA (e.g., wild-type or mutant BCMA) include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions or proliferative disorders associated with the expression of BCMA (e.g., wild-type or mutant BCMA), such as the cancers described herein, such as prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0106] Non-cancer-related conditions associated with BCMA (e.g., wild-type or mutant BCMA) include viral infections, e.g., HIV; fungal infections, e.g., C. neoformans; autoimmune diseases, e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris and Sjögren's syndrome; inflammatory bowel disease; ulcerative colitis; transplant-related allospecific immunodeficiencies related to mucosal immunity; and undesirable immune responses to biologics (e.g., factor VIII) where humoral immunity is important. In embodiments, non-cancer-related signs associated with BCMA expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express or have expressed mRNA encoding a tumor antigen at some point in time. In some embodiments, tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In one embodiment, tumor antigen-expressing cells initially produced detectable levels of tumor antigen protein, but subsequently ceased to produce substantially detectable levels of tumor antigen protein.

[0107] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of the antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues in the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0108] The term "stimulus" refers to a primary response that occurs when a stimulating molecule (e.g., the TCR / CD3 complex) binds to its cognitive ligand, thereby mediating a signaling event, including, but not limited to, signaling mediated by the TCR / CD3 complex. Stimuli can mediate changes in the expression of certain molecules, such as the downregulation of TGF-β, and / or recognition of cytoskeletal structures.

[0109] The term “stimulating molecule” refers to a molecule expressed by a T cell that provides one or more primary cytoplasmic signaling sequences that modulate primary activation of the TCR complex in a stimulating manner with respect to at least some aspects of the T cell signaling pathway. In some embodiments, the ITAM-containing domain within the CAR replicates primary TCR signaling independently of the endogenous TCR complex. In one embodiment, primary signaling is induced, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to the mediation of T cell responses, including, but not limited to, proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences that function in a stimulating manner (also referred to as “primary signaling domains”) may include signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in the present invention include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In certain CARs of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, such as the primary signaling sequence of CD3 zeta. The term “antigen-presenting cell” or “APC” refers to immune system cells (e.g., B cells, dendritic cells, etc.) such as accessory cells that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptor (TCR). APCs process the antigens and present them to T cells.

[0110] When this term is used herein, “intracellular signaling domain” refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transmits effector functional signals, prompting cells to perform specialized functions. The entire intracellular signaling domain may be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain, insofar as it transmits effector functional signals. Thus, the term “intracellular signaling domain” is intended to include any truncated portion of an intracellular signaling domain sufficient to transmit effector functional signals.

[0111] The intracellular signaling domain generates signals that promote immune effector functions in CAR-containing cells, such as CART cells. Examples of immune effector functions (e.g., in CART cells) include cytolytic activity, including cytokine secretion, and helper activity.

[0112] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain may be derived from a molecule involved in the primary stimulus or antigen-dependent stimulus. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. An exemplary co-stimulatory intracellular signaling domain may be derived from a molecule involved in the co-stimulatory signal or antigen-independent stimulus. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0113] The primary intracellular signaling domain may contain signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 beta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0114] The terms “Zeta” or alternatively “Zeta chain,” “CD3 zeta,” or “TCR zeta” refer to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3 zeta amino acid sequence. “Zeta-stimulating domain” or alternatively “CD3 zeta-stimulating domain” or “TCR zeta-stimulating domain” refer to the stimulating domain of CD3 zeta or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the cytoplasmic domain of zeta includes residues 52-164 of GenBank accession number BAG36664.1 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, “Zeta-stimulating domain” or “CD3 zeta-stimulating domain” refer to the sequence provided as SEQ ID NO: 9 or 10 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions).

[0115] The term "costimulatory molecule" refers to a cognitive-binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by T cells, including, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD 49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, IT GB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, L This includes ligands that specifically bind to y9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, and CD83.

[0116] The co-stimulatory intracellular signaling domain refers to the intracellular portion of a co-stimulatory molecule.

[0117] An intracellular signaling domain may include the entire intracellular portion of its originating molecule, the entire intrinsic intracellular signaling domain, or a functional fragment thereof.

[0118] The term "4-1BB" refers to CD137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot accession number P20963 provides an exemplary human 4-1BB amino acid sequence. "4-1BB costimulatory domain" refers to the costimulatory domain of 4-1BB or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, "4-1BB costimulatory domain" refers to the sequence provided as Sequence ID No. 7 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions).

[0119] When used herein, “immune effector cells” refer to cells involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.

[0120] When used herein, “immune effector function or immune effector response” refers to the function or response of, for example, immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response refers to the properties of T cells or NK cells that promote the death or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0121] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cell-lytic activity or helper activity, including cytokine secretion.

[0122] The term “coding” refers to the inherent properties of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties it provides, which serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a transcriptional template for a gene or cDNA, can be said to code for a protein or other product of the cDNA of that gene.

[0123] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that encode the same amino acid sequence, including degenerate versions of each other. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, insofar as the nucleotide sequence encoding that protein may contain one or more introns in any version.

[0124] The terms “effective dose” or “therapeutic dose” are used synonymously herein and refer to the amount of a compound, formulation, material or composition that is effective in achieving a particular biological outcome as described herein.

[0125] The term "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0126] The term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

[0127] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into a cell.

[0128] The term "transfer vector" refers to a composition containing isolated nucleic acid that can be used for the delivery of isolated nucleic acid into a cell. In the art, many vectors are known, including, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes self-replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentiviral vectors.

[0129] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, with other expression elements being supplied by a host cell or an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0130] The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0131] The term "lentiviral vector" refers specifically to vectors derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors as provided in Milone et al., Mol.Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors available for clinical use include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX® vector system from Lentigen. Non-clinical lentiviral vectors are also available and will be known to those skilled in the art.

[0132] The terms "homologous" or "identical" refer to the subunit sequence identity between two polymer molecules, between two nucleic acid molecules, or between two polypeptide molecules, such as two DNA molecules or two RNA molecules. When the subunit positions in both molecules are occupied by the same monomeric subunit, for example, when each position in two DNA molecules is occupied by adenine, they are homologous or identical at that position. Homologousity between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences (e.g., five positions in a polymer of 10 subunits) are homologous, then those two sequences are 50% homologous. If 90% of the positions (e.g., nine out of ten) are matching or homologous, then those two sequences are 90% homologous.

[0133] Humanized non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as the antibody's Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences) containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies and their antibody fragments are those in which residues from the recipient's complementarity-determining region (CDR) in a human immunoglobulin (recipient antibody or antibody fragment) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues not found in the recipient antibody or the transferred CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or antibody fragments contain substantially all of at least one and typically two variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin and all or most of the FR region being a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc), typically the Fc of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0134] "Completely human" refers to immunoglobulins such as antibodies or antibody fragments whose entire molecule is of human origin, or whose amino acid sequence is identical to that of human antibodies or immunoglobulins.

[0135] The term "isolated" means that something has been modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from the coexisting material in their natural state are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as in a host cell.

[0136] In connection with the present invention, the following abbreviations are commonly used for commonly existing nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0137] The terms "operably linked" or "transcriptional regulation" refer to a functional link between a regulatory sequence and a heterogeneous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, when a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Operafully linked DNA sequences can be contiguous and, for example, within the same reading frame when it is necessary to link two protein-coding regions together.

[0138] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0139] The terms “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” or “polynucleotide molecule” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded forms and polymers thereof. Unless otherwise specified, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. In some embodiments, “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” or “polynucleotide molecule” include nucleotide / nucleoside derivatives or analogs. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions, e.g., conserved substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions, such as conservative substitutions, can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0140] The terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in a protein sequence or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains of many types, commonly referred to in the art as proteins. “Polypeptides” particularly include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0141] The term "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism, which is necessary to initiate the specific transcription of a polynucleotide sequence.

[0142] The term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product that is operably ligated to that promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, while in others, it may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. A promoter / regulatory sequence may, for example, be responsible for tissue-specific expression of a gene product.

[0143] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designating a gene product, causes the cell to produce the gene product under most or all physiological conditions.

[0144] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designating a gene product, causes the production of a gene product in a cell only if a substantially equivalent inducer is present in the cell.

[0145] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designated by a gene, causes the production of a gene product in a cell only if the cell is substantially the tissue type corresponding to the promoter.

[0146] The term “cancer-associated antigen” or “tumor antigen” synonymously refers to a molecule (typically a protein, carbohydrate, or lipid) that is expressed entirely or instead as a fragment (e.g., MHC / peptide) on the surface of cancer cells and is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, the tumor antigen is a marker expressed on both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells (e.g., 1x overexpression, 2x overexpression, 3x overexpression or more compared to normal cells). In some embodiments, the tumor antigen is a cell surface molecule that is improperly synthesized on cancer cells, e.g., a molecule containing deletions, additions, or mutations compared to a molecule expressed on normal cells. In some embodiments, the tumor antigen is expressed only on the cell surface of cancer cells, entirely or instead as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the present invention comprises a CAR containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presenting peptide. Typically, peptides derived from endogenous proteins fit into the pocket of a major histocompatibility complex (MHC) class I molecule and are recognized by the T cell receptor (TCR) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes constitute a unique class of cell surface targets for immunotherapy.TCR-like antibodies targeting viral or tumor antigen-derived peptides in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries, such as human scFv phage display libraries.

[0147] The terms “tumor-supporting antigen” or “cancer-supporting antigen” are interchangeable and refer to molecules (typically proteins, carbohydrates, or lipids) expressed on the surface of cells that are not cancerous themselves but support cancer cells, for example by promoting proliferation or survival, or resistance to immune cells. Exemplary cells of this type include stromal cells and bone marrow-derived suppressor cells (MDSCs). Tumor-supporting antigens themselves do not necessarily have to play a supporting role for tumor cells, as long as the antigen is present on cells that support cancer cells.

[0148] The term “mobile polypeptide linker” or “linker,” when used in relation to scFv, refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In some embodiments, the mobile polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Ser)n (wherein n is a positive integer of 1 or more, e.g., n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10) (SEQ ID NO: 42). In some embodiments, the mobile polypeptide linker includes, but is not limited to, (Gly4 Ser)4 (SEQ ID NO: 27) or (Gly4 Ser)3 (SEQ ID NO: 28). In other embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Furthermore, the linker described in International Publication No. 2012 / 138475 (incorporated herein by reference) is also within the scope of the present invention.

[0149] When used herein, the 5' cap (RNA cap, RNA7-methylguanosine cap or RNA m) 7 A 5' cap (also known as a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group that is linked to the first transcription nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is linked to transcription and occurs synchronously, with each influencing the other. Immediately after transcription initiation, a cap synthesis complex associated with RNA polymerase binds to the 5' end of the synthesized mRNA. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping region can be modified to adjust the function of the mRNA, such as its stability or translation efficiency.

[0150] As used herein, “in vitro transcription RNA” refers to RNA synthesized in vitro, preferably mRNA. Generally, in vitro transcription RNA is prepared from an in vitro transcription vector. An in vitro transcription vector contains a template used to prepare in vitro transcription RNA.

[0151] As used herein, "poly(A)" refers to a series of adenosines added to mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, poly(A) is present in 50 to 5000 (SEQ ID NO: 30), preferably more than 64, more preferably more than 100, and most preferably more than 300 or 400 sequences. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA function such as localization, stability, or translation efficiency.

[0152] As used herein, “polyadenylation” refers to the covalent bonding of a polyadenylyle moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' end. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to the mRNA precursor by the action of the enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to the transcript containing a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription from DNA to RNA, but can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0153] As used herein, “transient” refers to the expression of a non-integrated transgene over a period of several hours, several days, or several weeks, the duration of which is shorter than the duration of gene expression when integrated into the genome in a host cell or contained in a stable plasmid replicon.

[0154] As used herein, the terms “to treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder or improvement in one or more symptoms (preferably one or more recognizable symptoms) of a proliferative disorder, brought about by the administration of one or more therapies (e.g., one or more therapeutic agents such as CARs of the present invention). In specific embodiments, the terms “to treat,” “treatment,” and “treating” refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily recognizable to the patient. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to either or both physical inhibition of the progression of a proliferative disorder, e.g., by stabilization of recognizable symptoms, or physiological inhibition, e.g., by stabilization of physical parameters. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to a reduction or stabilization of tumor size or the number of cancerous cells.

[0155] The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of signals from one part of a cell to another. The term "cell surface receptor" includes molecules and molecular complexes that receive signals and have the ability to transmit those signals across the cell membrane.

[0156] The term "subject" is intended to include living organisms capable of eliciting an immune response (e.g., mammals, humans).

[0157] The term "substantially purified" refers to cells that essentially contain no other cell types. Substantially purified cells also refer to cells that are isolated from the other cell types to which they normally bind in their natural state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In other examples, the term simply refers to cells that are isolated from the cells to which they naturally bind in their natural state. In some embodiments, these cells are cultured in vitro. In other embodiments, these cells are not cultured in vitro.

[0158] The term "therapeutic," as used herein, means treatment. A therapeutic effect is achieved by reducing, suppressing, relieving, or eradicating a disease state.

[0159] As used herein, the term "prevention" means the prevention of a disease or disease condition or the preventive treatment thereof.

[0160] In relation to the present invention, "tumor antigen," "hyperproliferative disorder antigen," or "antigen associated with hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In a particular embodiment, the hyperproliferative disorder antigen of the present invention is an adenocarcinoma such as primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer and breast cancer, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), ovarian cancer, pancreatic cancer, etc., or plasma cell proliferation disorder, such as asymptomatic myeloma (smoldering multiple myeloma or painless myeloma). Cancers originating from cancers including, but not limited to, myeloma, monoclonal hypergammaglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasmacytoplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome).

[0161] The terms "transfected," "transformed," or "transduced" refer to the process of introducing or transferring exogenous nucleic acids into host cells. A "transfected," "transformed," or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary target cells and their offspring.

[0162] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognitive-binding partner protein (e.g., a stimulating and / or co-stimulating molecule present on T cells) in the sample, but which does not substantially recognize or bind to other molecules in the sample.

[0163] As used herein, “modulated chimeric antigen receptor (RCAR)” refers to a set of polypeptides, typically two polypeptides in its simplest embodiment, that, when present in an immune effector cell, confer specificity to target cells, typically cancer cells, and intracellular signaling to the cell. In some embodiments, the RCAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) comprising a functional signaling domain derived from stimulating and / or co-stimulating molecules as defined herein in relation to the CAR molecule. In some embodiments, the set of polypeptides of the RCAR are not contiguous and, for example, are on different polypeptide chains. In some embodiments, the RCAR comprises a dimerization switch that, in the presence of a dimerizing molecule, can couple polypeptides to each other, for example, the antigen-binding domain to the intracellular signaling domain. In some embodiments, the RCAR is expressed in cells as described herein (e.g., immune effector cells), for example, RCAR-expressing cells (also referred to herein as “RCARX cells”). In some embodiments, the RCARX cells are T cells and are referred to as RCART cells. In one embodiment, RCARX cells are NK cells and are referred to as RCARN cells. RCAR can confer specificity to target cells, typically cancer cells, and regulated intracellular signaling or proliferation to RCAR-expressing cells, which can optimize the immunoeffector properties of RCAR-expressing cells. In an embodiment, RCAR cells rely at least in part on an antigen-binding domain to confer specificity to target cells containing antigens to which the antigen-binding domain is bound.

[0164] When used herein, “membrane anchor” or “membrane tethering domain” refers to a polypeptide or moiety sufficient to anchor an extracellular or intracellular domain to the cell membrane, such as a myristoyl group.

[0165] When the term “switch domain” is used herein, for example, to refer to RCAR, it refers to an entity, typically a polypeptide-based entity, that associates with another switch domain in the presence of a dimerizing molecule. This association results in a functional coupling between a first entity linked, e.g., fused to, the first switch domain, and a second entity linked, e.g., fused to, the second switch domain. The first and second switch domains are collectively referred to as a dimerizing switch. In embodiments, the first and second switch domains are identical to each other, for example, polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerizing switch. In embodiments, the first and second switch domains are different to each other, for example, polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerizing switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerizing molecule is a small molecule, e.g., a rapalog. In one embodiment, the switch domain is a polypeptide-based entity, such as an scFv that binds to a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide polymer, such as a myc ligand or a myc ligand polymer that binds to one or more myc scFvs. In another embodiment, the switch domain is a polypeptide-based entity, such as a myc receptor, and the dimerizing molecule is an antibody or a fragment thereof, such as a myc antibody.

[0166] When the term “dimerizing molecule” is used herein, for example, to refer to RCAR, it refers to a molecule that facilitates the association of the first switch domain and the second switch domain. In embodiments, the dimerizing molecule is either not naturally present in the subject or is not present in concentrations that can result in significant dimerization. In embodiments, the dimerizing molecule is a small molecule, such as rapamycin or rapalog, for example, RAD001.

[0167] The term “low immunoenhancing dose” refers to a dose of an mTOR inhibitor, used in conjunction with an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor, that partially, but not completely, inhibits mTOR activity, as measured, for example, by inhibition of P70 S6 kinase activity. Methods for evaluating mTOR activity, for example, by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to produce complete immunosuppression but sufficient to enhance the immune response. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor results in a decrease in the number of PD-1-positive T cells and / or an increase in the number of PD-1-negative T cells or an increase in the PD-1-negative T cell / PD-1-positive T cell ratio. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor results in one or more of the following: For example, the following marker on memory T cells, or on memory T cell precursors: CD62L high CD127 high CD27 + and increased expression of one or more BCL2 For example, a decrease in KLRG1 expression on memory T cells, for example on memory T cell precursors, and Memory T cell precursors, for example, with the following properties: CD62L high Increase, CD127 high Increase, CD27 + An increase in the number of cells having one or a combination of the following: an increase in KLRG1, a decrease in KLRG1, and an increase in BCL2. Here, all of the changes described above occur, for example, in comparison to an untreated group, and are at least transient.

[0168] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In some embodiments, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancer is also referred to as resistant cancer.

[0169] When used herein, “relapsed” or “recurrent” refers to the recurrence or reappearance of signs and symptoms of a disease (e.g., cancer) or a disease such as cancer after a period of improvement or response, for example, after prior treatment with a therapy, for example, cancer therapy. The initial response period may involve cancer cells at levels below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include cancer cells at levels above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance may include, for example, the reappearance of blast cells in the blood, bone marrow (>5%), or any extramedullary location after a complete response. In this context, a complete response may include <5% BM blast cells. More generally, in some embodiments, a response (e.g., complete response or partial response) may include the absence of detectable MRD (minimal residual disease). In one embodiment, the initial response period lasts at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 6, 8, 10, or 12 months, or at least 1, 2, 3, 4, or 5 years.

[0170] Scope: Throughout this disclosure, various aspects of the invention may be presented in the form of scope. It should be understood that descriptions in the form of scope are merely for convenience and conciseness and should not be interpreted as a definitive limitation to the scope of the invention. Accordingly, a scope description should be understood to have all possible sub-scopes specifically disclosed and the individual numerical values ​​within those scopes. For example, a scope description such as 1-6 should be understood to have specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6 and the individual numerical values ​​within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95-99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity, and also includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the range width.

[0171] When used herein, "gene editing system" refers to a system, e.g., one or more molecules, that induces and carries out modification, e.g., deletion, of one or more nucleic acids at or near a site of a gene DNA targeted by the system. Gene editing systems are well known in the art and are more fully described below.

[0172] As used herein, “combined” administration means the delivery of two or more different treatments to a subject during the course of the subject’s illness, for example, two or more treatments delivered after the subject has been diagnosed with the illness and before the illness is cured or eliminated or before the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment is still taking place at the start of the delivery of the second treatment, such that there is an overlap in the duration of administration. This may be referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective for combined administration. For example, the second treatment is more effective, for example, an equal effect is seen with less of the second treatment, or the second treatment alleviates symptoms to a greater extent than is seen when the second treatment is administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery results in a greater reduction of the symptoms or other parameters related to the disorder than is observed with the delivery of one treatment in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effect of the delivered first treatment is still detectable at the time of delivery of the second treatment.

[0173] Various embodiments of the compositions and methods described herein will be further described below. Further definitions are provided throughout this specification.

[0174] explanation This specification provides for compositions of substances and methods of use for treating diseases such as cancer, using cells expressing BCMA chimeric antigen receptors (CARs), such as CART-BCMA.

[0175] In one embodiment, the present invention provides cells (e.g., immune effector cells, e.g., T cells or NK cells) engineered to express CAR, and CART cells ("CART") or CAR NK cells exhibit antitumor properties. In one embodiment, cells are transformed with CAR, and CAR is expressed on the cell surface. In some embodiments, cells (e.g., immune effector cells, e.g., T cells or NK cells) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, cells can stably express CAR. In another embodiment, cells (e.g., immune effector cells, e.g., T cells or NK cells) are transfected with nucleic acids encoding CAR, e.g., mRNA, cDNA, DNA. In some such embodiments, cells can transiently express CAR.

[0176] In one embodiment, the CAR of the present invention combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some embodiments, the intracellular signaling molecule includes, but is not limited to, CD3-zeta chains, 4-1BB and CD28 signaling modules, and combinations thereof. In one embodiment, the antigen-binding domain binds to BCMA.

[0177] Furthermore, the present invention provides BCMA CAR compositions and agents or methods for treating cancer, any malignant tumor, or autoimmune disease in which cells or tissues expressing BCMA are involved.

[0178] In one aspect, the CAR of the present invention can be used to eradicate BCMA-expressing normal cells and is therefore applicable to use as a cell conditioning therapy before cell transplantation. In one aspect, BCMA-expressing normal cells are BCMA-expressing normal stem cells, and cell transplantation is stem cell transplantation.

[0179] In one embodiment, the present invention provides cells (e.g., T cells or NK cells) engineered to express a chimeric antigen receptor (CAR), where the CAR T cells ("CART") or CAR NK cells exhibit antitumor properties. A preferred antigen is BCMA. In one embodiment, the antigen-binding domain of the CAR comprises a human anti-BCMA antibody fragment. In another embodiment, the antigen-binding domain of the CAR comprises a human anti-BCMA antibody fragment containing scFv. Accordingly, the present invention provides BCMA-CARs engineered into cells, e.g., T cells or NK cells, comprising a human anti-BCMA binding domain, and methods for using them for adoptive therapy.

[0180] In one embodiment, BCMA-CAR comprises at least one intracellular domain selected from the group consisting of, for example, a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta-signaling domain, and any combination thereof. In one embodiment, BCMA-CAR comprises at least one intracellular signaling domain, which is derived from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.

[0181] Chimeric antigen receptor (CAR) The present invention provides a CAR (e.g., a CAR polypeptide) comprising an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain as described herein), a transmembrane domain, and an intracellular signaling domain, wherein the anti-BCMA binding domain comprises heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of any anti-BCMA heavy chain binding domain amino acid sequence listed in Tables 2 to 13. The anti-BCMA binding domain of the CAR may further comprise light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of any anti-BCMA light chain binding domain amino acid sequence listed in Tables 2 to 13.

[0182] The present invention also provides nucleic acid molecules encoding CARs as described herein, comprising, for example, an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain as described herein), a transmembrane domain, and an intracellular signaling domain, wherein the anti-BCMA binding domain comprises HC CDR1, HC CDR2, and HC CDR3 of any anti-BMCA heavy chain binding domain amino acid sequences listed in Tables 2 to 13. In some embodiments, the anti-BCMA binding domain of the encoded CAR may further comprise LC CDR1, LC CDR2, and LC CDR3 of any anti-BMCA light chain binding domain amino acid sequences listed in Tables 2 to 13.

[0183] In one embodiment, an exemplary BCMA CAR construct comprises an arbitrary leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulant domain. An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplary nucleic acid sequence encoding the leader sequence is provided as SEQ ID NO: 12. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 2, 3, 4, or 5. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. A sequence of the intracellular signaling domain of an exemplary 4-1BB protein is provided as SEQ ID NO: 7. A sequence of the intracellular signaling domain of an exemplary CD27 is provided as SEQ ID NO: 8. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 9 or 10. In certain embodiments, the domains are adjacent to or within the same reading frame to form a single fusion protein. In other embodiments, the domains are in separate polypeptides, such as in the RCAR molecule described herein.

[0184] A CAR construct may contain a Gly / Ser linker having one or more of the following sequences: GGGGS (sequence number 25); containing 1-6 "Gly Gly Gly Gly Ser" iteration units, e.g., GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS (sequence number 26); GGGGSGGGGS GGGGSGGGGS (sequence number 27); GGGGSGGGGS GGGGS (sequence number 28); GGGS (sequence number 29); or containing 1-10 "Gly Gly Gly Ser" iteration units, e.g., GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (sequence number 42).

[0185] In embodiments, the CAR construct includes a sequence containing a poly-A sequence, for example, 50-5000 or 100-5000 adenines (sequences 30 and 33, respectively) (e.g., sequence 30, sequence 33, sequence 34, or sequence 35) or a sequence containing 50-5000 thymines (sequence 32) (e.g., sequence 31, sequence 32). Alternatively, the CAR construct may include a linker containing, for example, the sequence GSTSGSGKPGSGEGSTKG (sequence 43).

[0186] In certain embodiments, the full-length BCMA CAR molecule comprises an amino acid sequence of R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03, or Hy52, or a sequence substantially (e.g., 95-99%) identical thereto, or is encoded by its nucleotide sequence. In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen-binding domain comprises an scFv amino acid sequence of R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03, or Hy52, or a sequence substantially (e.g., 95-99%) identical thereto, as provided in Tables 2, 6, and 10. In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen-binding domain includes heavy chain variable regions and / or light chain variable regions of sequences substantially (e.g., 95-99%) identical thereto, such as R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03, or Hy52, as provided in Tables 2, 6, and 10. In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen-binding domain comprises one, two, or three CDRs from the heavy chain variable region (e.g., HCDR1, HCDR2, and / or HCDR3) of R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03, or Hy52 provided in Tables 2-13, or a sequence substantially (e.g., 95-99%) identical thereto, and / or one, two, or three CDRs from the light chain variable region (e.g., LCDR1, LCDR2, and / or LCDR3).

[0187] Table 1 lists non-exclusive examples of sequences of various components that may be part of the CAR molecules described herein. In the table, "aa" represents an amino acid, and "na" represents a nucleic acid encoding the corresponding peptide.

[0188] [Table 1]

[0189] [Table 2]

[0190] [Table 3]

[0191] [Table 4]

[0192] [Table 5]

[0193] [Table 6]

[0194] CAR antigen-binding domain In one embodiment, a portion of the CAR containing an antigen-binding domain includes an antigen-binding domain that targets a tumor antigen, such as a tumor antigen as described herein. In one embodiment, the CAR of the present invention includes a binding domain that specifically binds to BCMA (e.g., human BCMA).

[0195] The antigen-binding domain may be any protein that binds to an antigen, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and their functional fragments, single-domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), and recombinant fibronectin domains, and alternative scaffolds known in the art to function as antigen-binding domains.

[0196] Exemplary anti-BCMA binding domain amino acid sequences are provided in Tables 2-13. In one embodiment, the antigen-binding domain comprises a human antibody or human antibody fragment. In some embodiments, the human anti-BCMA binding domain comprises one or more (e.g., all three) of the human anti-BCMA binding domains LC CDR1, LC CDR2, and LC CDR3 described herein (e.g., Tables 2-13) and / or one or more (e.g., all three) of the human anti-BCMA binding domains HC CDR1, HC CDR2, and HC CDR3 described herein (e.g., Tables 2-13). In some embodiments, the human anti-BCMA binding domain comprises human VL and / or human VH described herein (e.g., Tables 2, 6, and 10). In some embodiments, the anti-BCMA binding domain is an scFv comprising the VL and VH of the amino acid sequences in Tables 2, 6, and 10. In one embodiment, the anti-BCMA binding domain (e.g., scFv) includes VL, which has at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences provided in Tables 2, 6, and 10, but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10, or a sequence that is 95-99% identical to the amino acid sequences in Tables 2, 6, and 10; and / or VH, which has at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences provided in Tables 2, 6, and 10, but the number of modifications (e.g., substitutions, e.g., conservative substitutions) does not exceed 30, 20, or 10, or a sequence that is 95-99% identical to the amino acid sequences in Tables 2, 6, and 10.

[0197] Table 7

[0198] Table 8

[0199] Table 9

[0200] Table 10

[0201] Table 11

[0202] Table 12

[0203] Table 13

[0204] Table 14

[0205] Table 15

[0206] Table 16

[0207] Table 17

[0208] Table 18

[0209] Table 19

[0210] Table 20

[0211] Table 21

[0212] Table 22

[0213] Table 23

[0214] Table 24

[0215] Table 25

[0216] Table 26

[0217] Table 27

[0218] Table 28

[0219] Table 29

[0220] Table 30

[0221] Table 31

[0222] Table 32

[0223] Table 33

[0224] Table 34

[0225] Table 35

[0226] Table 36

[0227] Table 37

[0228] [Table 38]

[0229] [Table 39]

[0230] [Table 40]

[0231] [Table 41]

[0232] [Table 42]

[0233] [Table 43]

[0234] [Table 44]

[0235] [Table 45]

[0236] [Table 46]

[0237] In some embodiments, the human anti-BCMA binding domain includes HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3.

[0238] In certain embodiments, the CAR molecule described herein or the anti-BCMA binding domain described herein is (1) One, two, or three light chain (LC) CDRs selected from the following: (i) LC CDR1 of SEQ ID NO: 54, LC CDR2 of SEQ ID NO: 55, and LC CDR3 of SEQ ID NO: 56; and / or (2) One, two, or three heavy chain (HC) CDRs from the following: (i) HC CDR1 of sequence number 44, HC CDR2 of sequence number 45, and HC CDR3 of sequence number 84; (ii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 46; (iii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 68; or (iv) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 76 Includes.

[0239] In some embodiments, the CAR molecule or anti-BCMA binding domain described herein is (1) One, two, or three light chain (LC) CDRs from the following: (i) LC CDR1 of sequence number 95, LC CDR2 of sequence number 131, and LC CDR3 of sequence number 132; (ii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 96, and LC CDR3 of SEQ ID NO: 97; (iii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 115; or (iv) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 97; and / or (2) One, two, or three heavy chain (HC) CDRs from the following: (i) HC CDR1 of sequence number 86, HC CDR2 of sequence number 130, and HC CDR3 of sequence number 88; (ii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 87, and HC CDR3 of SEQ ID NO: 88; or (iii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 109, and HC CDR3 of SEQ ID NO: 88 Includes.

[0240] In some embodiments, the CAR molecule or anti-BCMA binding domain described herein is (1) One, two, or three light chain (LC) CDRs from the following: (i) LC CDR1 of sequence number 147, LC CDR2 of sequence number 182, and LC CDR3 of sequence number 183; (ii) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 148, and LC CDR3 of SEQ ID NO: 149; or (iii) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 170, and LC CDR3 of SEQ ID NO: 171; and / or (2) One, two, or three heavy chain (HC) CDRs from the following: (i) HC CDR1 of sequence number 179, HC CDR2 of sequence number 180, and HC CDR3 of sequence number 181; (ii) HC CDR1 of SEQ ID NO: 137, HC CDR2 of SEQ ID NO: 138, and HC CDR3 of SEQ ID NO: 139; or (iii) HC CDR1 of SEQ ID NO: 160, HC CDR2 of SEQ ID NO: 161, and HC CDR3 of SEQ ID NO: 162 Includes.

[0241] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 84, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 68, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 76, 54, 55, and 56, respectively.

[0242] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 84, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 46, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 68, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 76, 57, 58, and 59, respectively.

[0243] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 85, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 51, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 69, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 77, 60, 58, and 56, respectively.

[0244] In some embodiments, the human anti-BCMA binding domain comprises an scFv containing VH (e.g., VH as described herein) and VL (e.g., VL as described herein). In some embodiments, VH is bound to VL via a linker, e.g., a linker as described herein, e.g., a linker as described in Table 1. In some embodiments, the human anti-BCMA binding domain comprises a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4) (SEQ ID NO: 26). The light chain variable region and heavy chain variable region of the scFv may be, for example, in either of the following directions: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0245] In one embodiment, the anti-BCMA binding domain is a fragment, for example, a single-chain variable fragment (scFv). In one embodiment, the anti-BCMA binding domain is Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one embodiment, the antibody and its fragments of the present invention bind to the BCMA protein with wild-type affinity or enhanced affinity.

[0246] In some cases, scFv can be produced by methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking the VH and VL regions together using a mobile polypeptide linker. The scFv molecule contains a linker of optimal length and / or amino acid composition (e.g., a Ser-Gly linker). Linker length can significantly influence how the variable regions of the scFv fold and interact. In fact, when using short polypeptide linkers (e.g., 5-10 amino acids), intrachain folding is inhibited. Intrachain folding is also necessary for the two variable regions to unite and form a functional epitope binding site. For examples of linker orientations and sizes, see, for example, Hollinger et al. 1993 Proc Natl Acad.Sci.USA90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and International Publication Brochures Nos. 2006 / 020258 and 2007 / 024715, which are incorporated herein by reference.

[0247] scFv may contain a linker between its VL and VH regions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues. The linker sequence may contain any naturally occurring amino acids. In one embodiment, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a series of glycine and serine repeats such as (Gly4Ser)n (where n is a positive integer of 1 or more) (SEQ ID NO: 25). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). Variations in linker length may maintain or enhance activity and may result in superior efficacy in activity testing.

[0248] transmembrane domain With respect to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain that binds to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular domain of the protein from which the transmembrane originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 from the extracellular domain) and / or one or more additional amino acids associated with the intracellular domain of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 from the intracellular domain). In one embodiment, the transmembrane domain binds to other domains of the CAR. In one example, the transmembrane domain can be selected or modified by amino acid substitution to minimize interactions with other members of the receptor complex, for example, to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein. In one embodiment, the transmembrane domain can homodimerize with another CAR on the surface of a CAR-expressing cell (e.g., a CART cell). In a different embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell, for example, CART.

[0249] The transmembrane domain may be of natural origin or recombinant origin. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain can always signal to the intracellular domain when the CAR binds to a target. Particularly useful transmembrane domains in the present invention may include, for example, the alpha, beta, or zeta chain of a T cell receptor, or at least the transmembrane regions of CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In some embodiments, the transmembrane domain is at least a co-stimulatory molecule (e.g., MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphoid activator molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a) / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD 49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, IT GB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile) It may include transmembrane domains of ligands that specifically bind to CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0250] In one embodiment, the transmembrane domain can bind to the extracellular region of the CAR, such as the antigen-binding domain of the CAR, via a hinge, such as a hinge from a human protein. For example, in some embodiments, the hinge may be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8a hinge. In some embodiments, the hinge or spacer comprises (for example, derived from) the amino acid sequence of SEQ ID NO: 2. In one embodiment, the transmembrane domain comprises (for example, derived from) the transmembrane domain of SEQ ID NO: 6.

[0251] In one embodiment, the hinge or spacer includes an IgG4 hinge. For example, in some embodiments, the hinge or spacer includes the hinge of SEQ ID NO: 3. In some embodiments, the hinge or spacer includes a hinge encoded by the nucleotide sequence of SEQ ID NO: 14.

[0252] In one embodiment, the hinge or spacer includes an IgD hinge. For example, in some embodiments, the hinge or spacer includes a hinge of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge or spacer includes a hinge encoded by the nucleotide sequence of SEQ ID NO: 15.

[0253] In one embodiment, the transmembrane domain may be recombinant, in which case it predominantly contains hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine can be seen at each end of the recombinant transmembrane domain.

[0254] Optionally, short oligo or polypeptide linkers of 2 to 10 amino acids in length may form a bond between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the linker is encoded by the nucleotide sequence of SEQ ID NO: 16.

[0255] In one embodiment, the hinge or spacer includes a KIR2DS2 hinge.

[0256] Cytoplasmic domain The cytoplasmic domain or region of the CAR of the present invention includes an intracellular signaling domain. The intracellular signaling domain is generally responsible for the activation of at least one normal effector function of the immune cell into which the CAR has been introduced.

[0257] Examples of intracellular signaling domains used in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that function in coordination to initiate signaling after antigen receptor binding, as well as any derivatives or variants thereof and any recombinant sequences having the same functional capabilities.

[0258] It is known that signals produced by the TCR alone are insufficient for complete T cell activation, and that secondary and / or costimulatory signals are also required. Therefore, T cell activation can be said to involve two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0259] Primary signaling domains regulate the primary activation of the TCR complex in either a stimulative or inhibitory manner. Primary intracellular signaling domains that act in a stimulative manner may contain immunoreceptor tyrosine-based activation motifs or signaling motifs known as ITAMs.

[0260] Examples of primary intracellular signaling domains containing ITAM that are particularly useful in the present invention include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d. In some embodiments, the CAR of the present invention includes an intracellular signaling domain, such as the primary signaling domain of CD3 zeta.

[0261] In some embodiments, the primary signaling domain includes a modified ITAM domain having altered (e.g., increased or decreased) activity compared to the natural ITAM domain, such as a mutant ITAM domain. In some embodiments, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs.

[0262] Further examples of molecules containing primary intracellular signaling domains particularly useful in the present invention include those of DAP10, DAP12, and CD32.

[0263] The intracellular signaling domain of a CAR may contain a primary signaling domain, such as a CD3 zeta signaling domain, by itself, or may be combined with any other desired intracellular signaling domain useful in relation to the CAR of the present invention. For example, the intracellular signaling domain of a CAR may contain a primary signaling domain, such as a CD3 zeta chain portion, and a costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. The costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand necessary for an efficient response of lymphocytes to an antigen.Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, C D18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), The present invention includes ligands that specifically bind to CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. For example, CD27 co-stimulation has been shown to enhance the proliferation, effector function, and survival of human CART cells in vitro, and to enhance human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012;119(3):696-706). The intracellular signaling sequences within the cytoplasm of the CARs of the present invention can be linked to each other randomly or in a specific order.Optionally, short oligo or polypeptide linkers of, for example, 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form links between intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid, such as alanine or glycine, can be used as a suitable linker.

[0264] In one embodiment, the intracellular signaling domain is designed to include two or more, for example, two, three, four, five, or more, co-stimulatory signaling domains. In one embodiment, the two or more, for example, two, three, four, five, or more, co-stimulatory signaling domains are separated by a linker molecule, for example, a linker molecule described herein. In some embodiments, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0265] In one embodiment, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a CD28 signaling domain. In one embodiment, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a 4-1BB signaling domain. In one embodiment, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 7. In one embodiment, the CD3 zeta signaling domain is the signaling domain of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human CD3 zeta).

[0266] In one embodiment, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a CD27 signaling domain. In one embodiment, the CD27 signaling domain includes the amino acid sequence of SEQ ID NO: 8. In one embodiment, the CD27 signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 19.

[0267] In one embodiment, the cell is designed to include a CD3 zeta signaling domain and a CD28 signaling domain. In one embodiment, the CD28 signaling domain includes the amino acid sequence of SEQ ID NO: 36. In one embodiment, the CD28 signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 37.

[0268] In one embodiment, the cell is designed to include the signaling domain of CD3 zeta and the signaling domain of ICOS. In one embodiment, the signaling domain of ICOS includes the amino acid sequence of SEQ ID NO: 38. In one embodiment, the signaling domain of ICOS is encoded by the nucleic acid sequence of SEQ ID NO: 39.

[0269] CAR configuration Multispecific CAR In one embodiment, the CAR of the present invention is a multispecific CAR. In some embodiments, the multispecific CAR is a bispecific CAR. In some embodiments, the bispecific CAR includes an antigen-binding domain that is a bispecific antibody molecule. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are the same antigen, e.g., the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule includes a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule comprises a semi-antibody having binding specificity to a first epitope and a semi-antibody having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule comprises a semi-antibody or fragment thereof having binding specificity to a first epitope and a semi-antibody or fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv or fragment thereof having binding specificity to a first epitope and an scFv or fragment thereof having binding specificity to a second epitope.

[0270] In certain embodiments, the CAR of the present invention comprises an antigen-binding domain that is a multispecific (e.g., bispecific or trispecific) antibody molecule. Protocols for producing bispecific or heterodimeric antibody molecules are known in the art, for example, the “nob-in-a-hole” approach described in U.S. Patent No. 5,731,168; for example, electrostatic steering Fc pair formation as described in International Publication Nos. 09 / 089004, 06 / 106905, and 2010 / 129304; for example, chain exchange domain (SEED) heterodimer formation as described in International Publication No. 07 / 110205; example For example, Fab arm exchange as described in International Publication No. 08 / 119353, International Publication No. 2011 / 131746, and International Publication No. 2013 / 060867; bi-antibody conjugates by antibody crosslinking for producing bispecific structures using heterobifunctional reagents having, for example, amine-reactive groups and sulfhydryl-reactive groups, as described in U.S. Patent No. 4433059; disulfide conjugates between two chains, as described in U.S. Patent No. 4444878. Dispecific antibody determinants produced by the recombination of half-antibodies (heavy-light chain pairs or Fab) from different antibodies through a compound reduction and oxidation cycle; for example, trifunctional antibodies, 3Fab' fragments crosslinked by sulfhydryl reactive groups, as described in U.S. Patent No. 5,273,743; for example, pairs of scFv crosslinked by biosynthetic binding proteins, e.g., C-terminal tails, preferably disulfide or amine-reactive chemical crosslinking, as described in U.S. Patent No. 5,534,254; for example, difunctional antibodies, e.g., Fab fragments with different binding properties dimerized by leucine zippers (e.g., c-fos and c-jun) having substituted constant domains, as described in U.S. Patent No. 5,582,996; for example, dispecific and oligospecific monovalent and oligovalent receptors, e.g., VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with a light chain, as described in U.S. Patent No. 5,591,828; for example,Dimers of polypeptides generally called dispecific antibodies (including higher-order structures for creating dispecific, trispecific, and tetraspecific molecules), such as dispecific DNA-antibody conjugates, e.g., crosslinking of an antibody or Fab fragment via a double-strand DNA section, as described in U.S. Patent No. 5,635602; dispecific fusion proteins, e.g., expression constructs containing two scFvs, e.g., containing a hydrophilic helical peptide linker and a fully constant region, as described in U.S. Patent No. 5,637481; polyvalent and polyspecific binding proteins, e.g., dimers of polypeptides having a first domain with a binding region for the Ig heavy chain variable region and a second domain with a binding region for the Ig light chain variable region, as described in U.S. Patent No. 5,837242; and peptide spacers further bound to the antibody hinge region and CH3 region, having linked VL and VH chains, which can be dimerized to form a dispecific / polyvalent molecule, as described in U.S. Patent No. 5,837821. Nibody constructs; VH and VL domains linked by a short peptide linker (e.g., 5 or 10 amino acids) in either direction, or without a linker, that can form dimers to form bispecific antibodies; trimers and tetramers, for example, as described in U.S. Patent No. 5,844,094; a series of VH domains (or VL domains in family members) linked by peptide bonds with a crosslinkable group at the C-terminus, further linked with a VL domain to form a series of FV (or scFv), as described in U.S. Patent No. 5,864,019; and single-chain linked polypeptides having both VH and VL domains linked by peptide linkers, which are combined into a multivalent structure by non-covalent or chemical crosslinking to form, for example, homodivalent, heterodivalent, trivalent, and tetravalent structures, using either scFV or bispecific antibody forms, as described in U.S. Patent No. 5,869,620. For further examples of polyspecific and bispecific molecules and methods for producing them, see, for example, U.S. Patent No. 5910573, U.S. Patent No. 5932448, U.S. Patent No. 5959083, U.S. Patent No. 5989830, U.S. Patent No. 6005079, U.S. Patent No. 6239259,U.S. Patent No. 6,294,353, U.S. Patent No. 6,333,396, U.S. Patent No. 6,476,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453, U.S. Patent No. 6,743,896, U.S. Patent No. 6,809,185, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Application Publication No. 2 Specifications 002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 2003211078A1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1 The following are the specifications of the U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005100543A1, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 200 Specifications 6204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1, U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1, U.S. Patent Application Publication No. 2007141049A1, U.S. Patent Application Publication No. 2007154901A1, U.S. Patent Application Publication No. 2007274985A1, U.S. Patent Application Publication No. 2008050370A1, U.S. Patent Application Publication No. 2008069820A1,U.S. Patent Application Publication No. 2008152645A1, U.S. Patent Application Publication No. 2008171855A1, U.S. Patent Application Publication No. 2008241884A1, U.S. Patent Application Publication No. 2008254512A1, U.S. Patent Application Publication No. 2008260738A1, U.S. Patent Application Publication No. 2009130106A1, U.S. Patent Application Publication No. 2009148905A1, U.S. Patent Application Publication No. 2009155275A1, U.S. National Patent Application Publication No. 2009162359A1, U.S. Patent Application Publication No. 2009162360A1, U.S. Patent Application Publication No. 2009175851A1, U.S. Patent Application Publication No. 2009175867A1, U.S. Patent Application Publication No. 2009232811A1, U.S. Patent Application Publication No. 2009234105A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent Specification No. 346087A2, International Publication No. 0006605A2, International Publication No. 02072635A2, International Publication No. 04081051A1, International Publication No. 06020258A2, International Publication No. 2007044887A2, International Publication No. 2007095338A2, International Publication No. 2007137760A2, International Publication No. 2008119353A1, International Publication This can be seen in brochures No. 2009021754A2, International Publication No. 2009068630A1, International Publication No. 9103493A1, International Publication No. 9323537A1, International Publication No. 9409131A1, International Publication No. 9412625A2, International Publication No. 9509917A1, International Publication No. 9637621A2, and International Publication No. 9964460A1. The contents of the applications cited above are incorporated herein by reference in their entirety.

[0271] In each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH can be located upstream or downstream of VL. In one embodiment, the upstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH1) upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH2) upstream of its VL (VL2), resulting in the overall bispecific antibody molecule having the configuration VH1-VL1-VL2-VH2. In another embodiment, the upstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL2) upstream of its VH (VH2), resulting in the overall bispecific antibody molecule having the configuration VL1-VH1-VH2-VL2. Optionally, the linker is positioned between two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2 when the construct is sequenced as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker may be one of the linkers described herein, for example, a (Gly4-Ser)n linker (SEQ ID NO: 26) (wherein n is 1, 2, 3, 4, 5, or 6, preferably 4). In general, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. Optionally, the linker is positioned between VL and VH of the first scFv. Optionally, the linker is positioned between VL and VH of the second scFv. In a construct having multiple linkers, any two or more linkers may be the same or different. Accordingly, in one embodiment, the two-specificity CAR comprises VL, VH and one or more linkers of the choice in the arrangement described herein.

[0272] In one embodiment, a bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, e.g., scFv, which has binding specificity to BCMA and includes, for example, scFv as described herein, e.g., Tables 2, 6, and 10, or a second immunoglobulin variable domain sequence having binding specificity to the light chain CDR and / or heavy chain CDR from the BCMA scFv described herein and to a second epitope, for different antigens. In some embodiments, the second immunoglobulin variable domain sequence has binding specificity to antigens expressed on AML cells, e.g., antigens other than BCMA. For example, the second immunoglobulin variable domain sequence has binding specificity to CD123. As another example, the second immunoglobulin variable domain sequence has binding specificity to CLL-1. As yet another example, the second immunoglobulin variable domain sequence has binding specificity to CD34. As yet another example, the second immunoglobulin variable domain sequence has binding specificity to FLT3. For example, the second immunoglobulin variable domain sequence has binding specificity to folate receptor β. In some embodiments, the second immunoglobulin variable domain sequence has binding specificity to antigens expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0273] Chimera TCR In one embodiment, the anti-BCMA antibody and antibody fragments of the present invention (e.g., those disclosed in Tables 2, 6, and 10) can be transplanted into one or more constant domains of a T cell receptor ("TCR") chain, such as the TCR alpha or TCR beta chain, to create a chimeric TCR that specifically binds to BCMA. While not theoretically bound, it is thought that the chimeric TCR signals through the TCR complex upon antigen binding. For example, a BCMA scFv, such as those disclosed herein, can be transplanted into the constant domains of a TCR chain, such as the TCR alpha chain and / or TCR beta chain, such as at least the extracellular constant domain, transmembrane domain, and cytoplasmic domain. As another example, a BCMA antibody fragment, such as the VL domain described herein, can be transplanted into the constant domain of the TCR alpha chain, and a BCMA antibody fragment, such as the VH domain described herein, can be transplanted into the constant domain of the TCR beta chain (or alternatively, the VL domain can be transplanted into the constant domain of the TCR beta chain and the VH domain into the TCR alpha chain). As another example, CDRs of anti-BCMA antibodies or antibody fragments, such as those listed in Tables 2-13, can be transplanted into the TCR alpha and / or beta chains to create a chimeric TCR that specifically binds to BCMA. For example, an LCDR disclosed herein may be transplanted into the variable domain of the TCR alpha chain, an HCDR disclosed herein may be transplanted into the variable domain of the TCR beta chain, or vice versa. Such chimeric TCRs can be produced by methods known in the art (e.g., Willemsen RA et al, Gene Therapy 2000;7:1369-1377; Zhang T et al, Cancer Gene Ther 2004;11:487-496; Aggen et al, Gene Ther. 2012 Apr;19(4):365-74).

[0274] Further Embodiments In one embodiment, the CAR-expressing cells described herein may further include, for example, different antigen-binding domains of the second CAR for a second CAR, for example, the same target (BCMA) or a different target (e.g., CD19, CD20, or CS-1, or other multiple myeloma targets, e.g., kappa light chain, CD138, Lewis Y antigen, or CD38 (Garfall et al., Discovery Medicine, 2014, 17(91):37-46)). In some embodiments, the CAR-expressing cells include a first CAR that targets a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain, and a second CAR that targets a second different antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. While we do not wish to be constrained by theory, the placement of a co-stimulatory signaling domain, e.g., 4-1BB, CD28, CD27 ICOS, or OX-40, and a primary signaling domain, e.g., CD3 zeta, on a second CAR to a first CAR may limit CAR activity in cells expressing both targets. In some embodiments, CAR-expressing cells include a first BCMA CAR comprising a BCMA-binding domain, a transmembrane domain, and a co-stimulatory domain, and a second CAR that targets a non-BCMA antigen (e.g., antigens expressed on leukemia or lymphoma cells, e.g., CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, the CAR-expressing cell comprises a first BCMA CAR including a BCMA-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets a non-BCMA antigen (e.g., antigens expressed on leukemia or lymphoma cells, e.g., CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38) and includes an antigen-binding domain, a transmembrane domain, and a co-stimulatory signaling domain. In some embodiments, the CAR-expressing cell comprises the BCMA CAR described herein and a CAR targeting CD19 (CD19 CAR).

[0275] In some embodiments, CAR-expressing cells include BCMA CARs and inhibitory CARs as described herein. In some embodiments, inhibitory CARs include an antigen-binding domain that binds to antigens found in normal cells but not in cancer cells. In some embodiments, inhibitory CARs include an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

[0276] In some embodiments, when a CAR-expressing cell contains two or more different CARs, the antigen-binding domains of the various CARs are arranged such that they do not interact with each other. For example, a cell expressing a first and a second CAR may have an antigen-binding domain of the first CAR, for example, as a fragment, such as scFv, which does not form a binding with the antigen-binding domain of the second CAR, such as VHH.

[0277] In one embodiment, the antigen-binding domain includes a single-domain antigen-binding (SDAB) molecule, where the complementary determinative region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain variable domains, naturally occurring binding molecules lacking a light chain, single domains derived from conventional four-chain antibodies, engineered domains, and single-domain scaffolds other than those derived from antibodies. The SDAB molecule may be a single-domain molecule of the Art or a future single-domain molecule. The SDAB molecule may be derived from any species, including but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cattle. This term also includes naturally occurring single-domain antibody molecules from species other than camelidae and sharks.

[0278] In one embodiment, the SDAB molecule may be derived from a variable region of an immunoglobulin found in fish, such as an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark serum. Methods for producing single-domain molecules derived from the variable region of a NAR ("IgNAR") are described in International Publication No. 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0279] In another embodiment, the SDAB molecule is a naturally occurring single-domain antigen-binding molecule known as a heavy-chain molecule lacking a light chain. Such single-domain molecules are described, for example, in International Publication No. 9404678 and in Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarification, this variable domain derived from a naturally occurring heavy-chain molecule lacking a light chain is known here as VHH or nanobody to distinguish it from the conventional VH of tetra-chain immunoglobulins. Such VHH molecules may originate from camelid species, such as camels, llamas, dromedaries, alpacas, and guanacos. Other species outside the camelid family may naturally produce heavy-chain molecules lacking a light chain, and such VHHs are within the scope of this invention.

[0280] SDAB molecules can be produced recombinantly, via CDR transplantation, humanization, camelization, deimmunization, and / or in vitro (e.g., by selection via phage display).

[0281] Cells having multiple chimeric membrane-embedded receptors, each containing antigen-binding domains that interact with each other, have been found to be undesirable, for example, because they prevent one or more antigen-binding domains from binding to their congener antigens. Disclosed herein are cells having first and second naturally occurring chimeric membrane-embedded receptors containing antigen-binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding first and second naturally occurring chimeric membrane-embedded receptors containing antigen-binding domains that minimize such interactions, as well as methods for producing and using such cells and nucleic acids. In one embodiment, one of the antigen-binding domains of the first and second naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0282] In one embodiment, the present invention comprises first and second CARs, wherein one of the antigen-binding domains of the first and second CARs does not include a variable light domain and a variable heavy domain. In one embodiment, one of the antigen-binding domains of the first and second CARs is an scFv, and the other is not an scFv. In one embodiment, one of the antigen-binding domains of the first and second CARs comprises a single VH domain, for example, a camelid, shark, or lamprey single VH domain or a single VH domain derived from a human or mouse sequence. In one embodiment, one of the antigen-binding domains of the first and second CARs comprises a nanobody. In one embodiment, one of the antigen-binding domains of the first and second CARs comprises a camelid VHH domain.

[0283] In one embodiment, one antigen-binding domain of the first CAR and the second CAR comprises an scFv, and the other comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain or a single VH domain derived from a human or mouse sequence. In one embodiment, one antigen-binding domain of the first CAR and the second CAR comprises an scFv, and the other comprises a nanobody. In one embodiment, one antigen-binding domain of the first CAR and the second CAR comprises an scFv, and the other comprises a camelid VHH domain.

[0284] In one embodiment, when present on the cell surface, the binding of the antigen-binding domain of the first CAR to its congener antigen is not substantially reduced by the presence of the second CAR. In one embodiment, the binding of the antigen-binding domain of the first CAR to its congener antigen in the presence of the second CAR is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of the first CAR to its congener antigen in the absence of the second CAR.

[0285] In one embodiment, when present on the cell surface, the antigen-binding domains of the first CAR and the second CAR bind to each other less than when both are scFv antigen-binding domains. In one embodiment, the antigen-binding domains of the first CAR and the second CAR bind to each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than when both are scFv antigen-binding domains.

[0286] In another embodiment, the CAR-expressing cells described herein may further express another agent, such as an agent that enhances the activity of the CAR-expressing cells. For example, in some embodiments, the agent may be an agent that inhibits an inhibitory molecule, such as an agent described herein. Inhibitory molecules, such as PD1, can reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFRβ. In some embodiments, the agent inhibiting the inhibitory molecule includes a second polypeptide that provides a positive signal to the cell, for example, a first polypeptide bound to an intracellular signaling domain as described herein, e.g., the inhibitory molecule. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFβ or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, ICOS, or CD28 as described herein) and / or a primary signaling domain (e.g., including the CD3 zeta signaling domain as described herein).In some embodiments, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least the extracellular domain portion of PD1) and a second polypeptide of an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein). In embodiments, the CAR-expressing cells described herein include, for example, switch-costimulatory receptors as described in International Publication No. 2013 / 019615, which is incorporated herein in whole by reference. PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed in activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int.Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation by binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

[0287] In some embodiments, an inhibitory molecule, such as a drug containing the extracellular domain (ECD) of programmed cell death 1 (PD1), can be fused to the transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (referred to herein as a PD1 CAR). In some embodiments, when used in combination with a BCMA CAR as described herein, the PD1 CAR improves the persistence of CAR-expressing cells, such as T cells or NK cells. In some embodiments, the CAR is a PD1 CAR containing the extracellular domain of PD1 shown underlined in SEQ ID NO: 24. In some embodiments, the PD1 CAR contains the amino acid sequence of SEQ ID NO: 24.

[0288] In some embodiments, PD1 CAR includes the following amino acid sequence (SEQ ID NO: 22).

[0289] In some embodiments, the drug comprises a nucleic acid sequence encoding PD1 CAR, for example, the PD1 CAR described herein. In some embodiments, the nucleic acid sequence of PD1 CAR is provided as SEQ ID NO: 23, with PD1 ECD underlined.

[0290] In another embodiment, the present invention provides a population of CAR-expressing cells, for example, CART cells or CAR-expressing NK cells. In one embodiment, the population of CAR-expressing cells comprises a mixture of cells expressing various CARs. For example, in some embodiments, the population of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells) may include a first cell expressing a CAR having an anti-BCMA binding domain as described herein and a second cell expressing a CAR having a different anti-BCMA binding domain, for example, an anti-BCMA binding domain as described herein that is different from the anti-BCMA binding domain of the CAR expressed by the first cell. As another example, the population of CAR-expressing cells may include, for example, a first cell expressing a CAR containing an anti-BCMA binding domain, such as as described herein, and a second cell expressing a CAR containing an antigen-binding domain for a target other than BCMA (e.g., CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38). In some embodiments, the population of CAR-expressing cells includes, for example, first cells expressing a CAR containing an anti-BCMA binding domain, as described herein, and second cells expressing a CAR containing a CD19-targeting antigen-binding domain (CD19 CAR). In some embodiments, the population of CAR-expressing cells includes, for example, first cells expressing a CAR containing a primary intracellular signaling domain and second cells expressing a CAR containing a secondary signaling domain.

[0291] In another embodiment, the present invention provides a population of cells in which at least one cell expresses a CAR having the anti-BCMA domain described herein, and a second cell expresses another agent, such as an agent that enhances the activity of the CAR-expressing cell. For example, in some embodiments, the agent may be an agent that inhibits an inhibitory molecule. The inhibitory molecule may, for example, in some embodiments, reduce the ability of the CAR-expressing cell to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. In some embodiments, the agent inhibiting the inhibitory molecule comprises a first polypeptide, e.g., the inhibitory molecule, bound to a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain as described herein. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFβ or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, ICOS, or CD28 as described herein) and / or a primary signaling domain (e.g., including the CD3 zeta signaling domain as described herein).In some embodiments, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0292] In one embodiment, the present invention provides a method comprising administering a population of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells), for example, a mixture of cells expressing various CARs, in combination with another agent, for example, a kinase inhibitor such as the kinase inhibitor described herein. In another embodiment, the present invention provides a method comprising administering a population of cells in which at least one cell expresses a CAR having an anti-cancer-associated antigen-binding domain as described herein, and a second cell expressing another agent, for example, an agent that enhances the activity of CAR-expressing cells, in combination with another agent, for example, a kinase inhibitor such as the kinase inhibitor described herein.

[0293] Natural killer cell receptor (NKR) CAR In one embodiment, the CAR molecule described herein comprises one or more components of natural killer cell receptors (NKRs), thereby forming an NKR-CAR. The NKR component may be a transmembrane domain, hinge domain, or cytoplasmic domain derived from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), e.g., KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR 3DL3, KIR2DP1 and KIR3DP1; native cytotoxic receptors (NCRs), e.g., NKp30, NKp44, NKp46; the SLAM family of immune cell receptor signaling lymphocyte-activating molecules, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10; Fc receptors (FcRs), e.g., CD16 and CD64; and Ly49 receptors, e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interact with adapter molecules or intracellular signaling domains, e.g., DAP12. Exemplary configurations and sequences of CAR molecules containing NKR elements are described in International Publication No. 2014 / 145252, which is incorporated herein by reference.

[0294] Strategies for controlling chimeric antigen receptors There are many ways in which CAR activity can be controlled. In one embodiment, controllable CARs (RCARs) whose CAR activity can be controlled are desirable to optimize the safety and efficacy of CAR therapy. For example, induction of apoptosis using a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3;365(18):1673-1683) can be used as a safety switch in CAR therapy according to the present invention. In another example, CAR-expressing cells can also express the inducible caspase-9 (iCaspase-9) molecule, which, upon administration of a dimerizing factor drug (e.g., rimiducid (also known as AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad))), leads to caspase-9 activation and apoptosis in cells. The iCaspase-9 molecule contains a chemoinducer of the dimerization (CID) binding domain, which mediates dimerization in the presence of CID. This leads to the inducible and selective depletion of CAR-expressing cells. In some cases, the iCaspase-9 molecule is encoded by the CAR coding vector and another nucleic acid molecule. In other cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR coding vector. iCaspase-9 can provide a safety switch to avoid some toxicity in CAR-expressing cells. For example, Song et al. Cancer Gene Ther. 2008;15(10):667-75;Clinical Trial See Id.No.NCT02107963 and Di Stasi et al.N.Engl.J.Med.2011;365:1673-83.

[0295] Alternative strategies for controlling CAR therapy according to the present invention include the use of small molecules or antibodies that deactivate or block CAR activity, for example, by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), or by eliminating CAR-expressing cells. For example, the CAR-expressing cells described herein may also express antigens that are recognized by molecules that can induce cell death, such as ADCC or complement-induced cell death. For example, the CAR-expressing cells described herein may also express receptors that can be targeted by antibodies or antibody fragments. Examples of such receptors include EpCAM, VEGFR, integrins (e.g., integrins ανβ3, α4, αI3 / 4β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a / LFA-1, CD1 5, including CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basidine, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, and EGFR and its cleavage versions (e.g., versions that retain one or more extracellular epitopes but lack one or more regions in the cytoplasmic domain). For example, the CAR-expressing cells described herein lack signaling ability but also express truncated epidermal growth factor receptor (EGFR) that holds an epitope recognized by molecules capable of inducing ADCC, such as cetuximab (Erbitux®). As a result, administration of cetuximab induces ADCC and subsequent depletion of CAR-expressing cells (see, for example, International Publication No. 2011 / 056894 and Jonnalagadda et al., Gene Ther. 2013;20(8)853-860).Another strategy involves the expression of a highly small marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in CAR-expressing cells described herein, conjugated to rituximab, resulting in selective depletion of CAR-expressing cells by ADCC (see, e.g., Philip et al., Blood. 2014;124(8)1277-1287). Other methods for depleting CAR-expressing cells described herein include, for example, the administration of CAMPATH®, a monoclonal anti-CD52 antibody that selectively binds to and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction by ADCC induction. In other embodiments, CAR-expressing cells may be selectively targeted using CAR ligands, e.g., anti-idiotype antibodies. In one embodiment, anti-idiotype antibodies can induce effector cell activity, e.g., ADCC or ADC activity, thereby reducing the number of CAR-expressing cells. In other embodiments, a CAR ligand, such as an anti-idiotype antibody, can be bound to a cell-lethal agent, such as a toxin, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecule itself can be configured so that its activity can be controlled, for example, activated or blocked, as described below.

[0296] In some embodiments, the RCAR comprises a series of typically simplest embodiments comprising two polypeptides, in which the elements of a standard CAR described herein, such as an antigen-binding domain and an intracellular signaling domain, are arranged on separate polypeptides or members. In some embodiments, the series of polypeptides includes a dimerization switch that can bind the polypeptides to each other in the presence of a dimerization molecule, for example, by binding the antigen-binding domain to the intracellular signaling domain. Further descriptions and exemplary arrangements of such controllable CARs are provided herein and in International Publication No. 2015 / 090229, which is incorporated herein by reference in its entirety.

[0297] In one embodiment, the RCAR comprises two polypeptides or members: 1) an intracellular signaling domain comprising, for example, a primary intracellular signaling domain and a first switch domain as described herein; and 2) an antigen-binding member comprising, for example, an antigen-binding domain and a second switch domain that target a tumor antigen, as described herein. Optionally, the RCAR comprises a transmembrane domain as described herein. In one embodiment, the transmembrane domain may be located on the intracellular signaling member, on the antigen-binding member, or on both. (Unless otherwise specified, when the members or elements of the RCAR are those described herein, the order may be as described, but other orders are also included. In other words, in one embodiment, the order is as described herein, but in other embodiments, the order may be different. For example, the order of the elements at one end of the transmembrane region may differ from the example, for example, the arrangement of the switch domain relative to the intracellular signaling domain may differ, for example, be reversed).

[0298] In one embodiment, the first and second switch domains can form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be, for example, a homodimerization switch in which the first and second switch domains are identical, or a heterodimerization switch in which the first and second switch domains are different from each other.

[0299] In embodiments, RCAR may include a “multiswitch.” The multiswitch may include a heterodimerized switch domain or a homodimerized switch domain. The multiswitch independently includes a plurality of switch domains, for example, two, three, four, five, six, seven, eight, nine, or ten, on a first member, for example, an antigen-binding member, and a second member, for example, an intracellular signaling member. In one embodiment, the first member may include a plurality of first switch domains, for example, an FKBP-based switch domain, and the second member may include a plurality of second switch domains, for example, an FRB-based switch domain. In one embodiment, the first member may include first and second switch domains, for example, an FKBP-based switch domain and an FRB-based switch domain, and the second member may include first and second switch domains, for example, an FKBP-based switch domain and an FRB-based switch domain.

[0300] In one embodiment, the intracellular signaling member includes one or more intracellular signaling domains, such as a primary intracellular signaling domain and one or more co-stimulatory signaling domains.

[0301] In one embodiment, the antigen-binding member may comprise one or more intracellular signaling domains, for example, one or more costimulatory signaling domains. In one embodiment, the antigen-binding member comprises a plurality of costimulatory signaling domains described herein, selected from, for example, two or three, 4-1BB, CD28, CD27, ICOS, and OX40, and in one embodiment, not comprising primary intracellular signaling domains. In one embodiment, the antigen-binding member comprises the following costimulatory signaling domains in the extracellular to intracellular direction: 4-1BB-CD27; 41-BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; CD28-4-1BB; or 4-1BB-CD28. In such embodiments, the intracellular binding member comprises a CD3 zeta domain. In such embodiments, the RCAR comprises (1) an antigen-binding member including an antigen-binding domain, a transmembrane domain, two costimulatory domains, and a first switch domain; and (2) an intracellular signaling domain including a transmembrane domain or membrane tethering domain, at least one primary intracellular signaling domain, and a second switch domain.

[0302] One embodiment provides an RCAR in which the antigen-binding member is not tethered to the CAR cell surface. This allows a cell having an intracellular signaling member to conveniently pair with one or more antigen-binding domains without the need to transform the cell with a sequence encoding the antigen-binding member. In such embodiments, the RCAR comprises 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; and 2) an antigen-binding member comprising an antigen-binding domain and a second switch domain, wherein the antigen-binding member does not include a transmembrane domain or a membrane tethering domain and optionally does not include an intracellular signaling domain. In one embodiment, the RCAR may further comprise a second antigen-binding member comprising 3) a second antigen-binding domain, e.g., a second antigen-binding domain that binds to a different antigen than that bound by the antigen-binding domain; and a second switch domain.

[0303] RCARs in which the antigen-binding member includes bispecific activation and targeting ability are also provided herein. In this embodiment, the antigen-binding member may include a plurality of antigen-binding domains, e.g., two, three, four, or five, e.g., scFv, where each proto-binding domain binds to a target antigen, e.g., a different antigen or the same antigen, e.g., the same or different epitopes of the same antigen. In one embodiment, the plurality of antigen-binding domains are tandem, and optionally, a linker or hinge region is positioned between each of the antigen-binding domains. Suitable linker and hinge regions are described herein.

[0304] One embodiment provides an RCAR having a configuration that allows for switching of proliferation. In this embodiment, the RCAR includes 1) an intracellular signaling member comprising, optionally, one or more costimulatory signaling domains selected from, for example, 4-1BB, CD28, CD27, ICOS, and OX40 and a switch domain; and 2) an antigen-binding member comprising an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, for example, a CD3 zeta domain, wherein the antigen-binding member does not include a switch domain or does not include a switch domain that dimerizes with a switch domain on the intracellular signaling member. In one embodiment, the antigen-binding member does not include a costimulatory signaling domain. In one embodiment, the intracellular signaling member comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second intracellular signaling member comprising a second switch domain of a heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

[0305] In any of the RCAR configurations described herein, the first and second switch domains include FKBP-FRB based switches as described herein.

[0306] Also provided herein are cells containing the RCAR described herein. Any cells manipulated to express RCAR can be used as RCARX cells. In one embodiment, the RCARX cells are T cells and are referred to as RCART cells. In one embodiment, the RCARX cells are NK cells and are referred to as RCARN cells.

[0307] Nucleic acids and vectors containing RCAR-coding sequences are also provided herein. Sequences encoding various elements of RCAR can be placed in the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, (i) a sequence encoding an antigen-binding member and (ii) a sequence encoding an intracellular signaling member can be present in the same nucleic acid, e.g., a vector. Production of the corresponding proteins can be achieved, for example, by using separate promoters or by using a bicistronic transcript (which may result in the production of two proteins by cleavage of a single translation product or translation of two separate protein products). In one embodiment, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is placed between (i) and (ii). In one embodiment, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is placed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, the first promoter is operably ligated to (i) and the second promoter is operably ligated to (ii) so that (i) and (ii) are transcribed as separate mRNAs.

[0308] Alternatively, sequences encoding various elements of RCAR can be placed in various nucleic acid molecules, such as different plasmids or vectors, such as viral vectors or lentiviral vectors. For example, (i) sequences encoding antigen-binding members can be placed in a first nucleic acid, such as a first vector, and (ii) sequences encoding intracellular signaling members can be present in a second nucleic acid, such as a second vector.

[0309] Dimerization switch Dimerization switches can be non-covalent or covalent. In non-covalent dimerization switches, the dimerizing molecule promotes non-covalent interactions between switch domains. In covalent dimerization switches, the dimerizing molecule promotes covalent interactions between switch domains.

[0310] In one embodiment, RCAR comprises an FKBP / FRAP or FKBP / FRB-based dimerization switch. FKBP12 (FKBP or FK506-binding protein) is an abundant cytoplasmic protein that serves as the primary intracellular target for the natural product immunosuppressant, rapamycin. Rapamycin binds to FKBP and its large PI3K homolog, FRAP (RAFT, mTOR). FRB is the 93-amino acid portion of FRAP sufficient for the binding of the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA 92:4947-51).

[0311] In the embodiment, the FKBP / FRAP, for example, the FKBP / FRB-based switch can use a dimerized molecule, such as rapamycin or a rapamycin analog.

[0312] The following is an example amino acid sequence of FKBP. [ka]

[0313] In some embodiments, the FKBP switch domain may include a fragment of FKBP having the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or rapalog. In some embodiments, the FKBP switch domain includes the following amino acid sequence. [ka]

[0314] The amino acid sequence of FRB is as follows: [ka]

[0315] As used herein, the term “FKBP / FRAP, e.g., FKBP / FRB-based switch” includes an FKBP fragment or analogue having the ability to bind to FRB or its fragment or analogue in the presence of rapamycin or rapalog, e.g., RAD001, and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the FKBP sequence of SEQ ID NO: 275 or 276, or 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid A dimerized switch comprises a first switch domain that is no different beyond a residue; and an FRB fragment or analogue having the ability to bind to an FRB or its fragment or analogue in the presence of rapamycin or rapalog, and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the FRB sequence of SEQ ID NO: 277, or comprising a second switch domain that is no different beyond 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue. In one embodiment, the RCAR described herein comprises one switch domain containing the amino acid residue disclosed in SEQ ID NO: 275 (or SEQ ID NO: 589) and one switch domain containing the amino acid residue disclosed in SEQ ID NO: 277.

[0316] In one embodiment, the FKBP / FRB dimerization switch comprises an enhanced modified FRB switch domain in which the complex formation between the FKBP-based switch domain and the dimerization molecule, e.g., rapamycin or rapalog, e.g., RAD001, is altered. In one embodiment, the modified FRB switch domain comprises one or more mutations selected from amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, e.g., two, three, four, five, six, seven, eight, nine, ten, or more mutations, where the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the mutant FRB includes a mutation in E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 278, or leucine (E2032L), e.g., SEQ ID NO: 279. In one embodiment, the mutant FRB includes a mutation in T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 280. In one embodiment, the mutant FRB includes mutations in E2032 and T2098, where E2032 is mutated to any amino acid, and T2098 is mutated to any amino acid, e.g., SEQ ID NO: 281. In one embodiment, the mutant FRB includes the E2032I and T2098L mutations, for example, SEQ ID NO: 282. In another embodiment, the mutant FRB includes the E2032L and T2098L mutations, for example, SEQ ID NO: 283.

[0317] [Table 47]

[0318] Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / binder dimerization switches, and halotag / snaptag dimerization switches. Such switches and associated dimerizing molecules will be apparent to those skilled in the art, following the guidance provided herein.

[0319] dimerization molecule The binding between switch domains is facilitated by the dimerizing molecule. The interaction or binding between switch domains in the presence of the dimerizing molecule enables signal transduction between a polypeptide bound to the first switch domain, e.g., fused to it, and a polypeptide bound to the second switch domain, e.g., fused to it. When measured in the system described herein in the presence of a non-rate-limiting level of the dimerizing molecule, the signal transduction increases by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 5x, 10x, 50x, and 100x.

[0320] Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), such as RAD001, can be used as dimerizing molecules in the FKBP / FRB-based dimerizing switches described herein. In one embodiment, the dimerizing molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridafololimus), biolimus, and AP21967. Further rapamycin analogs suitable for use in FKBP / FRB-based dimerizing switches are described further in the section titled “Combination Therapies” or “Combinations with Low Immunoenhancing Dose mTOR Inhibitors.”

[0321] Split Car In one embodiment, CAR-expressing cells utilize a split CAR. The split CAR approach is described in detail in International Publication No. 2014 / 055442 and International Publication No. 2014 / 055657, which are incorporated herein by reference. Briefly, the split CAR system comprises cells expressing a first CAR having a first antigen-binding domain and a costimulatory domain (e.g., 41BB), and the cells also express a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cells encounter the first antigen, the costimulatory domain is activated, and the cells proliferate. When the cells encounter the second antigen, the intracellular signaling domain is activated, and cytotoxic activity is initiated. Thus, the CAR-expressing cells are fully activated only in the presence of both antigens. In embodiments, the first antigen-binding domain recognizes BCMA and includes, for example, the antigen-binding domain described herein, and the second antigen-binding domain recognizes antigens expressed in acute myeloid leukemia cells, such as CD123, CLL-1, CD34, FLT3, or folate receptor beta. In embodiments, the first antigen-binding domain recognizes BCMA and includes, for example, the antigen-binding domain described herein, and the second antigen-binding domain recognizes antigens expressed in B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0322] Stability and mutation The stability of anti-BCMA binding domains, such as scFv molecules (e.g., soluble scFv), can be evaluated by referring to the biophysical properties (e.g., thermal stability) of conventional control scFv molecules or full-length antibodies.

[0323] The improved thermal stability of the anti-BCMA binding domain, e.g., scFv, subsequently contributes to the overall CART-BCMA construct, leading to an improvement in the therapeutic properties of the CART-BCMA construct. The thermal stability of the anti-BCMA binding domain, e.g., scFv, can be improved by at least about 2°C or 3°C compared to conventional antibodies. In some embodiments, the anti-BCMA binding domain, e.g., scFv, has a thermal stability that is 1°C improved compared to conventional antibodies. In another embodiment, the anti-BCMA binding domain, e.g., scFv, has a thermal stability that is 2°C improved compared to conventional antibodies. In yet another embodiment, scFv has a thermal stability that is 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, and 15°C improved compared to conventional antibodies. Comparisons can be made, for example, between the scFv molecules disclosed herein and the scFv molecules or Fab fragments of antibodies from which scFv VH and VL are derived. Thermal stability can be measured using methods known in the art. For example, Tm can be measured in some embodiments. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

[0324] Mutations in scFv (resulting from humanization of soluble scFv or direct mutagenesis) alter the stability of scFv and improve the overall stability of scFv and the CART33 construct. The stability of human scFv can be compared to that of mouse scFv using measures such as Tm, denaturation temperature, and aggregation temperature.

[0325] The binding ability of the mutant scFv can be determined using the assay described in the examples.

[0326] In some embodiments, the anti-BCMA binding domain, e.g., scFv, includes at least one mutation derived from the humanization process, such that the mutant scFv provides improved stability of the CART-BCMA construct. In another embodiment, the anti-BCMA binding domain, e.g., scFv, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations derived from the humanization process, such that the mutant scFv provides improved stability of the CART-BCMA construct.

[0327] Methods for evaluating protein stability The stability of the antigen-binding domain can be evaluated, for example, using the following methods. Such methods allow for the determination of multiple thermal denaturation transitions that limit the overall stability threshold of a multidomain unit (e.g., a multidomain protein exhibiting a single denaturation transition) in which the least stable domain denatures first or cooperatively. The least stable domain can be identified by numerous further methods. Mutagenesis can be performed to explore which domain limits overall stability. Furthermore, the protease resistance of multidomain proteins can be performed by DSC or other spectroscopic methods under conditions in which the least stable domain is known to denature (Fontana, et al., (1997) Fold. Des., 2:R17-26; Dimasi et al. (2009) J. Mol. Biol. 393:672-692). Once the least stable domain is identified, the sequence (or portion thereof) encoding this domain can be used as the test sequence in this method.

[0328] a) Thermal stability The thermal stability of a composition can be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by analytical spectroscopy.

[0329] An example of analytical spectroscopy is differential scanning calorimetry (DSC). DSC uses a calorimeter that is sensitive to the heat absorption associated with the denaturation of most proteins or protein domains (see, for example, Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, a protein sample is placed in a calorimeter and the temperature is raised until the Fab or scFv denaturates. The temperature at which the protein denatures is an indicator of the overall protein stability.

[0330] Another example of analytical spectroscopy is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed polarizations due to structural asymmetry. Damaged or denatured structures yield CD spectra that are quite different from those of regular or folded structures. CD spectra reflect the sensitivity of proteins to the denaturing effect of increasing temperature and are therefore an indicator of protein thermal stability (see van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000).

[0331] Another example of analytical spectroscopy for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, cited above). Yet another example of analytical spectroscopy for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma, cited above).

[0332] The thermal stability of a composition can be measured biochemically. An example of a biochemical method for evaluating thermal stability is a thermal loading assay. In a thermal loading assay, the composition is subjected to a certain range of high temperatures for a set period of time. For example, in some embodiments, a test scFv molecule or a molecule containing scFv molecules is subjected to a certain range of temperature increases for, for example, 1 to 1.5 hours. Subsequently, the protein activity is assayed using the relevant biochemical assay. For example, if the protein is a binding protein (e.g., scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

[0333] Such assays can be performed in a high-throughput format and are disclosed in examples using Escherichia coli (E. coli) and high-throughput screening. Libraries of anti-BCMA binding domains (e.g., scFv variants) can be prepared using methods known in the art. Expression of anti-BCMA binding domains, e.g., scFv, can be induced, and these anti-BCMA binding domains, e.g., scFv, can be subjected to thermal loading. Loaded samples can be assayed for binding, and stable anti-BCMA binding domains, e.g., scFv, can be scaled up and further characterized.

[0334] Thermal stability is evaluated by measuring the melting temperature (Tm) of the composition using one of the above techniques (e.g., analytical spectroscopy). The melting temperature is the temperature at the midpoint of the temperature transition curve where 50% of the composition's molecules are folded (see, for example, Dimasi et al. (2009) J. Mol Biol. 393:672-692). In some embodiments, the Tm values ​​of the anti-BCMA binding domain, e.g., scFv, are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, The temperatures are 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C. In some embodiments, the Tm value of IgG is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C. The temperatures are 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C. In some embodiments, the Tm value of the polyvalent antibody is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C. The temperatures are 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C.

[0335] Thermal stability is also assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetry techniques (e.g., DSC). The specific heat of a composition is the energy (e.g., in kcal / mol) required to raise the temperature of 1 mol of water by 1°C. A large Cp is characteristic of denatured or inactive protein compositions. The change in the heat capacity (ΔCp) of the composition is measured by determining the specific heat of the composition before and after a temperature transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, including the Gibbs free energy of denaturation (ΔG), the enthalpy of denaturation (ΔH), or the entropy of denaturation (ΔS). Using one or more of the above biochemical assays (e.g., heat load assays), the temperature at which 50% of the composition retains its activity (e.g., binding activity) (i.e., T) is determined. C Determine the value.

[0336] Furthermore, mutations in the anti-BCMA binding domain, e.g., scFv, alter the thermal stability of the anti-BCMA binding domain, e.g., scFv, compared to the unmutated anti-BCMA binding domain, e.g., scFv. When a human or humanized anti-BCMA binding domain, e.g., scFv, is incorporated into a BCMA construct, the anti-BCMA binding domain, e.g., humanized scFv, confers thermal stability to the entire anti-BCMA CART construct. In some embodiments, the anti-BCMA binding domain, e.g., scFv, comprises a single mutation that confers thermal stability to the anti-BCMA binding domain, e.g., scFv. In other embodiments, the anti-BCMA binding domain, e.g., scFv, comprises multiple mutations that confer thermal stability to the anti-BCMA binding domain, e.g., scFv. In some embodiments, multiple mutations in the anti-BCMA binding domain, e.g., scFv, have an additive effect on the thermal stability of the anti-BCMA binding domain, e.g., scFv.

[0337] b) % flocculation The stability of a composition can be determined by measuring its aggregation tendency. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, the aggregation of a composition can be evaluated using chromatography, such as size exclusion chromatography (SEC). SEC separates molecules based on size. A column is filled with semi-solid polymer gel beads that allow ions and small molecules to enter on the inside but not larger ones. When a protein composition is applied to the top of the column, small, folded proteins (i.e., non-aggregated proteins) are dispersed in a larger volume of solvent where large protein aggregates are available. As a result, larger aggregates move faster through the column, and in this way the mixture can be separated or fractionated into its elements. Each fraction can be quantified separately as it elutes from the gel (e.g., by light scattering). Thus, the aggregation percentage of a composition can be determined by comparing the concentration of the fraction with the total concentration of the protein applied to the gel. Stable compositions elute from the column essentially as a single fraction and appear essentially as a single peak in the elution profile or chromatogram.

[0338] c) binding affinity The stability of a composition can be evaluated by determining its target binding affinity. A wide range of methods for determining binding affinity are known in the art. An example of a method for determining binding affinity is the use of surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that enables the analysis of real-time biomolecular-specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further information, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0339] In one embodiment, the antigen-binding domain of the CAR comprises an amino acid sequence homologous to the antigen-binding domain amino acid sequence described herein, and the antigen-binding domain retains the desired functional properties of the anti-BCMA antibody fragment described herein. In a particular embodiment, the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises an scFv.

[0340] In various embodiments, the antigen-binding domain of the CAR is manipulated by modification of one or more amino acids within one or both variable regions (e.g., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. In a particular embodiment, the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises an scFv.

[0341] Those skilled in the art will understand that the antibodies or antibody fragments of the present invention may be further modified so that their amino acid sequences differ (e.g., from the wild type), but the desired activity will not be modified. For example, conservative substitutions resulting in further nucleotide substitutions, such as amino acid substitutions, such as conservative substitutions at "non-essential" amino acid residues, can be made to proteins. For example, non-essential amino acid residues in a molecule can be replaced with other amino acid residues from the same side-chain family. In another embodiment, a series of amino acids can be replaced with a series of structurally similar amino acids that differ in the order and / or composition of their side-chain family members, for example, a conservative substitution can be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0342] A family of amino acid residues having similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), is defined in the art.

[0343] In relation to two or more nucleic acid or polypeptide sequences, identity percentage refers to two or more sequences that are identical. Two sequences are "substantially identical" if, when compared and aligned across a comparison window or designated region using one of the following sequence comparison algorithms or by manual alignment and visual inspection, they have a specific percentage of identical amino acid residues or nucleotides (for example, 60% identity, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity across the entire sequence, or, if not specified). By choice, identity exists over a region of at least approximately 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0344] For sequence comparison, typically one sequence acts as a control sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test and control sequences are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the control sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, the similarity method for searching of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0345] Two examples of algorithms suitable for determining sequence identity and sequence similarity percentages are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0346] The percentage of identity between two amino acid sequences can also be determined using the E. Meyers and W. Miller ((1988) Comput.Appl.Biosci.4:11-17) algorithm incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm incorporated into the GAP program of the GCG software package (available at www.gcg.com), using the Blossom 62 matrix or PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6.

[0347] In one embodiment, the present invention aims to modify the amino acid sequence of a starting antibody or fragment (e.g., scFv) to produce a functionally homogeneous molecule. For example, the VH or VL of an anti-BCMA binding domain (e.g., scFv) contained in a CAR can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity of the starting VH or VL framework region of the anti-BCMA binding domain (e.g., scFv). The present invention aims to modify the entire CAR construct, for example, modifying the amino acid sequence of one or more domains in the CAR construct to produce a functionally homogeneous molecule. The CAR construct can be modified to retain at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the identity of the starting CAR construct.

[0348] Nucleic acid constructs encoding CAR The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one embodiment, the nucleic acid molecule is provided as a messenger RNA transcript. In one embodiment, the nucleic acid molecule is provided as a DNA construct.

[0349] Accordingly, in one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain), a transmembrane domain, and a stimulatory domain, e.g., a co-stimulatory signaling domain and / or a primary signaling domain, e.g., an intracellular signaling domain including a zeta chain. In some embodiments, the anti-BCMA binding domain is the anti-BCMA binding domain described herein or a sequence that identifies it with 95-99% accuracy. In some embodiments, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In some embodiments, the isolated nucleic acid molecule further comprises a sequence encoding a primary signaling domain. In some embodiments, the primary signaling domain comprises a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain.In some embodiments, the co-stimulatory domain is, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD 137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITG B7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9( This is a functional signaling domain of a protein selected from the group consisting of ligands that specifically bind to CD229, CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0350] In another embodiment, the present invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising the leader sequence of SEQ ID NO: 1.

[0351] In another embodiment, the present invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule.

[0352] Nucleic acid sequences encoding a desired molecule can be obtained using standard techniques, for example, by derivatizing the gene from a vector known to contain it, by screening a library from cells expressing the gene, or by directly isolating it from cells and tissues known to contain it, using methods known in the recombinant art. Alternatively, the gene of interest can be produced by synthesis rather than cloning.

[0353] The present invention also provides vectors into which the DNA of the present invention is inserted. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable long-term stable integration and transmission to daughter cells of the transgene. Lentivirus vectors have further advantages over onco-retrovirus-derived vectors, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of being low immunogenic. Retrovirus vectors can also be, for example, gamma-retrovirus vectors. Gamma-retrovirus vectors may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., 2)-terminal repeat sequences (LTRs), and the target transgene, such as a gene encoding a CAR. Gamma-retrovirus vectors may lack viral structural genes such as gag, pol, and env. Exemplary gamma-retrovirus vectors include mouse leukemia virus (MLV), splenic fociform virus (SFFV), myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun;3(6):677-713.

[0354] In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the present invention is an adenovirus vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as sleeping beauty, CRISPR, CAS9, and zinc finger nucleases. See June et al. 2009 Nature Reviews Immunology 9.10:704-716, incorporated herein by reference.

[0355] In general, the expression of native or synthetic nucleic acids encoding CARs is typically achieved by manipulating a nucleic acid or portion thereof encoding a CAR polypeptide onto a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, start sequences, and promoters useful for controlling the expression of a desired nucleic acid sequence.

[0356] The expression of constructs of the present invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0357] Nucleic acids can be cloned into numerous types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Of particular interest are expression vectors, replication vectors, probe-producing vectors, and sequencing vectors.

[0358] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., International Publication No. 01 / 96584; International Publication No. 01 / 29058; and U.S. Patent No. 6,326,193).

[0359] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells in vivo or ex vivo. Numerous retroviral systems are known in the art. In one embodiment, an adenovirus vector is used. Numerous adenovirus vectors are known in the art. In some embodiments, a lentiviral vector is used.

[0360] Further promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in a region 30–110 bp upstream of the initiation site, although many promoters have been shown to also contain functional elements downstream of the initiation site. The space between promoter elements is often mobile, so that promoter function is maintained when elements are reversed or moved. In the thymidine kinase (TK) promoter, the space between promoter elements can increase to 50 bp apart before activity begins to decline. With promoters, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0361] An example of a promoter capable of expressing CAR transgenes in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the α subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. See, for example, Milone et al., Mol.Ther. 17(8):1453-1464 (2009). In one embodiment, the EF1a promoter includes the sequence provided as Sequence ID No. 11.

[0362] Another example of a promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences, including but not limited to the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, and Roussarcoma virus promoter, as well as human gene promoters, including but not limited to the actin promoter, myosin promoter, elongation factor-1α promoter, hemoglobin promoter, and creatine kinase promoter, may also be used. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended to be part of the present invention. The use of inducible promoters provides a molecular switch that can activate the expression of an operably linked polynucleotide sequence when expression is desired and block it when expression is not desired. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0363] Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a cleaved PGK promoter (for example, a PGK promoter having one or more nucleotide deletions, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, compared to the wild-type PGK promoter sequence) may be desirable. Nucleotide sequences of example PGK promoters are provided below. WT PGK Promoter [ka] Exemplary cleavage-type PGK promoter: PGK100: [ka] PGK200: [ka] PGK300: [ka] PGK400: [ka]

[0364] The vector may also include, for example, signal sequences to promote secretion, polyadenylation signals and transcriptional terminators (e.g., from the bovine growth hormone (BGH) gene), elements that enable episomal replication and replication in prokaryotes (e.g., from SV40 and ColE1 or others known in the art), and / or elements that enable selection (e.g., ampicillin resistance genes and / or zeosin markers).

[0365] To evaluate the expression of CAR polypeptides or parts thereof, an expression vector is introduced into cells. To facilitate the identification and selection of expressing cells from a cell population being explored for gene transfer or viral vector infection, a selectable marker gene or a reporter gene, or both, may be included. In other embodiments, the selectable marker is supported on a different cross-section of DNA and used in co-transfection methods. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0366] Reporter genes are likely used to identify the cells to which the gene has been introduced and to evaluate the functionality of the regulatory sequence. Generally, a reporter gene is a gene that encodes a polypeptide that is not present or expressed in the recipient organism or tissue, but whose expression is manifested by an easily detectable characteristic, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be manufactured by known techniques or are commercially available. Generally, a construct with a minimum 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as a promoter. Such promoter regions can be ligated to the reporter gene and used to evaluate drugs for their ability to regulate promoter-driven transcription.

[0367] In some embodiments, the vector may further include a nucleic acid encoding a second CAR. In some embodiments, the second CAR includes an antigen-binding domain for targets expressed on acute myeloid leukemia cells, such as CD123, CD34, CLL-1, folate receptor beta, or FLT3; or targets expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. In some embodiments, the vector includes a nucleic acid sequence encoding a first CAR, which specifically binds to a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain; and a nucleic acid sequence encoding a second CAR, which specifically binds to a second different antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. In some embodiments, the vector comprises a nucleic acid encoding a first BCMA CAR, including a BCMA-binding domain, a transmembrane domain, and a costimulatory domain, and a nucleic acid encoding a second CAR, including an antigen-binding domain, a transmembrane domain, and a primary signaling domain, targeting an antigen other than BCMA (e.g., antigens expressed on AML cells, e.g., CD123, CD34, CLL-1, folate receptor beta, or FLT3; or antigens expressed on B cells, e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a). In another embodiment, the vector comprises a nucleic acid encoding a first BCMA CAR including a BCMA-binding domain, a transmembrane domain, and a primary signaling domain, and a nucleic acid encoding a second CAR that specifically binds to an antigen other than BCMA (e.g., antigens expressed on AML cells, e.g., CD123, CD34, CLL-1, folate receptor beta, or FLT3; or antigens expressed on B cells, e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a) and includes an antigen-binding domain, a transmembrane domain, and a costimulatory signaling domain for the antigen.

[0368] In some embodiments, the vector comprises a nucleic acid encoding the BCMA CAR described herein and a nucleic acid encoding an inhibitory CAR. In some embodiments, the inhibitory CAR includes an antigen-binding domain that binds to an antigen found in normal cells, for example, normal cells that also express BCMA, but not in cancer cells. In some embodiments, the inhibitory CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domains of inhibitory CARs may be the intracellular domains of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFRβ.

[0369] In embodiments, the vector may include two or more nucleic acid sequences encoding a CAR, for example, the BCMA CAR described herein and a second CAR, for example, an inhibitory CAR or a CAR that specifically binds to an antigen other than BCMA (e.g., an antigen expressed on AML cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta; or an antigen-expressing B cell, e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a). In such embodiments, the nucleic acid sequences encoding the two or more CARs are encoded by a single nuclear molecule and as a single polypeptide chain in the same frame. In this embodiment, the two or more CARs may be separated by, for example, one or more peptide cleavage sites (e.g., autocleavage sites or substrates for intracellular proteases). Examples of peptide cleavage sites include, where the GSG residue is optional: T2A:(GSG)EGRGSLLTCGDVEENPGP(Sequence ID 194) P2A:(GSG)ATNFSLLKQAGDVEENPGP(Sequence ID 195) E2A:(GSG)QCTNYALLKLAGDVESNPGP(Sequence ID 196) F2A:(GSG)VKQTLNFDLLKLAGDVESNPG P(Sequence ID 197)

[0370] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0371] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. (See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0372] Biological methods for introducing a target polynucleotide into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for gene insertion into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-related viruses, for example. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0373] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system for use as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods of targeted delivery of nucleic acids are available, such as the delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0374] When non-viral delivery systems are used, the typical delivery medium is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids can be bound to lipids. Lipid-bound nucleic acids can be bound to lipids by encapsulation in the aqueous interior of liposomes, dispersion within the lipid bilayer of liposomes, binding to liposomes via linking molecules that bind to both liposomes and oligonucleotides, encapsulation in liposomes, complexation with liposomes, dispersion in lipid-containing solutions, mixing with lipids, combination with lipids, inclusion as a suspension in lipids, inclusion in micelles or complexation, or by other means. Lipids, lipid / DNA, or lipid / expression vector-bound compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure as micelles or in a "disintegrated" structure. They may also be simply dispersed in solution and possibly form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a group of compounds containing long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0375] Suitable lipids can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Use chloroform as the sole solvent because it volatilizes more readily than methanol. "Liposome" is a general term encompassing a variety of monolayer and multilayer lipid media formed by the production of encapsulated lipid bilayers or aggregates. Liposomes can be characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayered liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid elements self-aggregate before forming a closed structure, encapsulating water and dissolved solute between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures in solution different from the usual vesicle structure are also included. For example, lipids may be thought to exist in micelle structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0376] Regardless of the method by which exogenous nucleic acids are introduced into host cells or otherwise exposed to the inhibitors of the present invention, a variety of assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as those used for drug identification, that detect the presence or absence of specific peptides by immunological means (ELISA and Western blotting); or assays described herein that fall within the scope of the present invention.

[0377] The present invention further provides vectors comprising CAR-encoding nucleic acid molecules. In one embodiment, a CAR vector can be directly transduced into cells, such as T cells or NK cells. In one embodiment, the vector is a vector comprising, but not limited to, cloning or expression vectors, such as one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviruses, and lentiviral vector constructs. In one embodiment, the vector can express a CAR construct in mammalian T cells or NK cells. In one embodiment, the mammalian T cells are human T cells. In one embodiment, the mammalian NK cells are human NK cells.

[0378] RNA transfection A method for producing in vitro transcribed RNA CARs is disclosed herein. The invention also includes CARs encoding RNA constructs that can be directly transfected into cells. A method for producing mRNA for use in transfection comprises in vitro transcription (IVT) of a template using specifically designed primers, followed by poly-A addition, which can produce a construct, typically 50 to 2000 nucleotides long (SEQ ID NO: 35), comprising 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or intra-sequence ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail. The RNA thus produced can be efficiently transfected into cells of different species. In one embodiment, the template comprises the sequence of the CAR.

[0379] In one embodiment, anti-BCMA CAR is encoded by messenger RNA (mRNA). In one embodiment, mRNA encoding anti-BCMA CAR is introduced into immune effector cells, such as T cells or NK cells, for the production of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells).

[0380] In some embodiments, an in vitro transcription RNA CAR can be introduced into cells in the form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) production template. The DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, the RNA CAR template includes an extracellular region containing a single-chain variable domain of an antitumor antibody; a hinge region, a transmembrane domain (e.g., the CD8a transmembrane domain); and a cytoplasmic region containing intracellular signaling domains, such as the CD3-zeta signaling domain and the 4-1BB signaling domain.

[0381] In some embodiments, the DNA used for PCR includes an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the nucleic acid may include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid may include exons and introns. In some embodiments, the DNA used for PCR is a human nucleic acid sequence. In another embodiment, the DNA used for PCR is a human nucleic acid sequence including the 5' and 3' UTRs. The DNA may, alternatively, be a naturally occurring artificial DNA sequence not normally expressed in organisms. An exemplary artificial DNA sequence includes portions of genes ligated together to form an open reading frame encoding a fusion protein. The ligated portions of DNA may be from a single organism or from two or more organisms.

[0382] PCR is used to produce templates for in vitro transcription of mRNA to be used for transfection. Methods for performing PCR are well known in the art. Primers used in PCR are designed to have a region substantially complementary to the region of DNA used as the template for PCR. As used herein, “substantially complementary” refers to a nucleotide sequence in which most or all of the residues of the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, a primer can be designed to amplify a portion of nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of nucleic acid that codes for a specific domain of interest. In some embodiments, primers are designed to amplify the coding region of human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR can be produced by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides substantially complementary to the nucleotides on a DNA template that is upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5th position relative to the coding strand of the DNA sequence to be amplified. A "reverse primer" is a primer that contains a region of nucleotides substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position relative to the coding strand of the DNA sequence to be amplified.

[0383] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from numerous suppliers.

[0384] Chemical structures that have the ability to enhance stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In some embodiments, the 5' UTR is 1 to 3000 nucleotides long. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be varied by different methods, including, but not limited to, the design of primers for PCR to anneal different regions of the UTR. Using this approach, those skilled in the art can modify the 5' and 3' UTR lengths after transfection of the transcription RNA to achieve optimal translation efficiency.

[0385] The 5' and 3' UTRs may be naturally occurring, endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, non-endogenous UTR sequences for the nucleic acid of interest can be added by incorporating the UTR sequence into forward and reverse primers or by any other modification of the template. The use of non-endogenous UTR sequences for the nucleic acid of interest may be useful for modifying RNA stability and / or translation efficiency. For example, AU-rich elements in 3' UTR sequences are known to reduce mRNA stability. Therefore, 3' UTRs can be selected and designed to increase the stability of transcribed RNA based on UTR properties well known in the art.

[0386] In some embodiments, the 5'UTR may contain a Kozak sequence of an endogenous nucleic acid. Alternatively, when a non-endogenous 5'UTR is added to the nucleic acid of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. While Kozak sequences can increase the translation efficiency of some RNA transcripts, they do not appear to be necessary for all RNA to enable efficient translation. The need for Kozak sequences for many mRNAs is well known in the art. In other embodiments, the 5'UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to inhibit exonuclease degradation of mRNA.

[0387] To enable RNA synthesis from a DNA template without the need for gene cloning, the transcription promoter should be bound to the DNA template upstream of the sequence to be transcribed. When a sequence functioning as an RNA polymerase promoter is added to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is the T7 polymerase promoter, as described elsewhere in this specification. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0388] In a preferred embodiment, the mRNA has caps at both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On a circular DNA template, such as plasmid DNA, RNA polymerase produces long concatemer products that are both unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'UTR end, even after post-transcriptional polyadenylation, yields normal-sized mRNA that is not effective for eukaryotic transfection.

[0389] In a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of both templates (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur.J.Biochem., 270:1485-65 (2003)).

[0390] The conventional method for incorporating poly(A / T) extensions into DNA templates is molecular cloning. However, the integration of poly(A / T) sequences into plasmid DNA can lead to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning process not only difficult and time-consuming, but also often unreliable. This is why a method that allows for the production of DNA templates with poly(A / T) 3' stretches without cloning is highly desirable.

[0391] The poly(A) tail of the transcription DNA template can be produced during PCR using reverse primers containing poly(T) tails, such as 100T tails (SEQ ID NO: 31) (sizes can range from 50 to 5000T (SEQ ID NO: 32)), or after PCR by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA, reducing its degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In some embodiments, the poly(A) tail is 100 to 5000 adenosines (SEQ ID NO: 33).

[0392] The RNA poly(A) tail can be further elongated after in vitro transcription using poly(A) polymerases such as E. coli poly(A) polymerase (E-PAP). In some embodiments, extending the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) results in approximately a twofold increase in RNA translation efficiency. Furthermore, the addition of different chemical groups to the 3' end can increase mRNA stability. Such additions may include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerases. ATP analogs can further enhance RNA stability.

[0393] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is known in the art and is provided using the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0394] RNA produced by the methods disclosed herein may also include intra-sequence ribosome entry sites (IRES) sequences. IRES sequences may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. They may also contain any solute suitable for cell electroporation, which may include factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0395] RNA can be introduced into target cells using a number of different methods, such as any commercially available method, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Cologne.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or particulate gun particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0396] Non-viral delivery methods In some embodiments, nonviral methods can be used to deliver the nucleic acids encoding the CARs described herein to cells, tissues, or subjects.

[0397] In one embodiment, the nonviral method involves the use of transposons (also called transposition factors). In some embodiments, a transposon is a DNA fragment that can be inserted into a genomic location, for example, a self-replicating DNA fragment that can insert a copy of itself into the genome, or a DNA fragment that is spliced ​​outside a long nucleic acid and can be inserted into another location in the genome. For example, a transposon comprises a DNA sequence consisting of an inverted repeat flanking gene for transposition.

[0398] Nucleic acid delivery methods using exemplary transposons include the Sleeping Beauty transposon system (SBTS) and the piggyBac (PB) transposon system. For example, Aronovich et al.Hum.Mol.Genet.20.R1(2011):R14-20;Singh et al.Cancer Res.15(2008):2961-2971;Huang et al.Mol.Ther.16(2008):580-589;Grabundzija et al. al.Mol.Ther.18(2010):1200-1209;Kebriaei et al.Blood.122.21(2013):166;Williams.Molecular Therapy 16.9(2008):1515-16;Bell et al.Nat.Protoc.2.12(2007):3153-65; and Ding et al. See al.Cell.122.3(2005):473-83 (all of which are incorporated herein by reference).

[0399] SBTS consists of two components: 1) a transposon containing the transgene, and 2) a source of transposase enzymes. Transposases can transpose the transposon from a carrier plasmid (or other donor DNA) to target DNA, such as host cell chromosomes / genome. For example, a transposase binds to the carrier plasmid / donor DNA, excises the transposon (containing the transgene) from the plasmid, and inserts it into the host cell's genome. See, for example, Aronovich et al. (see above).

[0400] Exemplary transposons include pT2-based transposons. See, for example, Grabundzija et al., Nucleic Acids Res. 41.3 (2013): 1829-47; and Singh et al., Cancer Res. 68.8 (2008): 2961-2971, all incorporated herein by reference. Exemplary transposases include Tc1 / mariner-type transposases, such as SB10 transposase or SB11 transposase (e.g., hyperfunctional transposases that can be expressed from a cytomegalovirus promoter). See, for example, Aronovich et al., Kebriaei et al., and Grabundzija et al., all incorporated herein by reference.

[0401] The use of SBTS enables the efficient incorporation and expression of transgenes, such as nucleic acids encoding the CAR described herein. For example, a method is provided herein for producing cells, such as T cells or NK cells, that stably express the CAR described herein, using a transposon system such as SBTS.

[0402] In some embodiments, one or more nucleic acids, such as plasmids, containing SBTS components are delivered to cells (e.g., T cells or NK cells) by the methods described herein. For example, the nucleic acids are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, such as the methods described herein, such as electroporation, transfection, or lipofection. In some embodiments, the nucleic acids include a transposon containing a transgene, such as a nucleic acid encoding a CAR as described herein. In some embodiments, the nucleic acids include a transposon containing a transgene (e.g., a nucleic acid encoding a CAR as described herein) and a nucleic acid sequence encoding a transposase enzyme. In other embodiments, for example, a dual plasmid system is provided, for example, a system of two nucleic acids, where a first plasmid includes a transposon containing a transgene and a second plasmid includes a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to a host cell.

[0403] In some embodiments, cells expressing the CARs described herein, such as T cells or NK cells, are produced using a combination of SBTS and gene editing using nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), CRISPR / Cas systems, or modified meganuclease reworked homing endonucleases).

[0404] In some embodiments, the use of nonviral delivery methods allows for the reprogramming of cells, such as T cells or NK cells, and their direct injection into the target. Advantages of nonviral vectors include, but are not limited to, ease and relatively low cost of production in sufficient quantities to meet the needs of the patient population, stability during storage, and lack of immunogenicity.

[0405] Cell supply source Cells, such as immune effector cells (e.g., T cells or NK cells), can be obtained from a source of subject before proliferation and genetic modification or other modification. The term “subject” is intended to include a viable organism (e.g., mammal) from which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats and their transgenic species. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors.

[0406] In one aspect of the present invention, any number of immunoeffector cell lines (e.g., T cells or NK cells) available in the art may be used. In one aspect of the present invention, T cells may be obtained from units of blood collected from a subject using any technique known to those skilled in the art, such as Ficoll® isolation. In one preferred aspect, cells are obtained from the circulating blood of an individual by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes, as well as erythrocytes and platelets. In one aspect, the cells obtained by apheresis are washed to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for the subsequent processing steps. In one aspect of the present invention, the cells are washed with phosphate-buffered saline (PBS). In another aspect, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations.

[0407] The initial activation process in the absence of calcium can lead to enhanced activation. As will be readily apparent to those skilled in the art, the washing step can be achieved by semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as saline solutions with or without Ca-free, Mg-free PBS, PlasmaLyte A, or other buffers. Alternatively, undesirable elements of the apheresis sample can be removed, and the cells can be resuspended directly in the culture medium.

[0408] The method of this application can utilize culture medium conditions containing 5% or less, for example, 2%, of human AB serum, and it is recognized that known culture medium conditions and compositions, such as those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement” Clinical & Translational Immunology (2015) 4,e31;doi:10.1038 / cti.2014.31, can be used.

[0409] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes by, for example, centrifugation with a PERCOLLTM gradient or countercurrent centrifugation elution. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugate beads such as DYNABEADS® M-450 CD3 / CD28 T for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is about 30 minutes. In a further embodiment, the time is in the range of 30 minutes to 36 hours or longer and any integer value in between. In a further embodiment, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In another preferred embodiment, the time is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. Longer incubation times may be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times may increase the capture efficiency of CD8+ T cells. Therefore, subpopulations of T cells may be preferentially selected at the start of culture or at other points in the process simply by shortening or lengthening the time that T cells are bound to CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio (as further described herein). Furthermore, subpopulations of T cells may be preferentially selected at the start of culture or at other points in the process by increasing or decreasing the anti-CD3 and / or anti-CD28 antibody ratio on the beads or other surfaces. Those skilled in the art will recognize that multiple selections may also be used in the present invention. In one embodiment, it may be desirable to carry out a selection process and use "unselected" cells in the activation and proliferation process. "Unselected" cells may also be subjected to further selection.

[0410] Negative selection of T cell populations can be achieved by a combination of antibodies that target surface markers specific to cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies that target cell surface markers present in cells to be negatively selected. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In one embodiment, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in one embodiment, regulatory T cells are depleted by anti-C25...

Claims

1. A pharmaceutical composition comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain and a co-stimulatory signaling domain, the anti-BCMA binding domain comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region 1 (HC CDR1), heavy chain complementarity-determining region 2 (HC CDR2), and heavy chain complementarity-determining region 3 (HC CDR3), and a light chain variable region (VL) including light chain complementarity-determining region 1 (LC CDR1), light chain complementarity-determining region 2 (LC CDR2), and light chain complementarity-determining region 3 (LC CDR3), and the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are Sequence numbers 86, 87, 88, 95, 96, and 97 respectively It contains the amino acid sequence, and The aforementioned co-stimulus signaling domain includes a functional signaling domain derived from 4-1BB, and the aforementioned primary signaling domain includes a functional signaling domain derived from CD3 zeta. Pharmaceutical composition.

2. A pharmaceutical composition comprising an isolated CAR comprising an anti-BCMA binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain and a co-stimulus signaling domain, wherein the anti-BCMA binding domain comprises a heavy chain variable region (VH) including heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3), and a light chain variable region (VL) including light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 are Sequence numbers 86, 87, 88, 95, 96, and 97 respectively It contains the amino acid sequence, and The aforementioned co-stimulus signaling domain includes a functional signaling domain derived from 4-1BB, and the aforementioned primary signaling domain includes a functional signaling domain derived from CD3 zeta. Pharmaceutical composition.

3. (i) The VH comprises the amino acid sequence of SEQ ID NO: 93; and (ii) The VL contains the amino acid sequence of SEQ ID NO: 102, The pharmaceutical composition according to claim 1 or 2.

4. (i) comprising a nucleic acid sequence encoding the VH, wherein the nucleic acid sequence comprises the nucleic acid sequence of sequence number 260 or 94; and / or (ii) comprising a nucleic acid sequence encoding the VL, wherein the nucleic acid sequence comprises the nucleic acid sequence of sequence number 261 or 103, A pharmaceutical composition according to any one of claims 1 to 3.

5. The aforementioned VH and VL are, Sequence IDs 93 and 102, respectively. A pharmaceutical composition according to any one of claims 1 to 4, comprising the amino acid sequence.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-BCMA binding domain comprises a single-chain variable fragment (scFv) containing the amino acid sequence of SEQ ID NO:

105.

7. The pharmaceutical composition according to claim 1, wherein the anti-BCMA binding domain comprises scFv, the nucleic acid molecule comprises a nucleic acid sequence encoding scFv, and the nucleic acid sequence comprises the nucleic acid sequence of sequence number 253 or 106.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

107.

9. The pharmaceutical composition according to claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence of sequence number 259, 258, or 108.

10. (i) The VH and VL are connected by a linker, (ii) The VH and VL are connected by a linker, the linker comprising the amino acid sequence of SEQ ID NO: 63 or 104, A pharmaceutical composition according to any one of claims 1 to 9.

11. (i) The transmembrane domain includes the transmembrane domain of a protein selected from the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154; (ii) The transmembrane domain contains the amino acid sequence of SEQ ID NO: 6; or (iii) The nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence comprises the nucleic acid sequence of Sequence ID No.

17. The pharmaceutical composition according to claim 1.

12. (i) The anti-BCMA binding domain is connected to the transmembrane domain by a hinge region (ii) The anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, the hinge region comprising the amino acid sequence of SEQ ID NOs: 2, 3, or 4; or (iii) The anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, the nucleic acid molecule includes a nucleic acid sequence encoding the hinge region, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 13, 14, or 15. The pharmaceutical composition according to claim 1.

13. (i) The functional signaling domain derived from CD3 zeta contains the amino acid sequence of SEQ ID NO: 9 or 10; or (ii) The nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO:

256. The pharmaceutical composition according to claim 1.

14. (i) The signaling domain derived from 4-1BB contains the amino acid sequence of SEQ ID NO: 7; or (ii) The nucleic acid molecule comprises a nucleic acid sequence encoding the co-stimulus signaling domain, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO:

255. The pharmaceutical composition according to claim 1.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the functional signaling domain derived from 4-1BB comprises the amino acid sequence of SEQ ID NO: 7, and / or the functional signaling domain derived from CD3 zeta comprises the amino acid sequence of SEQ ID NO: 9 or 10.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the CAR further comprises a leader sequence containing the amino acid sequence of SEQ ID NO:

1.

17. The aforementioned CAR has the following characteristics: (i) When expressed in cells, the CAR activates NFAT signaling in BCMA-expressing cells in the presence of such cells; (ii) When the CAR is expressed in cells, it induces cytotoxicity in BCMA-expressing cells; and (iii) When the CAR is expressed in cells, it induces cytokine expression in the cells in the presence of BCMA-expressing cells. A pharmaceutical composition according to any one of claims 1 to 16, comprising one or more of the above.

18. A pharmaceutical composition comprising an isolated polypeptide molecule encoded by a nucleic acid molecule according to any one of claims 1 or 3 to 17.

19. (i) A vector comprising a nucleic acid molecule according to any one of claims 1 or 3 to 17, a nucleic acid molecule encoding a CAR according to any one of claims 2, 3, 5, 6, 8 and 10 to 17, or a nucleic acid molecule encoding a polypeptide molecule according to claim 18. (ii) A vector comprising a nucleic acid molecule according to any one of claims 1 or 3 to 17, a nucleic acid molecule encoding a CAR according to any one of claims 2, 3, 5, 6, 8 and 10 to 17, or a nucleic acid molecule encoding a polypeptide molecule according to claim 18, wherein the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenovirus vector or a retroviral vector, or (iii) A vector comprising a nucleic acid molecule according to any one of claims 1 or 3 to 17, a nucleic acid molecule encoding a CAR according to any one of claims 2, 3, 5, 6, 8 and 10 to 17, or a nucleic acid molecule encoding a polypeptide molecule according to claim 18, wherein the vector comprises an EF-1 promoter containing the nucleic acid sequence of SEQ ID NO:

11. A pharmaceutical composition containing the following:

20. A pharmaceutical composition comprising a cell containing a nucleic acid molecule according to any one of claims 1 or 3 to 17, a CAR according to any one of claims 2, 3, 5, 6, 8, and 10 to 17, a polypeptide molecule according to claim 18, or a vector according to claim 19.

21. A pharmaceutical composition according to claim 20, for use in inducing antitumor immunity in a subject.

22. The pharmaceutical composition according to claim 20, for use in treating subjects having a disease related to BCMA expression.

23. The pharmaceutical composition according to claim 22, wherein the cells are autologous T cells or allogeneic T cells.

24. The aforementioned diseases related to BCMA expression are (i) cancer or malignant tumor, or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or preleukemia, (ii) Non-cancer-related indications associated with BCMA expression The pharmaceutical composition according to claim 22 or 23.

25. The pharmaceutical composition according to any one of claims 22 to 24, wherein the disease is a blood cancer or a solid tumor.

26. The aforementioned diseases include acute leukemia, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and bone marrow A pharmaceutical composition according to any one of claims 22 to 25, selected from dysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, prostate cancer, pancreatic cancer, lung cancer, plasma cell proliferation disorder, monoclonal hypergammaglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma, systemic amyloid light chain amyloidosis, or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome) or a combination thereof.

27. The pharmaceutical composition according to any one of claims 22 to 26, wherein the disease is multiple myeloma.

28. (i) The pharmaceutical composition is formulated for administration in combination with a second therapeutic agent, (ii) The pharmaceutical composition is formulated for administration in combination with a PD-1 inhibitor. (iii) The pharmaceutical composition is formulated for administration in combination with a PD-L1 inhibitor. (iv) The pharmaceutical composition is formulated for administration in combination with a LAG-3 inhibitor. (v) The pharmaceutical composition is formulated for administration in combination with a TIM-3 inhibitor. (vi) The pharmaceutical composition is formulated for administration in combination with a CTLA-4 inhibitor. (vii) The pharmaceutical composition is formulated for administration in combination with interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both IL-15 polypeptide and IL-15Ra polypeptide. (viiii) The pharmaceutical composition is formulated for administration in combination with an interleukin-12 (IL-12) polypeptide, or (ix) The pharmaceutical composition is formulated for administration in combination with an mTOR inhibitor. A pharmaceutical composition according to any one of claims 25 to 27.

29. From the N-terminus to the C-terminus: (i) Leader array of sequence number 1; (ii) HC CDR1 of sequence number 86; (iii) HC CDR2 of sequence number 87; (iv) HC CDR3 of sequence number 88; (v) LC CDR1 of Sequence ID No. 95; (vi) LC CDR2 of sequence number 96; (vii) LC CDR3 of sequence number 97; (viiii) CD8 transmembrane domain and hinge of Sequence ID No. 202; (ix) Functional signaling domain of SEQ ID NO: 7, 4-1BB; and (x) Functional signaling domain of CD3 zeta in Sequence ID No. 10 A pharmaceutical composition comprising a chimeric antigen receptor (CAR) that binds to BCMA.

30. The pharmaceutical composition according to claim 29, comprising the VH region of SEQ ID NO: 93, the VL region of SEQ ID NO: 102, and / or the scFv of SEQ ID NO:

105.

31. A pharmaceutical composition according to claim 29 or 30, comprising the amino acid sequence of SEQ ID NO: 257 or SEQ ID NO:

107.

32. A pharmaceutical composition comprising an isolated nucleic acid molecule encoding the CAR molecule described in any one of claims 29 to 31.

33. A pharmaceutical composition comprising a vector containing a nucleic acid molecule encoding a chimeric antigen receptor (CAR) that binds to BCMA, wherein the CAR is arranged from the N-terminus to the C-terminus: (i) Leader array of sequence number 1; (ii) HC CDR1 of sequence number 86; (iii) HC CDR2 of sequence number 87; (iv) HC CDR3 of sequence number 88; (v) LC CDR1 of Sequence ID No. 95; (vi) LC CDR2 of sequence number 96; (vii) LC CDR3 of sequence number 97; (viiii) CD8 transmembrane domain and hinge of Sequence ID No. 202; (ix) Functional signaling domain of SEQ ID NO: 7, 4-1BB; and (x) Functional signaling domain of CD3 zeta in Sequence ID No. 10 A pharmaceutical composition containing the following:

34. A pharmaceutical composition comprising cells containing a chimeric antigen receptor (CAR) that binds to BCMA, wherein the CAR is oriented from the N-terminus to the C-terminus: (i) Leader array of sequence number 1; (ii) HC CDR1 of sequence number 86; (iii) HC CDR2 of sequence number 87; (iv) HC CDR3 of sequence number 88; (v) LC CDR1 of Sequence ID No. 95; (vi) LC CDR2 of sequence number 96; (vii) LC CDR3 of sequence number 97; (viiii) CD8 transmembrane domain and hinge of Sequence ID No. 202; (ix) Functional signaling domain of SEQ ID NO: 7, 4-1BB; and (x) Functional signaling domain of CD3 zeta in Sequence ID No. 10 A pharmaceutical composition containing the following:

35. A pharmaceutical composition comprising a cell containing a CAR molecule according to any one of claims 29 to 31, a nucleic acid molecule according to claim 32, or a vector according to claim 33.

36. A pharmaceutical composition according to any one of claims 29 to 31, 34, or 35, for use in treating diseases related to BCMA expression.

37. The pharmaceutical composition according to claim 36, wherein the disease associated with the expression of BCMA is multiple myeloma.

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